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

Genome-wide identification, characterization, and expression analysis of BZR transcription factor family in Gerbera hybrida | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genome-wide identification, characterization, and expression analysis of BZR transcription factor family in Gerbera hybrida Qishan Luo, Gan Huang, Xiaohui Lin, Xiaojing Wang, Yaqin Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5257183/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Feb, 2025 Read the published version in BMC Plant Biology → Version 1 posted 4 You are reading this latest preprint version Abstract Background: The BZR family genes encode plant-specific transcription factors that serve as pivotal regulators of plant BR signaling pathway, critically influencing plant growth and development. Results: In this study, we performed a genome-wide investigation of BZR family gene in gerbera in order to identify the key components of 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 structure. Phylogenetic analysis clustered these genes into two distinct subgroups. Analysis of cis-acting elementsrevealed their involvement in hormone response, stress response and growth regulation. Subcellular localization analysis indicated nuclear localization for GhBEH1 and GhBEH2, while the remainingfive genes exhibited dual localization in the nucleus and cytoplasm. Transactivation assay indicated GhBEH1 and GhBEH2 may function as transcriptional repressors, contrasting with the transcriptional activation observed for the other five genes. Notably, seven GhBEHs exhibits various expression patterns under different growth stages of ray florets and BR treatment conditions. While, GhBEH1 and GhBEH2 showed pronounced responsiveness to BR stimulation. Conclusion: Our work explains genome-wide identification, characterization, and expression analysis of BZR transcription factor family in gerbera, and hinted these seven GhBEHs is involved in regulating petal growth and development. These findings provide a basis for further studies on further research on the biological function of the BZR gene family in petal growth and a theoretical basis for future horticultural application in gerbera. BZR transcription factor Expression pattern Genome-wide analysis Petal growth Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 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 – 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 trigger a phosphorylation cascade that inhibit 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, BZR1 and 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 in specific cellular developmental processes [ 14 , 15 ]. As crucial phytohormones, BRs are essential for regulating various aspects of plant growth and development, primarily through mediating BZR transcription factors [ 2 , 4 , 5 , 8 , 10 , 16 – 18 ]. In Arabidopsis, the BZR gene family is divided into four 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 respectively [ 19 , 21 , 22 ], play pivotal roles as 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 their role in modulating downstream gene expression throughout various stages of plant growth and development [ 19 , 21 ]. Although current research has identified numerous BR-responsive genes [ 23 – 25 ], only a subset have been confirmed as direct targets of BZR1 or BZR2/BES1[ 26 , 27 ]. In rice, BZR1 collaborates with the RLA1/SMOS1 transcription factors 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, which are 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 have yet to be investigated. This suggests that BZR1 and BZR2/BES1 may regulate other transcription factors to modulate secondary BR-responsive genes and perform additional functions. Therefore, the identification and characterization of new BZR genes from various plant species represent 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 has been extensively employed as a model organism for investigating the organogenesis and developmental processes within the Asteraceae family [ 32 – 34 ]. Although numerous TFs governing petal growth have been extensively investigated in gerbera [ 35 – 41 ], research on the BZR TFs in gerbera remains limited, particularly 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 provide a thorough investigation of this gene family within the context of gerbera. Initially, the GhBEH (BZR/BES homologs) genes were 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 framework 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 the molecular mechanisms governing petal growth in gerbera. Methods Plant material and growth conditions Gerbera hybrida cultivar (cv.) Terra Regina and Shenzhen No. 5 were used in this study. 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 temperature of 22–28°C and relative humidity of 60–80%. Identification of 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 BZR gene family was annotated based on prior investigations [ 42 , 43 ], and redundant sequences were filtered out via sequence alignment conducted using DNA MAN software. Phylogenetic analysis and multiple sequence alignment The seven BZR proteins from gerbera 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 BZR gene family was constructed using MEGA5.1 software based on the neighbour-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 were obtained from the gerbera database by 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. Additionally, the gene structure was 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 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. To specifically label the nucleus, the nuclear localization marker NLS-mCherry was co-transfected into gerbera protoplasts. 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. Subsequently, the effector-GAL4 vector, REN vector, and reporter vector carrying 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. 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 along with 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) Quantitative RT-PCR (qRT-PCR) RNA extraction was performed followed by reverse transcription using the Toyobo reverse transcription kit, following the protocols described in a previous study [ 44 ]. qRT-PCR was conducted using a Bio-Rad Real-Time PCR system. The expression levels of the GhBEHs ( GhBEH1 , GhBEH2 , GhBEH3 , GhBEH4 , GhBEH5 , GhBEH6 , GhBEH7 ) were normalized to GhACTIN ( AJ763915 ) gene expression, as described in gerbera studies [ 38 , 45 ]. Relative expression levels were calculated by 2 −△△CT [ 46 ]. Each sample was analyzed with three biological 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 hours. 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 by 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 (GACN01037487.1) was designated GhBEH1 ( BES1/BZR1 homolog 1), while the remaining six homologs were designated GhBEH2 (GACN01006390.1), GhBEH3 (GACN0100383.1), GhBEH4 (GACN01030557.1), GhBEH5 (GACN01023011.1), GhBEH6 (GACN01045166.1), and GhBEH7 (GACN01022598.1) (Additional file 1). The full 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 (Figure 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. To elucidate the structural characteristics of GhBEH genes, we conducted an analysis of their exon-intron structure. And the results indicated that all GhBEH genes have a length of within 5 kb, with each gene containing a single intron, as illustrated in Figure 1B. 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 (Figure 2). The analysis 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. While 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. 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 (Figure 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 similar to those found in homologous sequences from Asteraceae plants, and contain conserved PEST motifs and proline residues. This residue plays a crucial role in the functionality of Arabidopsis BZR1 and BES1 [13]. 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 (Figure 4A), suggesting potential localization in both the nucleus and cytoplasm. 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 (Figure 5A). It has been reported that the mutation of 234th proline to leucine in Arabidopsis significantly enhances nuclear localization signal of BZR1, a pivotal regulator in its function [19, 47]. Therefore, we constructed YFP-GhBEH1 P192L , YFP-GhBEH2 P209L , YFP-GhBEH3 P219L , and YFP-GhBEH4 P206L vectors (designated as GhBEH1 P192L , GhBEH2 P209L , GhBEH3 P219L , and GhBEH4 P206L , respectively, upon introduction of the proline-to-leucine mutation) (Figure 4B), and transformed them into protoplasts to assess their subcellular localization. Our observations revealed that YFP-GhBEH1 P192L and YFP-GhBEH2 P209L maintained their nuclear localization (Figure 4C and Figure 5B). In contrast, the nuclear localization of YFP-GhBEH3 P219L and YFP-GhBEH4 P206L 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, it has been demonstrated that 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]. To investigate the transcriptional activity of the BZR family member in gerbera, transactivation assays were initially performed in yeast. The seven GhBEH genes fused with 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. While, the remaining five members exhibited normal growth on SD/-LTH and SD/-LTHA, consistent with the positive control, indicating t 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 (Figure 6A). These experiments collectively demonstrate that GhBEH1 and GhBEH2 lack self-activation activity, consistent with observations in Arabidopsis BZR1 and BES1, thus suggesting functional homology. In contrast, GhBEH3, GhBEH4, GhBEH5, GhBEH6, and GhBEH7 all exhibit strong self-activation activity. Meanwhile, dual-luciferase assays was conducted to further substantiate the transcriptional activity of GhBEHs (Figure 6B). The results showed that GhBEH1 and GhBEH2 had significantly lower ratios compared to the control group. Conversely, GhBEH3 through GhBEH7 demonstrated ratios significantly higher than the control (Figure 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. A total of 147 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 (Figure 7). Predominantly represented were 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 stress processes, thereby potentially influencing plant growth and stress resistance. Notably, different genes not only contain different numbers of phytohormone response elements, such as GhBEH1 (4 elements) and GhBEH4 (3 elements), but also include elements responsive to different types of 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. Expression analysis of GhBEHs under BR treatment The BZR family genes are the key transcription factors in plant BR signaling pathway. In the 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 responds rapidly to BL, showing significant upregulation at 0.5 h post-treatment, gradually declining by 4 h (Figure 8A). A corresponding heatmap provides a visual representation of these expression dynamics. The varied changes in GhBEH expression levels post-BR treatment suggest diverse response timings and intensities to BR. 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 both up-regulation and down-regulation (Figure 9). These findings suggest that GhBEH1 and GhBEH2 exhibit obvious response to BR, indicating their strong capability to respond to both endogenous and exogenous BR stimuli. Expression pattern of GhBEHs in different petal growth stages The early study divided the petal growth and developmental stages into 11 phases in gerbera [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 (Figure 10A, 10B). In contrast, GhBEH3 , GhBEH4 , GhBEH5 , and GhBEH6 consistently exhibited significant downregulation expression throughout stages 1-11. While, GhBEH7 did not show significant changes in expression levels across these stages. 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 (Figure 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 approximately 1/20th of GhBEH1 . These data suggest different degrees of influence of these genes on petal growth of ray floret. Discussion The BZR family comprises pivotal transcription factors in plants that play a crucial role in the BR signaling pathway, governing plant growth, development, and participating in abiotic stress responses. [ 50 – 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. This distinction suggests potential functional specialization among these genes, indicating their specific roles within distinct cellular compartments in vivo. BR have been documented to induce the dephosphorylation of Arabidopsis BES1 and BZR1, thereby promoting their nuclear accumulation [ 11 , 42 , 43 ]. Furthermore, studies on Arabidopsis BZR1 have demonstrated that the substitution of proline with leucine at position 234 enhances its nuclear localization signal, thereby influencing its functional activity [ 19 , 47 ]. In our study, we observed that GhBEH1 P192L and GhBEH2 P209L maintained their nuclear localization patterns unchanged. In contrast, the nuclear localization of GhBEH3 P219L and GhBEH4 P206L 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 their functional roles. Cis-acting elements are non-coding DNA sequences situated within promoter regions, exerting influence over gene expression and gene function [ 55 ]. In this study, a comprehensive analysis identified a total of 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 7 GhBEH s possess motifs related to stress responsiveness, suggesting that their role in modulating gerbera's adaptation to 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 [ 56 ]. 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 [ 57 ]. 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 greatest 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 mechanisms of cell proliferation and expansion. Early stages of petal growth are characterized by changes in cell number, while middle and late stages emphasize cell size dynamics [ 58 – 60 ]. BR play a pivotal role in promoting cell elongation and expansion, thereby exerting significant influence on overall petal growth [ 8 , 61 , 62 ]. 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 in the later stage. These observations suggest that GhBEH3 , GhBEH4 , GhBEH5 and GhBEH6 likely contribute to early petal growth, potentially through involvement in petal cells proliferation. Conversely, GhBEH1 and GhBEH2 may play roles in the middle and late stages of petal development, potentially participating in processes associated with cell elongation and expansion. Nevertheless, further experimental evidence is necessary to confirm these hypotheses. Conclusions In this study, the whole genome of the gerbera BZR family gene was investigated and seven BZR genes named GhBEH1-7 were identified. Researches 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 the middle and late stages of petal growth. It is noteworthy that all seven GhBEHs respond to BR, especially GhBEH1 and GhBEH2 , may play 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. 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 Declarations Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/ Acknowledgements We thank Pro. Paula Elomaa and Dr. Teng Zhang for instructing some experiments and giving advices for manuscript. We thank Dr. Fan Li for providing the genome of gerbera. We are grateful to Cambridge Academic Manuscripts (www. Cambridge Academic Manuscripts. com) for manuscript editing. Author 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. Funding This work was supported by Natural Science Foundation of Guangdong Province (2023A1515012864, 2021A1515012479), 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). Availability of data and materials All data generated or analysed during this study are included in this published article. Ethics approval and consent to participate Not applicable. Clinical trial number Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests Author details 1 Guangdong Provincial Key Laboratory of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou 510631, China. 2 College of Landscape Architecture and Art, Henan Agricultural University, Zhengzhou 450002, Henan Province, China. References Li J,J C. A putative leucine-rich repeat receptor kinase involved in brassinosteroid signal transduction. Cell. 1997; 90(5): 929-938. https://doi.org/10.1016/s0092-8674(00)80357-8. Clouse S. Molecular genetic studies confirm the role of brassinosteroids in plant growth and development. Plant J. 1996; 10(1): 1-8. https://doi.org/10.1046/j.1365-313X.1996.10010001.x. Mandava N B. Plant growth-promoting brassinosteroids. Annu rev plant physiol plant mol biol. 1988; 39: 23-52. Clouse S. Brassinosteroid Signal Transduction: From Receptor Kinase Activation to Transcriptional Networks Regulating Plant Development. Plant Cell. 2011; 23(4): 1219-1230. https://doi.org/10.1105/tpc.111.084475. Sun Y, Fan X, Cao D, Tang W, He K, Zhu J, He J, Bai M, Zhu S, Oh E, et al. Integration of Brassinosteroid Signal Transduction with the Transcription Network for Plant Growth Regulation in Arabidopsis. Dev Cell. 2010; 19(5): 765-777. https://doi.org/10.1016/j.devcel.2010.10.010. Oh E, Zhu J, Bai M, Arenhart R, Sun Y,Wang Z. Cell elongation is regulated through a central circuit of interacting transcription factors in the Arabidopsis hypocotyl. ELife. 2014; 3: e03031. https://doi.org/10.7554/eLife.03031. Thummel C,Chory J. Steroid signaling in plants and insects--common themes, different pathways. Genes Dev. 2002; 16(24): 3113-3129. https://doi.org/10.1101/gad.1042102. Kim T,Wang Z. Brassinosteroid Signal Transduction from Receptor Kinases to Transcription Factors. Annu Rev Plant Biol. 2010; 61(1): 681-704. https://doi.org/10.1146/annurev.arplant.043008.092057. Zhu J-Y, Sae-Seaw J,Wang Z-Y. Brassinosteroid signalling. Dev. 2013; 140(8): 1615-1620. https://doi.org/10.1242/dev.060590. Kim T, Guan S, Sun Y, Deng Z, Tang W, Shang J, Sun Y, Burlingame A,Wang Z. Brassinosteroid signal transduction from cell-surface receptor kinases to nuclear transcription factors. Nat Cell Biol. 2009; 11(10): 1254-1260. https://doi.org/10.1038/ncb1970. He J, Gendron J, Yang Y, Li J,Wang Z. The GSK3-like kinase BIN2 phosphorylates and destabilizes BZR1, a positive regulator of the brassinosteroid signaling pathway in Arabidopsis. Proc Natl Acad Sci. 2002; 99(15): 10185-10190. https://doi.org/10.1073/pnas.152342599. Tang W, Yuan M, Wang R, Yang Y, Wang C, Oses-Prieto J A, Kim T-W, Zhou H-W, Deng Z, Gampala S S, et al. PP2A activates brassinosteroid-responsive gene expression and plant growth by dephosphorylating BZR1. Nat Cell Biol. 2011; 13(2): 124-131. https://doi.org/10.1038/ncb2151. Wang Z, Bai M, Oh E,Zhu J. Brassinosteroid Signaling Network and Regulation of Photomorphogenesis. Annu Rev Genet. 2012; 46(1): 701-724. https://doi.org/10.1146/annurev-genet-102209-163450. Youn J,Kim T. Functional Insights of Plant GSK3-like Kinases: Multi-Taskers in Diverse Cellular Signal Transduction Pathways. Mol Plant. 2015; 8(4): 552-565. https://doi.org/10.1016/j.molp.2014.12.006. Li J,Nam K, Hee,. Regulation of Brassinosteroid Signaling by a GSK3/SHAGGY-Like Kinase. Science. 2002; 295(5558): 1299-1301. https://doi.org/10.1126/science.1065769. Clouse S,Sasse J. BRASSINOSTEROIDS: Essential Regulators of Plant Growth and Development. Annu Rev Plant Biol. 1998; 49: 427-451. https://doi.org/10.1146/annurev.arplant.49.1.427. Tang W, Tae-Wuk Kim, Juan A, Oses-Prieto, Yu Sun, Zhiping Deng, Shengwei Zhu, Ruiju Wang, Alma L. Burlingame,Wang Z-Y. BSKs Mediate Signal Transduction from the Receptor Kinase BRI1 in Arabidopsis. Science. 2008; 321(5888): 557-560. https://doi.org/10.1126/science.1156973. Ye H, Li L,Yin Y. Recent Advances in the Regulation of Brassinosteroid Signaling and Biosynthesis PathwaysF. J Integr Plant Biol. 2011; 53(6): 455-468. https://doi.org/10.1111/j.1744-7909.2011.01046.x. Yin Y, Vafeados D, Tao Y, Yoshida S, Asami T,Chory J. A New Class of Transcription Factors Mediates Brassinosteroid-Regulated Gene Expression in Arabidopsis. Cell. 2005; 120(2): 249-259. https://doi.org/10.1016/j.cell.2004.11.044. Yu X, Li L, Zola J, Aluru M, Ye H, Foudree A, Guo H, Anderson S, Aluru S, Liu P, et al. A brassinosteroid transcriptional network revealed by genome‐wide identification of BESI target genes in Arabidopsis thaliana. Plant J. 2011; 65(4): 634-646. https://doi.org/10.1111/j.1365-313X.2010.04449.x. He J, Gendron J, Sun Y, Gampala S, Gendron N, Sun C,Wang Z. BZR1 is a transcriptional repressor with dual roles in brassinosteroid homeostasis and growth responses. Science. 2005; 307(5715): 1634-1638. https://doi.org/10.1126/science.1107580. Oh E, Zhu J, Ryu H, Hwang I,Wang Z. TOPLESS mediates brassinosteroid-induced transcriptional repression through interaction with BZR1. Nat Commun. 2014; 5(1): 4140. https://doi.org/10.1038/ncomms5140. Goda H, Shinichiro S, Tadao A, Shozo F, Yukihisa S,Shigeo Y. Comprehensive Comparison of Auxin-Regulated and Brassinosteroid-Regulated Genes in Arabidopsis. Plant Physiol. 2004; 134(4): 1555-1573. https://doi.org/10.1104/pp.103.034736. Nemhauser J, Mockler T,Chory J. Interdependency of Brassinosteroid and Auxin Signaling in Arabidopsis. PLoS Biol. 2004; 2(9): e258. https://doi.org/10.1371/journal.pbio.0020258. Vert G, Nemhauser J, Geldner N, Hong F,Chory J. Molecular Mechanisms of Steroid Hormone Signaling in Plants. Annual Review of Cell and Developmental Biology. 2005; 21(1): 177-201. https://doi.org/10.1146/annurev.cellbio.21.090704.151241. Zhang L, Bai M, Wu J, Zhu J, Wang H, Zhang Z, Wang W, Sun Y, Zhao J, Sun X, et al. Antagonistic HLH/bHLH Transcription Factors Mediate Brassinosteroid Regulation of Cell Elongation and Plant Development in Rice andArabidopsis Plant Cell. 2009; 21(12): 3767-3780. https://doi.org/10.1105/tpc.109.070441. Li L, Yu X, Thompson A, Guo M, Yoshida S, Asami T, Chory J,Yin Y. Arabidopsis MYB30 is a direct target of BES1 and cooperates with BES1 to regulate brassinosteroid‐induced gene expression. Plant J. 2009; 58(2): 275-286. https://doi.org/10.1111/j.1365-313X.2008.03778.x. Qiao S, Sun S, Wang L, Wu Z, Li C, Li X, Wang T, Leng L, Tian W, Lu T, et al. The RLA1/SMOS1 Transcription Factor Functions with OsBZR1 to Regulate Brassinosteroid Signaling and Rice Architecture. Plant Cell. 2017; 29(2): 292-309. https://doi.org/10.1105/tpc.16.00611. Zhang X, Guo W, Du D, Pu L,Zhang C. Overexpression of a maize BR transcription factor ZmBZR1 in Arabidopsis enlarges organ and seed size of the transgenic plants. Plant Sci. 2020; 292: 110378. https://doi.org/10.1016/j.plantsci.2019.110378. Cheng P, Liu Y, Yang Y, Chen H, Cheng H, Hu Q, Zhang Z, Gao J, Zhang J, Ding L, et al. CmBES1 is a regulator of boundary formation in chrysanthemum ray florets. Hortic Res. 2020; 7(1): 129. https://doi.org/10.1038/s41438-020-00351-8. Ma X, Yuan Y, Li C, Wu Q, He Z, Li J,Zhao M. Brassinosteroids suppress ethylene-induced fruitlet abscission through LcBZR1/2-mediated transcriptional repression of LcACS1/4 and LcACO2/3 in litchi. Hortic Res. 2022; 8: 105. https://doi.org/10.1038/s41438-021-00540-z. Laitinen R A E, Broholm S, Albert V A, Teeri T H,Elomaa P. Patterns of MADS-box gene expression mark flower-type development in Gerbera hybrida(Asteraceae). BMC Plant Biol. 2006; 6(1): 11. https://doi.org/10.1186/1471-2229-6-11. Teeri T H, Uimari A, Kotilainen M, Laitinen R, Help H, Elomaa P,Albert V A. Reproductive meristem fates in Gerbera. J Exp Bot. 2006; 57(13): 3445-3455. https://doi.org/10.1093/jxb/erl181. Zhang T, Cieslak M, Owens A, Wang F, Broholm S K, Teeri T H, Elomaa P,Prusinkiewicz P. Phyllotactic patterning of gerbera flower heads. Proc Natl Acad Sci. 2021; 118(13): e2016304118. https://doi.org/10.1073/pnas.2016304118. Lin X, Huang S, Huang G, Chen Y, Wang X,Wang Y. 14-3-3 Proteins Are Involved in BR-Induced Ray Petal Elongation in Gerbera hybrida. Front Plant Sci. 2021; 12: 718091. https://doi.org/10.3389/fpls.2021.718091. Huang G, Han M, Jian L, Chen Y, Sun S, Wang X,Wang Y. An ETHYLENE INSENSITIVE3-LIKE1 Protein Directly Targets the GEG Promoter and Mediates Ethylene-Induced Ray Petal Elongation in Gerbera hybrida. Front Plant Sci. 2020; 10: 01737. https://doi.org/10.3389/fpls.2019.01737. Ren G, Li L, Patra B, Li N, Zhou Y, Zhong C, Wang Y, Yuan L, Wang X,Melzer R. The transcription factor GhTCP7 suppresses petal expansion by interacting with the WIP-type zinc finger protein GhWIP2 in Gerbera hybrida. J Exp Bot. 2023; 74(14): 4093-4109. https://doi.org/10.1093/jxb/erad152. Ren G, Li L, Huang Y, Wang Y, Zhang W, Zheng R, Zhong C,Wang X. GhWIP2, a WIP zinc finger protein, suppresses cell expansion in Gerbera hybrida by mediating crosstalk between gibberellin, abscisic acid, and auxin. New Phytol. 2018; 219(2): 728-742. https://doi.org/10.1111/nph.15175. Han M, Jin X, Yao W, Kong L, Huang G, Tao Y, Li L, Wang X,Wang Y. A Mini Zinc-Finger Protein (MIF) from Gerbera hybrida Activates the GASA Protein Family Gene, GEG, to Inhibit Ray Petal Elongation. Front Plant Sci. 2017; 8: 01649. https://doi.org/10.3389/fpls.2017.01649. Zhong C, Tang Y, Pang B, Li X, Yang Y, Deng J, Feng C, Li L, Ren G, Wang Y, et al. The R2R3-MYB transcription factor GhMYB1a regulates flavonol and anthocyanin accumulation in Gerbera hybrida. Hortic Res. 2020; 7(1): 78. https://doi.org/10.1038/s41438-020-0296-2. Jiang R, Yuan W, Yao W, Jin X, Wang X,Wang Y. A regulatory GhBPE-GhPRGL module maintains ray petal length in Gerbera hybrida. Mol Hortic. 2022; 2(1): 9. https://doi.org/10.1186/s43897-022-00030-3. Wang Z, Nakano T, Gendron J, He J, Chen M, Vafeados D, Yang Y, Fujioka S, Yoshida S, Asami T, et al. Nuclear-localized BZR1 mediates brassinosteroid-induced growth and feedback suppression of brassinosteroid biosynthesis. Dev Cell. 2002; 2(4): 505-513. https://doi.org/10.1016/s1534-5807(02)00153-3. Yin Y, Wang Z, Mora-Garcia S, Li J, Yoshida S, Asami T,Chory J. BES1 accumulates in the nucleus in response to brassinosteroids to regulate gene expression and promote stem elongation. Cell. 2002; 109(2): 181-191. https://doi.org/10.1016/s0092-8674(02)00721-3. Su M, Huang G, Zhang Q, Wang X, Li C, Tao Y, Zhang S, Lai J, Yang C,Wang Y. The LEA protein, ABR, is regulated by ABI5 and involved in dark-induced leaf senescence in Arabidopsis thaliana. Plant Sci. 2016; 247: 93-103. https://doi.org/10.1016/j.plantsci.2016.03.009. Kuang Q, Li L, Peng J, Sun S,Wang X. Transcriptome Analysis of Gerbera hybrida Ray Florets: Putative Genes Associated with Gibberellin Metabolism and Signal Transduction. PLoS ONE. 2013; 8(3): e57715. https://doi.org/10.1371/journal.pone.0057715. Livak K,Schmittgen T. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2 −ΔΔCT Method. Methods. 2001; 25(4): 402-408. https://doi.org/10.1006/meth.2001.1262. Ryu H, Kim K, Cho H, Park J, Choe S,Hwang I. Nucleocytoplasmic Shuttling of BZR1 Mediated by Phosphorylation Is Essential inArabidopsisBrassinosteroid Signaling. Plant Cell. 2007; 19(9): 2749-2762. https://doi.org/10.1105/tpc.107.053728. Huang G, Han M, Yao W,Wang Y. Transcriptome analysis reveals the regulation of brassinosteroids on petal growth in Gerbera hybrida. PeerJ. 2017; 5: e3382. https://doi.org/10.7717/peerj.3382. Helariutta Y, Elomaa P, Mika K, Seppänen P,H. Teeri T. Cloning of cDNA coding for dihydroflavonol-4-reductase (DFR) and characterization of dfr expression in the corollas of Gerbera hybrida var. Regina (Compositae). Plant Mol. Biol. 1993; 22: 183-193. https://doi.org/10.1007/bf00014927. Saha G, Park J, Jung H, Ahmed N, Kayum M, Kang J,Nou I. Molecular characterization of BZR transcription factor family and abiotic stress induced expression profiling in Brassica rapa. Plant Physiol Biochem. 2015; 92: 92-104. https://doi.org/10.1016/j.plaphy.2015.04.013. Sun F, Yu H, Qu J, Cao Y, Ding L, Feng W, Khalid M, Li W,Fu F. Maize ZmBES1/BZR1-5 Decreases ABA Sensitivity and Confers Tolerance to Osmotic Stress in Transgenic Arabidopsis. Int J Mol Sci. 2020; 21(3): 996. https://doi.org/10.3390/ijms21030996. Yin Y, Qin K, Song X, Zhang Q, Zhou Y, Xia X,Yu J. BZR1 Transcription Factor Regulates Heat Stress Tolerance Through FERONIA Receptor-Like Kinase-Mediated Reactive Oxygen Species Signaling in Tomato. Plant Cell Physiol. 2018; 59(11): 2239–2254 https://doi.org/10.1093/pcp/pcy146. Tsang T, Hsueh Y, Wei E, Lundy D, Cheng B, Chen Y, Wang S,Hsieh P. Subcellular Localization of Survivin Determines Its Function in Cardiomyocytes. Theranostics. 2017; 7(18): 4577-4590. https://doi.org/10.7150/thno.20005. Plotnikov A, Zehorai E, Procaccia S,Seger R. The MAPK cascades: Signaling components, nuclear roles and mechanisms of nuclear translocation. Acta Bioch Bioph Sin. 2011; 1813(9): 1619-1633. https://doi.org/10.1016/j.bbamcr.2010.12.012. Zhao Q, Luo Z, Chen J, Jia H, Ai P, Chen A, Li Y,Xu G. Characterization of two cis-acting elements, P1BS and W-box, in the regulation of OsPT6 responsive to phosphors deficiency. Plant Growth Regul. 2021; 93(3): 303-310. https://doi.org/10.1007/s10725-020-00688-z. He G, Liu J, Dong H,Sun J. The Blue-Light Receptor CRY1 Interacts with BZR1 and BIN2 to Modulate the Phosphorylation and Nuclear Function of BZR1 in Repressing BR Signaling in Arabidopsis. Mol Plant. 2019; 12(5): 689-703. https://doi.org/10.1016/j.molp.2019.02.001. Galstyan A,Nemhauser J L. Auxin promotion of seedling growth via ARF5 is dependent on the brassinosteroid‐regulated transcription factors BES1 and BEH4. Plant Direct. 2019; 3(9). https://doi.org/10.1002/pld3.166. Zhang L, Li L, Wu J, Peng J, Zhang L,Wang X. Cell expansion and microtubule behavior in ray floret petals of Gerbera hybrida: Responses to light and gibberellic acid. Photochem Photobiol Sci. 2012; 11(2): 279-288. https://doi.org/10.1039/c1pp05218g. Xie Z, Nolan T, Jiang H, Tang B, Zhang M, Li Z,Yin Y. The AP2/ERF Transcription Factor TINY Modulates Brassinosteroid-Regulated Plant Growth and Drought Responses in Arabidopsis. Plant Cell. 2019; 31(8): 1788-1806. https://doi.org/10.1105/tpc.18.00918. Yang Y, Chen B, Dang X, Zhu L, Rao J, Ren H, Lin C, Qin Y,Lin D. Arabidopsis IPGA1 is a microtubule-associated protein essential for cell expansion during petal morphogenesis. J Exp Bot. 2019; 70(19): 5231-5243. https://doi.org/10.1093/jxb/erz284. Wang F, Bai M, Deng Z, Oses-Prieto J, Burlingame A, Lu T, Chong K,Wang Z. Proteomic Study Identifies Proteins Involved in Brassinosteroid Regulation of Rice Growth. J Integr Plant Biol. 2010; 52(12): 1075-1085. https://doi.org/10.1111/j.1744-7909.2010.00992.x. Chen W, Lv M, Wang Y, Wang P, Cui Y, Li M, Wang R, Gou X,Li J. BES1 is activated by EMS1-TPD1-SERK1/2-mediated signaling to control tapetum development in Arabidopsis thaliana. Nat Commun. 2019; 10(1): 4164. https://doi.org/10.1038/s41467-019-12118-4. Additional Declarations No competing interests reported. Supplementary Files Additionalfile1.docx Additional file 1: Full uncropped Gels and Blots image of GhBEH1-7 . Additionalfile2.docx Additional file 2: cDNA sequence and protein sequence of GhBEH1-7 . Cite Share Download PDF Status: Published Journal Publication published 04 Feb, 2025 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Revision requested 29 Oct, 2024 Editor assigned by journal 28 Oct, 2024 Submission checks completed at journal 28 Oct, 2024 First submitted to journal 13 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5257183","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":371728885,"identity":"0d370aea-b465-4752-9b63-425d612e3366","order_by":0,"name":"Qishan Luo","email":"","orcid":"","institution":"South China Normal University","correspondingAuthor":false,"prefix":"","firstName":"Qishan","middleName":"","lastName":"Luo","suffix":""},{"id":371728886,"identity":"804f1876-b6ea-447b-b353-2e7d0f0a5a41","order_by":1,"name":"Gan Huang","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Gan","middleName":"","lastName":"Huang","suffix":""},{"id":371728887,"identity":"63c7c771-3a81-4d36-8992-b79b6194d4c7","order_by":2,"name":"Xiaohui Lin","email":"","orcid":"","institution":"South China Normal University","correspondingAuthor":false,"prefix":"","firstName":"Xiaohui","middleName":"","lastName":"Lin","suffix":""},{"id":371728888,"identity":"a8860877-e314-4707-ae99-0dd7715b7b99","order_by":3,"name":"Xiaojing Wang","email":"","orcid":"","institution":"South China Normal University","correspondingAuthor":false,"prefix":"","firstName":"Xiaojing","middleName":"","lastName":"Wang","suffix":""},{"id":371728889,"identity":"903144bd-4ef6-4dca-91a0-feb8002ae4a2","order_by":4,"name":"Yaqin Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYBADHgb2BjjHgEgtPAdI1MLAIJFApBaD42cPv+apsZYxl3ydwPij5k5iA3vzNgmGmju4tZzJS7PmOZbOYzk7dwMzz7FniQ08x8okGI49w6nF7ECOmTEP22Eeg9tALYwNhxMbJHLMJIAM3FrOvwFq+QfUcvPsBsafIC3ybwhouZFj/Ji3DajlBu8GBl6wLTz4tdjfeGPGOLcvncfgTO6GwzzHDhu38aQVWyQcw61Fsj/H+MObb9b2wKDb+PBHzWHZfvbDG298qMGtBQjYpHgYmMGsA2AuiEjAp4GBgfnjD6iWUTAKRsEoGAVYAQDOmlbpXxvOqgAAAABJRU5ErkJggg==","orcid":"","institution":"South China Normal University","correspondingAuthor":true,"prefix":"","firstName":"Yaqin","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-10-14 01:08:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5257183/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5257183/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-025-06177-7","type":"published","date":"2025-02-04T15:56:58+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":68555894,"identity":"5cc157db-7082-419b-87f5-868228abcd14","added_by":"auto","created_at":"2024-11-08 13:29:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":225533,"visible":true,"origin":"","legend":"\u003cp\u003eChromosomal distribution and gene structures of \u003cem\u003eGhBEHs\u003c/em\u003e. (\u003cstrong\u003eA\u003c/strong\u003e) Chromosomal distribution of seven \u003cem\u003eGhBEHs\u003c/em\u003e in the gerbera genome. Red line and arrow indicate the position and the direction of seven \u003cem\u003eGhBEH\u003c/em\u003e genes respectively. (\u003cstrong\u003eB\u003c/strong\u003e) The structure of\u003cem\u003e GhBEHs\u003c/em\u003e visualized by GSDS 2.0. The exons are displayed in yellow colors and the lines between boxes represent introns\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5257183/v1/05dc648040d1b8b9dc085c18.png"},{"id":68556218,"identity":"b1886a3e-9de5-4b9d-b271-ff32afd16679","added_by":"auto","created_at":"2024-11-08 13:37:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5997907,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogeny 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 the blue dots. The scale bar represented 0.1 substitutions per site\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5257183/v1/6622766383b971fb58f3306c.png"},{"id":68554753,"identity":"1b1f672c-d5e1-4ad1-b5bf-8764cf02b1e1","added_by":"auto","created_at":"2024-11-08 13:21:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":15620856,"visible":true,"origin":"","legend":"\u003cp\u003eMultiple sequence alignment analysis of the BZRs in gerbera and other species. \u003cem\u003eArabidopsis thaliana\u003c/em\u003e: AtBZR1, AtBES1, AtBEH1, AtBEH2, AtBEH3, AtBEH4. \u003cem\u003eGerbera hybrida\u003c/em\u003e: GhBEH1, GhBEH2, GhBEH3, GhBEH4, GhBEH5, GhBEH6, GhBEH7. \u003cem\u003eHelianthus annuus\u003c/em\u003e: HaBEH1. \u003cem\u003eTanacetum cinerariifolium\u003c/em\u003e: TcBEH1. \u003cem\u003eMikania micrantha\u003c/em\u003e: McBEH1. \u003cem\u003eLactuca sativa\u003c/em\u003e: LsBEH1. PEST motifs, putative phosphorylation regions, nuclear localization sequences (NLS) and DNA binding domains (DB) are marked with underline\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5257183/v1/717beec0571c835b8a322a5d.png"},{"id":68555898,"identity":"4753451b-3e3b-4df2-8152-1faf10b140f3","added_by":"auto","created_at":"2024-11-08 13:29:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":929487,"visible":true,"origin":"","legend":"\u003cp\u003eSubcellular localization analysis of GhBEH proteins. (\u003cstrong\u003eA\u003c/strong\u003e) Subcellular localization of GhBEH proteins. Gerbera protoplasts transiently expressed GhBEH-YFP fusion proteins were observed through the laser scanning confocal microscope. (\u003cstrong\u003eB\u003c/strong\u003e) The schematic representation of proline mutation in GhBEH1, GhBEH2, GhBEH3 and GhBEH4 (GhBEH1\u003csup\u003eP192L\u003c/sup\u003e, GhBEH2\u003csup\u003eP209L\u003c/sup\u003e, GhBEH3\u003csup\u003eP219L\u003c/sup\u003e, GhBEH4\u003csup\u003eP206L\u003c/sup\u003e). (\u003cstrong\u003eC\u003c/strong\u003e) Subcellular localization of GhBEH1\u003csup\u003eP192L\u003c/sup\u003e, GhBEH2\u003csup\u003eP209L\u003c/sup\u003e, GhBEH3\u003csup\u003eP219L\u003c/sup\u003e and GhBEH4\u003csup\u003eP206L\u003c/sup\u003e. Scale bar = 10 μm\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5257183/v1/e28815d96a122c8e6871f052.png"},{"id":68554747,"identity":"2e746a85-0db8-4fa4-b059-cd36249e9b67","added_by":"auto","created_at":"2024-11-08 13:21:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":776867,"visible":true,"origin":"","legend":"\u003cp\u003eThe nuclear localization confirmation of GhBEH proteins. (\u003cstrong\u003eA\u003c/strong\u003e) 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. (\u003cstrong\u003eB\u003c/strong\u003e) The nuclear localization of GhBEH1\u003csup\u003eP192L\u003c/sup\u003e, GhBEH2\u003csup\u003eP209L\u003c/sup\u003e, GhBEH3\u003csup\u003eP219L\u003c/sup\u003e and GhBEH4\u003csup\u003eP206L\u003c/sup\u003e. Scale bar = 10 μm\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5257183/v1/fae33cceaea63b2dc92691ee.png"},{"id":68555897,"identity":"a4ad501b-7be1-421d-9f1a-4f319890a245","added_by":"auto","created_at":"2024-11-08 13:29:45","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":507205,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptional activity analysis of GhBEHs. (\u003cstrong\u003eA\u003c/strong\u003e) 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. (\u003cstrong\u003eB\u003c/strong\u003e) The vector construction of effector and reporters used in the dual-luciferase activity assay. (\u003cstrong\u003eC\u003c/strong\u003e) Transcriptional activity analysis of GhBEHs by dual-luciferase assay. Values were the means ± SD from three biological replicates; ∗ indicated a significant difference at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 by a Student’s \u003cem\u003et\u003c/em\u003e-test\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5257183/v1/e20e572567e1541256460bce.png"},{"id":68554748,"identity":"c02e9bff-7eb1-4f34-adc7-80fba55b9c27","added_by":"auto","created_at":"2024-11-08 13:21:45","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":560031,"visible":true,"origin":"","legend":"\u003cp\u003eCis-element analysis in the promoters of \u003cem\u003eGhBEHs\u003c/em\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5257183/v1/0d25bc7d06a0f707ae88ffe3.png"},{"id":68554746,"identity":"c2f54249-425b-494f-8c32-0acd458166e6","added_by":"auto","created_at":"2024-11-08 13:21:45","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":443353,"visible":true,"origin":"","legend":"\u003cp\u003eDynamic expression changes of \u003cem\u003eGhBEHs\u003c/em\u003e with different time of BL treatments. (\u003cstrong\u003eA\u003c/strong\u003e) The expression level of \u003cem\u003eGhBEHs\u003c/em\u003e under different time of BL treatments by qRT-PCR. The expression level of \u003cem\u003eGhBEHs\u003c/em\u003e at 0h was normalized to 1. (\u003cstrong\u003eB\u003c/strong\u003e) Heatmap of the expression level of \u003cem\u003eGhBEHs\u003c/em\u003e under different time of BL treatments. 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\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5257183/v1/d7dc2556412bbd23ef0ab292.png"},{"id":68554751,"identity":"1e6024f8-aa16-4da0-8218-ccc26cfbbdd0","added_by":"auto","created_at":"2024-11-08 13:21:45","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":368998,"visible":true,"origin":"","legend":"\u003cp\u003eDynamic expression changes of \u003cem\u003eGhBEHs\u003c/em\u003e with different time of BRZ or PPZ treatments.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) The expression heatmap of \u003cem\u003eGhBEHs\u003c/em\u003e under different time of BRZ treatments by qRT-PCR.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eB\u003c/strong\u003e) The expression heatmap of \u003cem\u003eGhBEHs\u003c/em\u003e under different time of PPZ treatments by qRT-PCR.\u003c/p\u003e\n\u003cp\u003eThe expression levels mapped to a color gradient from low (green) to high expression (red) are shown at the right of the figure\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-5257183/v1/5ce5b12ea07a6be2929eee0b.png"},{"id":68554749,"identity":"6dc4fdad-d088-4bd1-9972-18f58502cacd","added_by":"auto","created_at":"2024-11-08 13:21:45","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":634234,"visible":true,"origin":"","legend":"\u003cp\u003eExpression patterns of \u003cem\u003eGhBEHs\u003c/em\u003e during different ray floret growth stages in gerbera. (\u003cstrong\u003eA\u003c/strong\u003e) Expression level of \u003cem\u003eGhBEHs\u003c/em\u003e during different floret growth stages by qRT-PCR. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11 represent differnt growth stages of ray florets respectively. The expression level of \u003cem\u003eGhBEHs\u003c/em\u003ein Ray1 was normalized to 1. Ray: ray floret. (\u003cstrong\u003eB\u003c/strong\u003e) The expression heatmap of \u003cem\u003eGhBEHs\u003c/em\u003e 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\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-5257183/v1/a5ec5ba7da43fe244edbe386.png"},{"id":68554745,"identity":"7d40953c-1ead-4201-a4c6-43f2389e3d6c","added_by":"auto","created_at":"2024-11-08 13:21:45","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":192530,"visible":true,"origin":"","legend":"\u003cp\u003eThe relative expression level of \u003cem\u003eGhBEHs\u003c/em\u003e during different ray floret growth stages. The expression of \u003cem\u003eGhBEH1\u003c/em\u003e in stage 1 was set as unite 1. Three independent experiments were replicated, and values are means ± SE\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-5257183/v1/9c0cb4e002c8f64882b2ec44.png"},{"id":75929961,"identity":"58fe22fa-f0c8-42f6-8be5-033334158ea3","added_by":"auto","created_at":"2025-02-10 16:07:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":36556440,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5257183/v1/74bd67cf-52d9-471c-b4a7-96566fe4a389.pdf"},{"id":68554755,"identity":"3b9d394f-ccb0-4217-b187-f6ad455d56bc","added_by":"auto","created_at":"2024-11-08 13:21:53","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":77580,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional file 1: Full uncropped Gels and Blots image of \u003cem\u003eGhBEH1-7\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Additionalfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5257183/v1/de985bceb3857912aa0ad73e.docx"},{"id":68555896,"identity":"ddc8b8c4-3095-4720-8988-d2889c4061e4","added_by":"auto","created_at":"2024-11-08 13:29:45","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":21008,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional file 2: cDNA sequence and protein sequence of \u003cem\u003eGhBEH1-7\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Additionalfile2.docx","url":"https://assets-eu.researchsquare.com/files/rs-5257183/v1/49e44cf39fffdb998665e32c.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome-wide identification, characterization, and expression analysis of BZR transcription factor family in Gerbera hybrida","fulltext":[{"header":"Background","content":"\u003cp\u003eBrassinosteroids (BRs), a class of plant steroidal hormones [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], 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 [\u003cspan additionalcitationids=\"CR3 CR4 CR5\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In plants, BRs are perceived by the cell membrane receptor brassinosteroids-insensitive 1 (BRI1) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. This perception leads to the activation of the BRI1 receptor kinase and trigger a phosphorylation cascade that inhibit the glycogen synthase kinase-3 (GSK3)-like kinase BIN2 [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. BIN2 undergoes phosphorylation and regulates other kinases as well as crucial transcription factors (TFs), including members of the brassinazole-resistant (BZR) gene family [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. BIN2 phosphorylates BZR1 family transcription factors, and after BIN2 inactivation, PP2A dephosphorylates BZR1 family transcription factors [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Upon dephosphorylation, BZR1 and BES1 translocate into the nucleus, where they regulate the expression of target genes, thereby influencing cell growth [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This intricate regulatory mechanism mediated by BRs plays a central role in gene expression and in specific cellular developmental processes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs crucial phytohormones, BRs are essential for regulating various aspects of plant growth and development, primarily through mediating BZR transcription factors [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In Arabidopsis, the BZR gene family is divided into four classes: brassinazole-resistant 1 (BZR1), BRI-EMSSUPPRESSOR1 (BZR2/BES1), and homologs 1\u0026ndash;4 (BEH1-BEH4) of BZR1/2, sharing high sequence identity with BZR1/2 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. BZR1 and BZR2/BES1, identified as transcriptional repressors and activators respectively [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], play pivotal roles as 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 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. \u003cem\u003eBZR\u003c/em\u003e genes are notable for their role in modulating downstream gene expression throughout various stages of plant growth and development [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Although current research has identified numerous BR-responsive genes [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], only a subset have been confirmed as direct targets of BZR1 or BZR2/BES1[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In rice, BZR1 collaborates with the RLA1/SMOS1 transcription factors to form a transcriptional complex that synergistically regulates downstream genes within the BR signaling pathway, thereby influencing growth and development [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Similarly, in maize, BZR1 directly regulates the expression of the \u003cem\u003eGRACE\u003c/em\u003e and \u003cem\u003eKRP6\u003c/em\u003e genes, which are involved in cell expansion and organ size determination [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In Chrysanthemum, CmBES1 binds directly to the promoter of \u003cem\u003eCUC2\u003c/em\u003e, suppressing its expression and facilitating the transformation of tubular flowers into tongue-shaped flowers [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Furthermore, in litchi, BRs mitigate ethylene-induced fruit abscission by repressing the transcription of \u003cem\u003eLcACS1/4\u003c/em\u003e and \u003cem\u003eLcACO2/3\u003c/em\u003e through \u003cem\u003eLcBZR1/2\u003c/em\u003e [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Despite extensive research on BZR proteins in Arabidopsis, rice, Chrysanthemum and litchi, the functions of BZR transcription factors in gerbera have yet to be investigated. This suggests that BZR1 and BZR2/BES1 may regulate other transcription factors to modulate secondary BR-responsive genes and perform additional functions. Therefore, the identification and characterization of new BZR genes from various plant species represent promising avenues for gaining novel insights into this gene family.\u003c/p\u003e \u003cp\u003e \u003cem\u003eGerbera hybrida\u003c/em\u003e, 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 has been extensively employed as a model organism for investigating the organogenesis and developmental processes within the Asteraceae family [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Although numerous TFs governing petal growth have been extensively investigated in gerbera [\u003cspan additionalcitationids=\"CR36 CR37 CR38 CR39 CR40\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], research on the BZR TFs in gerbera remains limited, particularly 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 provide a thorough investigation of this gene family within the context of gerbera. Initially, the \u003cem\u003eGhBEH\u003c/em\u003e (BZR/BES homologs) genes were 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 \u003cem\u003eGhBEH\u003c/em\u003e family. Furthermore, real-time quantitative reverse transcription (qRT) PCR analysis was employed to investigate the expression patterns of \u003cem\u003eGhBEHs\u003c/em\u003e in various growth stages of ray florets. Concurrently, the response of \u003cem\u003eGhBEHs\u003c/em\u003e to BR treatment was examined. These findings establish a foundation framework for further exploration into the function of \u003cem\u003eGhBEH\u003c/em\u003e genes and for identifying and characterizing BZR genes in diverse species. Moreover, this study provides a theoretical basis for the in-depth investigation of the molecular mechanisms governing petal growth in gerbera.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant material and growth conditions\u003c/h2\u003e \u003cp\u003eGerbera hybrida cultivar (cv.) Terra Regina and Shenzhen No. 5 were used in this study. Shoots were individually propagated in multiplication medium to induce bud clusters and subsequently maintained in a tissue-culture room at 24\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C under a long-day photoperiod (16 h light/8 h dark). Upon root establishment, the plants were transferred to a greenhouse with a temperature of 22\u0026ndash;28\u0026deg;C and relative humidity of 60\u0026ndash;80%.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIdentification of\u003c/b\u003e \u003cb\u003eGhBZR\u003c/b\u003e \u003cb\u003efamily gene\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe BZR gene family sequences from Arabidopsis, sourced from the Arabidopsis Information Resource (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.arabidopsis.org/\u003c/span\u003e\u003cspan address=\"https://www.arabidopsis.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), were subjected to a BLASTP search against NCBI databases using an e-value of 10\u003csup\u003e\u0026minus;\u0026thinsp;10\u003c/sup\u003e to identify potential candidate genes. The domain structure of BZR gene family was annotated based on prior investigations [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], and redundant sequences were filtered out via sequence alignment conducted using DNA MAN software.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePhylogenetic analysis and multiple sequence alignment\u003c/h3\u003e\n\u003cp\u003eThe seven BZR proteins from gerbera 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 BZR gene family was constructed using MEGA5.1 software based on the neighbour-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.\u003c/p\u003e\n\u003ch3\u003eChromosome localization analysis and gene structure\u003c/h3\u003e\n\u003cp\u003eThe chromosomal location of GhBZR genes were obtained from the gerbera database by 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. Additionally, the gene structure was visualized using the Gene Structure Display Server (GSDS 2.0) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gsds.cbi.pku.edu.cn/\u003c/span\u003e\u003cspan address=\"http://gsds.cbi.pku.edu.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePromoter sequence analysis of\u003c/b\u003e \u003cb\u003eGhBEH\u003c/b\u003e \u003cb\u003egenes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eApproximately 2000 bp of sequence upstream from the start codon (ATG) in the \u003cem\u003eGhBEH\u003c/em\u003e genes was identified as the putative regulatory promoter region retrieved from gerbera database. Subsequently, these promoter sequences were analyzed using the PlantCARE database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003ePrediction and analysis of subcellular localization\u003c/h3\u003e\n\u003cp\u003eThe subcellular localizations of seven gerbera BZR proteins were predicted by predictprotein (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://open.predictprotein.org/\u003c/span\u003e\u003cspan address=\"https://open.predictprotein.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). 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. To specifically label the nucleus, the nuclear localization marker NLS-mCherry was co-transfected into gerbera protoplasts.\u003c/p\u003e\n\u003ch3\u003eDual-luciferase reporter assay\u003c/h3\u003e\n\u003cp\u003eTo 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. Subsequently, the effector-GAL4 vector, REN vector, and reporter vector carrying luciferase gene were co-transfected into gerbera protoplasts. The luciferase assay was performed according to the manufacturer\u0026rsquo;s protocol (Dual-Luciferase\u0026reg; Reporter Assay, Promega, United States), with Firefly and Renilla luciferase activities quantified using an Enspire multimode microplate reader.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eYeast two-hybrid assay\u003c/h2\u003e \u003cp\u003eThe full-length cDNA sequences of \u003cem\u003eGhBEHs\u003c/em\u003e (\u003cem\u003eGhBEH1\u003c/em\u003e, \u003cem\u003eGhBEH2\u003c/em\u003e, \u003cem\u003eGhBEH3\u003c/em\u003e, \u003cem\u003eGhBEH4\u003c/em\u003e, \u003cem\u003eGhBEH5\u003c/em\u003e, \u003cem\u003eGhBEH6\u003c/em\u003e, \u003cem\u003eGhBEH7\u003c/em\u003e) were cloned into the pGBKT7 vector to generate the BD construct. These constructs were co-transferred to yeast AH109 cells along with pGADT7 respectively to test the transcriptional activation ability of the \u003cem\u003eGhBEHs\u003c/em\u003e. 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.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eQuantitative RT-PCR (qRT-PCR)\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eQuantitative RT-PCR (qRT-PCR)\u003c/div\u003e \u003cp\u003eRNA extraction was performed followed by reverse transcription using the Toyobo reverse transcription kit, following the protocols described in a previous study [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. qRT-PCR was conducted using a Bio-Rad Real-Time PCR system. The expression levels of the \u003cem\u003eGhBEHs\u003c/em\u003e (\u003cem\u003eGhBEH1\u003c/em\u003e, \u003cem\u003eGhBEH2\u003c/em\u003e, \u003cem\u003eGhBEH3\u003c/em\u003e, \u003cem\u003eGhBEH4\u003c/em\u003e, \u003cem\u003eGhBEH5\u003c/em\u003e, \u003cem\u003eGhBEH6\u003c/em\u003e, \u003cem\u003eGhBEH7\u003c/em\u003e) were normalized to \u003cem\u003eGhACTIN\u003c/em\u003e (\u003cem\u003eAJ763915\u003c/em\u003e) gene expression, as described in gerbera studies [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Relative expression levels were calculated by 2\u003csup\u003e\u0026minus;△△CT\u003c/sup\u003e [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Each sample was analyzed with three biological replicates.\u003c/p\u003e\n\u003ch3\u003eHormone treatments\u003c/h3\u003e\n\u003cp\u003ePetals 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 hours. Subsequently, morphological observations were conducted, and the resulting data were collected and analyzed. Each experiment was replicated at least three times.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eA Student's t-test was employed for statistical analysis to determine the significance of differences between the samples. The graphs were created by the GraphPad Prism software.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eIdentification of BZR family genes in gerbera\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeven BZR family homologous genes were identified through extensive alignment and analysis using the transcriptome database of gerbera. The homologous gene (GACN01037487.1) was designated \u003cem\u003eGhBEH1\u003c/em\u003e (\u003cem\u003eBES1/BZR1\u003c/em\u003e homolog 1), while the remaining six homologs were designated \u003cem\u003eGhBEH2\u003c/em\u003e (GACN01006390.1), \u003cem\u003eGhBEH3\u003c/em\u003e (GACN0100383.1), GhBEH4 (GACN01030557.1), \u003cem\u003eGhBEH5\u003c/em\u003e (GACN01023011.1), \u003cem\u003eGhBEH6\u003c/em\u003e (GACN01045166.1), and \u003cem\u003eGhBEH7\u003c/em\u003e (GACN01022598.1) (Additional file 1). The full 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.\u0026nbsp;The cDNA sequence and protein sequence are included in Additional file 2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChromosome locations and gene structures of \u003cem\u003eGhBEHs\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe chromosomal locations of 7 \u003cem\u003eGhBEH\u003c/em\u003e 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 (Figure 1A). Notably, most \u003cem\u003eGhBEH\u003c/em\u003e 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. To elucidate the structural characteristics of \u003cem\u003eGhBEH\u003c/em\u003e genes, we conducted an analysis of their exon-intron structure. And the results indicated that all \u003cem\u003eGhBEH\u0026nbsp;\u003c/em\u003egenes have a length of within 5 kb, with each gene containing a single intron, as illustrated in Figure 1B.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic analysis and multiple sequence alignment analysis of the GhBEHs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo 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 (Figure 2). The analysis 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. While 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\u003c/p\u003e\n\u003cp\u003eAsteraceae plants.\u003c/p\u003e\n\u003cp\u003eTo 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 (Figure 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 similar to those found in homologous sequences from Asteraceae plants, and contain conserved PEST motifs and proline residues. This residue plays a crucial role in the functionality of Arabidopsis BZR1 and BES1 [13].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSubcellular localization of GhBEH proteins\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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 (Figure 4A), suggesting potential localization in both the nucleus and cytoplasm.\u003c/p\u003e\n\u003cp\u003eTo 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 (Figure 5A).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt has been reported that the mutation of 234th proline to leucine in Arabidopsis significantly enhances nuclear localization signal of BZR1, a pivotal regulator in its function\u0026nbsp;[19, 47]. Therefore, we constructed YFP-GhBEH1\u003csup\u003eP192L\u003c/sup\u003e, YFP-GhBEH2\u003csup\u003eP209L\u003c/sup\u003e, YFP-GhBEH3\u003csup\u003eP219L\u003c/sup\u003e, and YFP-GhBEH4\u003csup\u003eP206L\u003c/sup\u003e vectors (designated as GhBEH1\u003csup\u003eP192L\u003c/sup\u003e, GhBEH2\u003csup\u003eP209L\u003c/sup\u003e, GhBEH3\u003csup\u003eP219L\u003c/sup\u003e, and GhBEH4\u003csup\u003eP206L\u003c/sup\u003e, respectively, upon introduction of the proline-to-leucine mutation) (Figure 4B), and transformed them into protoplasts to assess their subcellular localization. Our observations revealed that YFP-GhBEH1\u003csup\u003eP192L\u003c/sup\u003e and YFP-GhBEH2\u003csup\u003eP209L\u003c/sup\u003e maintained their nuclear localization (Figure 4C and Figure 5B). In contrast, the nuclear localization of YFP-GhBEH3\u003csup\u003eP219L\u003c/sup\u003e and YFP-GhBEH4\u003csup\u003eP206L\u003c/sup\u003e was significantly enhanced, suggesting that the proline-to-leucine mutation promotes the nuclear localization of GhBEH3 and GhBEH4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscriptional activity analysis of GhBEHs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to bioinformatics, structural predictions and subcellular localization, it has been demonstrated that 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\u0026nbsp;[38, 39]. To investigate the transcriptional activity of the BZR family member in gerbera, transactivation assays were initially performed in yeast. The seven \u003cem\u003eGhBEH\u003c/em\u003e genes fused with 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. While, the remaining five members exhibited normal growth on SD/-LTH and SD/-LTHA, consistent with the positive control, indicating t 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 (Figure 6A). These experiments collectively demonstrate that GhBEH1 and GhBEH2 lack self-activation activity, consistent with observations in Arabidopsis BZR1 and BES1, thus suggesting functional homology. In contrast, GhBEH3, GhBEH4, GhBEH5, GhBEH6, and GhBEH7 all exhibit strong self-activation activity.\u003c/p\u003e\n\u003cp\u003eMeanwhile, dual-luciferase assays was conducted to further substantiate the transcriptional activity of GhBEHs (Figure 6B). The results showed that GhBEH1 and GhBEH2 had significantly lower ratios compared to the control group. Conversely, GhBEH3 through GhBEH7 demonstrated ratios significantly higher than the control (Figure 6C). These findings strongly suggest that GhBEH1 and GhBEH2 may function as transcriptional repressors, while the remaining five members act as transcriptional activators.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of cis-acting elements in the promoters of \u003cem\u003eGhBEHs\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the potential function of GhBEHs, we employed PlantCARE to predict cis-acting elements within the promoter regions of seven \u003cem\u003eBEH\u003c/em\u003e genes. A total of 147 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 (Figure 7). Predominantly represented were elements associated with tissue-specific expression, followed by those involved in stress and phytohormone responses. These findings hint that \u003cem\u003eGhBEH\u003c/em\u003e genes likely participate in plant hormone response and various stress processes, thereby potentially influencing plant growth and stress resistance. Notably, different genes not only contain different numbers of phytohormone response elements, such as \u003cem\u003eGhBEH1\u003c/em\u003e (4 elements) and \u003cem\u003eGhBEH4\u003c/em\u003e (3 elements), but also include elements responsive to different types of phytohormones. For instance, \u003cem\u003eGhBEH7\u003c/em\u003e contains elements responsive to abscisic acid, gibberellin, ethylene and auxin, whereas \u003cem\u003eGhBEH5\u003c/em\u003e only exhibits gibberellin response elements. These results indicate that distinct \u003cem\u003eGhBEH\u003c/em\u003e genes may play diverse regulatory roles by responding to specific phytohormone signals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression analysis of \u003cem\u003eGhBEHs\u003c/em\u003e under BR treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe BZR family genes are the key transcription factors in plant BR signaling pathway. In the expression profile analysis, only one member of the \u003cem\u003eBZR\u003c/em\u003e family, \u003cem\u003eGhBEH1\u003c/em\u003e, was identified as differentially expressed following BL (brassinolide) treatment [48]. To investigate whether other \u003cem\u003eGhBEHs\u003c/em\u003e 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 \u003cem\u003eGhBEH1\u003c/em\u003e and \u003cem\u003eGhBEH2\u003c/em\u003e exhibit a consistent pattern of initial upregulation followed by downregulation over time. \u003cem\u003eGhBEH1\u0026nbsp;\u003c/em\u003ereaches peak expression at 2 h, while \u003cem\u003eGhBEH2\u003c/em\u003e peaks slightly earlier, at 1 h. \u003cem\u003eGhBEH3\u003c/em\u003e and \u003cem\u003eGhBEH4\u003c/em\u003e respond slower to BL treatment, showing increased expression trends after 8 h. In contrast, \u003cem\u003eGhBEH5\u003c/em\u003e shows downregulated expression, while \u003cem\u003eGhBEH6\u003c/em\u003e and \u003cem\u003eGhBEH7\u003c/em\u003e responds rapidly to BL, showing significant upregulation at 0.5 h post-treatment, gradually declining by 4 h (Figure 8A). A corresponding heatmap provides a visual representation of these expression dynamics. The varied changes in \u003cem\u003eGhBEH\u003c/em\u003e expression levels post-BR treatment suggest diverse response timings and intensities to BR.\u003c/p\u003e\n\u003cp\u003eTo assess the response of \u003cem\u003eGhBEHs\u003c/em\u003e 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 \u003cem\u003eGhBEH1\u003c/em\u003e and \u003cem\u003eGhBEH2\u003c/em\u003e 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 both up-regulation and down-regulation (Figure 9). These findings suggest that \u003cem\u003eGhBEH1\u003c/em\u003e and \u003cem\u003eGhBEH2\u003c/em\u003e exhibit obvious response to BR, indicating their strong capability to respond to both endogenous and exogenous BR stimuli.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression pattern of \u003cem\u003eGhBEHs\u003c/em\u003e in different petal growth stages\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe early study divided the petal growth and developmental stages into 11 phases in gerbera [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 \u003cem\u003eGhBEH\u003c/em\u003e expression profiles across these stages revealed distinct trends: \u003cem\u003eGhBEH1\u003c/em\u003e and \u003cem\u003eGhBEH2\u003c/em\u003e 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 (Figure 10A, 10B). In contrast, \u003cem\u003eGhBEH3\u003c/em\u003e, \u003cem\u003eGhBEH4\u003c/em\u003e, \u003cem\u003eGhBEH5\u003c/em\u003e, and \u003cem\u003eGhBEH6\u003c/em\u003e consistently exhibited significant downregulation expression throughout stages 1-11. While, \u003cem\u003eGhBEH7\u003c/em\u003e did not show significant changes in expression levels across these stages.\u003c/p\u003e\n\u003cp\u003eTo further elucidate the expression dynamics of \u003cem\u003eGhBEHs\u003c/em\u003e across different growth stages of ray floret, a relative expression level graph was\u0026nbsp;generated using \u003cem\u003eGhBEH1\u003c/em\u003e expression at stage 1 as a reference baseline (Figure 11). The analysis revealed that \u003cem\u003eGhBEH1\u003c/em\u003e displayed the highest relative expression level among ray petals, followed by \u003cem\u003eGhBEH7\u003c/em\u003e, while the expression levels of other genes were comparatively lower. \u0026nbsp;Notably, \u003cem\u003eGhBEH2\u003c/em\u003e and \u003cem\u003eGhBEH6\u003c/em\u003e exhibited expression levels approximately 1/20th of \u003cem\u003eGhBEH1\u003c/em\u003e. These data suggest different degrees of influence of these genes on petal growth of ray floret.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe BZR family comprises pivotal transcription factors in plants that play a crucial role in the BR signaling pathway, governing plant growth, development, and participating in abiotic stress responses. [\u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. 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 \u003cem\u003eBZR\u003c/em\u003e 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.\u003c/p\u003e \u003cp\u003eThe subcellular localization of proteins is closely related to their cellular functions [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Typically, transcription factors are predominantly located within the cell nucleus, although some TFs exhibit dual localization in both the nucleus and the cytoplasm [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. 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. This distinction suggests potential functional specialization among these genes, indicating their specific roles within distinct cellular compartments in vivo. BR have been documented to induce the dephosphorylation of Arabidopsis BES1 and BZR1, thereby promoting their nuclear accumulation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Furthermore, studies on Arabidopsis BZR1 have demonstrated that the substitution of proline with leucine at position 234 enhances its nuclear localization signal, thereby influencing its functional activity [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In our study, we observed that GhBEH1\u003csup\u003eP192L\u003c/sup\u003e and GhBEH2\u003csup\u003eP209L\u003c/sup\u003e maintained their nuclear localization patterns unchanged. In contrast, the nuclear localization of GhBEH3\u003csup\u003eP219L\u003c/sup\u003e and GhBEH4\u003csup\u003eP206L\u003c/sup\u003e 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 their functional roles.\u003c/p\u003e \u003cp\u003eCis-acting elements are non-coding DNA sequences situated within promoter regions, exerting influence over gene expression and gene function [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. In this study, a comprehensive analysis identified a total of 147 cis-acting elements in the promoters of 7 \u003cem\u003eGhBEH\u003c/em\u003e 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 7 \u003cem\u003eGhBEH\u003c/em\u003es possess motifs related to stress responsiveness, suggesting that their role in modulating gerbera's adaptation to environment. In addition, except for \u003cem\u003eGhBEH6\u003c/em\u003e, promoters of the remaining 6 \u003cem\u003eGhBEH\u003c/em\u003e genes contain elements responsive to hormones, implicating the \u003cem\u003eBZR\u003c/em\u003e 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 [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. To further elucidate the hormonal regulation of gerbera \u003cem\u003eBZR\u003c/em\u003e genes, we assessed the expression profiles of each gene under BR treatment. The results revealed diverse expression patterns among the seven \u003cem\u003eGhBEH\u003c/em\u003e genes in response to BL, with particular emphasis on GhBEH6 because of the greatest fluctuation, which may be attributed to the apparent lack of hormone-responsive elements in its promoter region.\u003c/p\u003e \u003cp\u003eThe growth of petals in gerbera involves complex mechanisms of cell proliferation and expansion. Early stages of petal growth are characterized by changes in cell number, while middle and late stages emphasize cell size dynamics [\u003cspan additionalcitationids=\"CR59\" citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. BR play a pivotal role in promoting cell elongation and expansion, thereby exerting significant influence on overall petal growth [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. We examined the expression profiles of \u003cem\u003eGhBEHs\u003c/em\u003e in ray floret at different growth stages, revealing distinct patterns among the seven members. \u003cem\u003eGhBEH3\u003c/em\u003e, \u003cem\u003eGhBEH4\u003c/em\u003e, \u003cem\u003eGhBEH5\u003c/em\u003e, and \u003cem\u003eGhBEH6\u003c/em\u003e exhibited high expression levels during the early stages of petal growth, whereas \u003cem\u003eGhBEH7\u003c/em\u003e maintained consistent expression throughout the entire growth period. In contrast, \u003cem\u003eGhBEH1\u003c/em\u003e and \u003cem\u003eGhBEH2\u003c/em\u003e displayed predominant expression in the middle and late stages, with \u003cem\u003eGhBEH2\u003c/em\u003e showing a pronounced increase during the middle stage and maintaining high expression levels in the later stage. These observations suggest that \u003cem\u003eGhBEH3\u003c/em\u003e, \u003cem\u003eGhBEH4\u003c/em\u003e, \u003cem\u003eGhBEH5\u003c/em\u003e and \u003cem\u003eGhBEH6\u003c/em\u003e likely contribute to early petal growth, potentially through involvement in petal cells proliferation. Conversely, \u003cem\u003eGhBEH1\u003c/em\u003e and \u003cem\u003eGhBEH2\u003c/em\u003e may play roles in the middle and late stages of petal development, potentially participating in processes associated with cell elongation and expansion. Nevertheless, further experimental evidence is necessary to confirm these hypotheses.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, the whole genome of the gerbera BZR family gene was investigated and seven BZR genes named \u003cem\u003eGhBEH1-7\u003c/em\u003e were identified. Researches 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 \u003cem\u003eGhBEH1\u003c/em\u003e and \u003cem\u003eGhBEH2\u003c/em\u003e may play roles in the middle and late stages of petal growth. It is noteworthy that all seven \u003cem\u003eGhBEHs\u003c/em\u003e respond to BR, especially \u003cem\u003eGhBEH1\u003c/em\u003e and \u003cem\u003eGhBEH2\u003c/em\u003e, may play 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 \u003cem\u003eBZR\u003c/em\u003e gene family in petal growth.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBR \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Brassinosteroid\u003c/p\u003e\n\u003cp\u003eBRZ \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Brassinazole\u003c/p\u003e\n\u003cp\u003eDfs \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Disc florets\u003c/p\u003e\n\u003cp\u003ePPZ \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Propiconazole\u003c/p\u003e\n\u003cp\u003eqRT-PCR \u0026nbsp; \u0026nbsp; \u0026nbsp;Real-time quantitative reverse transcription PCR\u003c/p\u003e\n\u003cp\u003eRfs \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Ray florets\u003c/p\u003e\n\u003cp\u003eTfs \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Trans florets\u003c/p\u003e\n\u003cp\u003eTF \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Transcription factor\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe online version contains supplementary material available at https://doi. org/10.1186/\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Pro. Paula Elomaa and Dr. Teng Zhang for instructing some experiments and giving advices for manuscript. We thank Dr. Fan Li for providing the genome of gerbera. We are grateful to Cambridge Academic Manuscripts (www. Cambridge Academic Manuscripts. com) for manuscript editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQishan 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\u0026nbsp;supervision, writing-review and editing. Yaqin Wang did\u0026nbsp;resources, supervision, funding acquisition, writing-review and editing.\u0026nbsp;All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Natural Science Foundation of Guangdong Province (2023A1515012864, 2021A1515012479), Guangdong Key Laboratory of Plant Adaptation and Molecular Design (2022B1212010013-7), Laboratory of Lingnan Modern Agriculture Project (NZ2021009) and National Key R\u0026amp;D Program of China (2018YFD1000404).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eGuangdong Provincial Key Laboratory of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou 510631, China.\u003csup\u003e2\u003c/sup\u003eCollege of Landscape Architecture and Art, Henan Agricultural University, Zhengzhou 450002, Henan Province, China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eLi J,J C. A putative leucine-rich repeat receptor kinase involved in brassinosteroid signal transduction. Cell. 1997; 90(5): 929-938. https://doi.org/10.1016/s0092-8674(00)80357-8.\u003c/li\u003e\n \u003cli\u003eClouse S. Molecular genetic studies confirm the role of brassinosteroids in plant growth and development. Plant J. 1996; 10(1): 1-8. https://doi.org/10.1046/j.1365-313X.1996.10010001.x.\u003c/li\u003e\n \u003cli\u003eMandava N B. Plant growth-promoting brassinosteroids. Annu rev plant physiol plant mol biol. 1988; 39: 23-52.\u003c/li\u003e\n \u003cli\u003eClouse S. Brassinosteroid Signal Transduction: From Receptor Kinase Activation to Transcriptional Networks Regulating Plant Development. Plant Cell. 2011; 23(4): 1219-1230. https://doi.org/10.1105/tpc.111.084475.\u003c/li\u003e\n \u003cli\u003eSun Y, Fan X, Cao D, Tang W, He K, Zhu J, He J, Bai M, Zhu S, Oh E, et al. Integration of Brassinosteroid Signal Transduction with the Transcription Network for Plant Growth Regulation in Arabidopsis. Dev Cell. 2010; 19(5): 765-777. https://doi.org/10.1016/j.devcel.2010.10.010.\u003c/li\u003e\n \u003cli\u003eOh E, Zhu J, Bai M, Arenhart R, Sun Y,Wang Z. Cell elongation is regulated through a central circuit of interacting transcription factors in the Arabidopsis hypocotyl. ELife. 2014; 3: e03031. https://doi.org/10.7554/eLife.03031.\u003c/li\u003e\n \u003cli\u003eThummel C,Chory J. Steroid signaling in plants and insects--common themes, different pathways. Genes Dev. 2002; 16(24): 3113-3129. https://doi.org/10.1101/gad.1042102.\u003c/li\u003e\n \u003cli\u003eKim T,Wang Z. Brassinosteroid Signal Transduction from Receptor Kinases to Transcription Factors. Annu Rev Plant Biol. 2010; 61(1): 681-704. https://doi.org/10.1146/annurev.arplant.043008.092057.\u003c/li\u003e\n \u003cli\u003eZhu J-Y, Sae-Seaw J,Wang Z-Y. Brassinosteroid signalling. Dev. 2013; 140(8): 1615-1620. https://doi.org/10.1242/dev.060590.\u003c/li\u003e\n \u003cli\u003eKim T, Guan S, Sun Y, Deng Z, Tang W, Shang J, Sun Y, Burlingame A,Wang Z. Brassinosteroid signal transduction from cell-surface receptor kinases to nuclear transcription factors. Nat Cell Biol. 2009; 11(10): 1254-1260. https://doi.org/10.1038/ncb1970.\u003c/li\u003e\n \u003cli\u003eHe J, Gendron J, Yang Y, Li J,Wang Z. The GSK3-like kinase BIN2 phosphorylates and destabilizes BZR1, a positive regulator of the brassinosteroid signaling pathway in Arabidopsis. Proc Natl Acad Sci. 2002; 99(15): 10185-10190. https://doi.org/10.1073/pnas.152342599.\u003c/li\u003e\n \u003cli\u003eTang W, Yuan M, Wang R, Yang Y, Wang C, Oses-Prieto J A, Kim T-W, Zhou H-W, Deng Z, Gampala S S, et al. PP2A activates brassinosteroid-responsive gene expression and plant growth by dephosphorylating BZR1. Nat Cell Biol. 2011; 13(2): 124-131. https://doi.org/10.1038/ncb2151.\u003c/li\u003e\n \u003cli\u003eWang Z, Bai M, Oh E,Zhu J. Brassinosteroid Signaling Network and Regulation of Photomorphogenesis. Annu Rev Genet. 2012; 46(1): 701-724. https://doi.org/10.1146/annurev-genet-102209-163450.\u003c/li\u003e\n \u003cli\u003eYoun J,Kim T. Functional Insights of Plant GSK3-like Kinases: Multi-Taskers in Diverse Cellular Signal Transduction Pathways. Mol Plant. 2015; 8(4): 552-565. https://doi.org/10.1016/j.molp.2014.12.006.\u003c/li\u003e\n \u003cli\u003eLi J,Nam K, Hee,. Regulation of Brassinosteroid Signaling by a GSK3/SHAGGY-Like Kinase. Science. 2002; 295(5558): 1299-1301. https://doi.org/10.1126/science.1065769.\u003c/li\u003e\n \u003cli\u003eClouse S,Sasse J. BRASSINOSTEROIDS: Essential Regulators of Plant Growth and Development. Annu Rev Plant Biol. 1998; 49: 427-451. https://doi.org/10.1146/annurev.arplant.49.1.427.\u003c/li\u003e\n \u003cli\u003eTang W, Tae-Wuk Kim, Juan A, Oses-Prieto, Yu Sun, Zhiping Deng, Shengwei Zhu, Ruiju Wang, Alma L. Burlingame,Wang Z-Y. BSKs Mediate Signal Transduction from the Receptor Kinase BRI1 in Arabidopsis. Science. 2008; 321(5888): 557-560. https://doi.org/10.1126/science.1156973.\u003c/li\u003e\n \u003cli\u003eYe H, Li L,Yin Y. Recent Advances in the Regulation of Brassinosteroid Signaling and Biosynthesis PathwaysF. J Integr Plant Biol. 2011; 53(6): 455-468. https://doi.org/10.1111/j.1744-7909.2011.01046.x.\u003c/li\u003e\n \u003cli\u003eYin Y, Vafeados D, Tao Y, Yoshida S, Asami T,Chory J. A New Class of Transcription Factors Mediates Brassinosteroid-Regulated Gene Expression in Arabidopsis. Cell. 2005; 120(2): 249-259. https://doi.org/10.1016/j.cell.2004.11.044.\u003c/li\u003e\n \u003cli\u003eYu X, Li L, Zola J, Aluru M, Ye H, Foudree A, Guo H, Anderson S, Aluru S, Liu P, et al. A brassinosteroid transcriptional network revealed by genome‐wide identification of BESI target genes in Arabidopsis thaliana. Plant J. 2011; 65(4): 634-646. https://doi.org/10.1111/j.1365-313X.2010.04449.x.\u003c/li\u003e\n \u003cli\u003eHe J, Gendron J, Sun Y, Gampala S, Gendron N, Sun C,Wang Z. BZR1 is a transcriptional repressor with dual roles in brassinosteroid homeostasis and growth responses. Science. 2005; 307(5715): 1634-1638. https://doi.org/10.1126/science.1107580.\u003c/li\u003e\n \u003cli\u003eOh E, Zhu J, Ryu H, Hwang I,Wang Z. TOPLESS mediates brassinosteroid-induced transcriptional repression through interaction with BZR1. Nat Commun. 2014; 5(1): 4140. https://doi.org/10.1038/ncomms5140.\u003c/li\u003e\n \u003cli\u003eGoda H, Shinichiro S, Tadao A, Shozo F, Yukihisa S,Shigeo Y. Comprehensive Comparison of Auxin-Regulated and Brassinosteroid-Regulated Genes in Arabidopsis. Plant Physiol. 2004; 134(4): 1555-1573. https://doi.org/10.1104/pp.103.034736.\u003c/li\u003e\n \u003cli\u003eNemhauser J, Mockler T,Chory J. Interdependency of Brassinosteroid and Auxin Signaling in Arabidopsis. PLoS Biol. 2004; 2(9): e258. https://doi.org/10.1371/journal.pbio.0020258.\u003c/li\u003e\n \u003cli\u003eVert G, Nemhauser J, Geldner N, Hong F,Chory J. Molecular Mechanisms of Steroid Hormone Signaling in Plants. Annual Review of Cell and Developmental Biology. 2005; 21(1): 177-201. https://doi.org/10.1146/annurev.cellbio.21.090704.151241.\u003c/li\u003e\n \u003cli\u003eZhang L, Bai M, Wu J, Zhu J, Wang H, Zhang Z, Wang W, Sun Y, Zhao J, Sun X, et al. Antagonistic HLH/bHLH Transcription Factors Mediate Brassinosteroid Regulation of Cell Elongation and Plant Development in Rice andArabidopsis Plant Cell. 2009; 21(12): 3767-3780. https://doi.org/10.1105/tpc.109.070441.\u003c/li\u003e\n \u003cli\u003eLi L, Yu X, Thompson A, Guo M, Yoshida S, Asami T, Chory J,Yin Y. Arabidopsis MYB30 is a direct target of BES1 and cooperates with BES1 to regulate brassinosteroid‐induced gene expression. Plant J. 2009; 58(2): 275-286. https://doi.org/10.1111/j.1365-313X.2008.03778.x.\u003c/li\u003e\n \u003cli\u003eQiao S, Sun S, Wang L, Wu Z, Li C, Li X, Wang T, Leng L, Tian W, Lu T, et al. The RLA1/SMOS1 Transcription Factor Functions with OsBZR1 to Regulate Brassinosteroid Signaling and Rice Architecture. Plant Cell. 2017; 29(2): 292-309. https://doi.org/10.1105/tpc.16.00611.\u003c/li\u003e\n \u003cli\u003eZhang X, Guo W, Du D, Pu L,Zhang C. Overexpression of a maize BR transcription factor ZmBZR1 in Arabidopsis enlarges organ and seed size of the transgenic plants. Plant Sci. 2020; 292: 110378. https://doi.org/10.1016/j.plantsci.2019.110378.\u003c/li\u003e\n \u003cli\u003eCheng P, Liu Y, Yang Y, Chen H, Cheng H, Hu Q, Zhang Z, Gao J, Zhang J, Ding L, et al. CmBES1 is a regulator of boundary formation in chrysanthemum ray florets. Hortic Res. 2020; 7(1): 129. https://doi.org/10.1038/s41438-020-00351-8.\u003c/li\u003e\n \u003cli\u003eMa X, Yuan Y, Li C, Wu Q, He Z, Li J,Zhao M. Brassinosteroids suppress ethylene-induced fruitlet abscission through LcBZR1/2-mediated transcriptional repression of LcACS1/4 and LcACO2/3 in litchi. Hortic Res. 2022; 8: 105. https://doi.org/10.1038/s41438-021-00540-z.\u003c/li\u003e\n \u003cli\u003eLaitinen R A E, Broholm S, Albert V A, Teeri T H,Elomaa P. Patterns of MADS-box gene expression mark flower-type development in Gerbera hybrida(Asteraceae). BMC Plant Biol. 2006; 6(1): 11. https://doi.org/10.1186/1471-2229-6-11.\u003c/li\u003e\n \u003cli\u003eTeeri T H, Uimari A, Kotilainen M, Laitinen R, Help H, Elomaa P,Albert V A. Reproductive meristem fates in Gerbera. J Exp Bot. 2006; 57(13): 3445-3455. https://doi.org/10.1093/jxb/erl181.\u003c/li\u003e\n \u003cli\u003eZhang T, Cieslak M, Owens A, Wang F, Broholm S K, Teeri T H, Elomaa P,Prusinkiewicz P. Phyllotactic patterning of gerbera flower heads. Proc Natl Acad Sci. 2021; 118(13): e2016304118. https://doi.org/10.1073/pnas.2016304118.\u003c/li\u003e\n \u003cli\u003eLin X, Huang S, Huang G, Chen Y, Wang X,Wang Y. 14-3-3 Proteins Are Involved in BR-Induced Ray Petal Elongation in Gerbera hybrida. Front Plant Sci. 2021; 12: 718091. https://doi.org/10.3389/fpls.2021.718091.\u003c/li\u003e\n \u003cli\u003eHuang G, Han M, Jian L, Chen Y, Sun S, Wang X,Wang Y. An ETHYLENE INSENSITIVE3-LIKE1 Protein Directly Targets the GEG Promoter and Mediates Ethylene-Induced Ray Petal Elongation in Gerbera hybrida. Front Plant Sci. 2020; 10: 01737. https://doi.org/10.3389/fpls.2019.01737.\u003c/li\u003e\n \u003cli\u003eRen G, Li L, Patra B, Li N, Zhou Y, Zhong C, Wang Y, Yuan L, Wang X,Melzer R. The transcription factor GhTCP7 suppresses petal expansion by interacting with the WIP-type zinc finger protein GhWIP2 in Gerbera hybrida. J Exp Bot. 2023; 74(14): 4093-4109. https://doi.org/10.1093/jxb/erad152.\u003c/li\u003e\n \u003cli\u003eRen G, Li L, Huang Y, Wang Y, Zhang W, Zheng R, Zhong C,Wang X. GhWIP2, a WIP zinc finger protein, suppresses cell expansion in Gerbera hybrida by mediating crosstalk between gibberellin, abscisic acid, and auxin. New Phytol. 2018; 219(2): 728-742. https://doi.org/10.1111/nph.15175.\u003c/li\u003e\n \u003cli\u003eHan M, Jin X, Yao W, Kong L, Huang G, Tao Y, Li L, Wang X,Wang Y. A Mini Zinc-Finger Protein (MIF) from Gerbera hybrida Activates the GASA Protein Family Gene, GEG, to Inhibit Ray Petal Elongation. Front Plant Sci. 2017; 8: 01649. https://doi.org/10.3389/fpls.2017.01649.\u003c/li\u003e\n \u003cli\u003eZhong C, Tang Y, Pang B, Li X, Yang Y, Deng J, Feng C, Li L, Ren G, Wang Y, et al. The R2R3-MYB transcription factor GhMYB1a regulates flavonol and anthocyanin accumulation in Gerbera hybrida. Hortic Res. 2020; 7(1): 78. https://doi.org/10.1038/s41438-020-0296-2.\u003c/li\u003e\n \u003cli\u003eJiang R, Yuan W, Yao W, Jin X, Wang X,Wang Y. A regulatory GhBPE-GhPRGL module maintains ray petal length in Gerbera hybrida. Mol Hortic. 2022; 2(1): 9. https://doi.org/10.1186/s43897-022-00030-3.\u003c/li\u003e\n \u003cli\u003eWang Z, Nakano T, Gendron J, He J, Chen M, Vafeados D, Yang Y, Fujioka S, Yoshida S, Asami T, et al. Nuclear-localized BZR1 mediates brassinosteroid-induced growth and feedback suppression of brassinosteroid biosynthesis. Dev Cell. 2002; 2(4): 505-513. https://doi.org/10.1016/s1534-5807(02)00153-3.\u003c/li\u003e\n \u003cli\u003eYin Y, Wang Z, Mora-Garcia S, Li J, Yoshida S, Asami T,Chory J. BES1 accumulates in the nucleus in response to brassinosteroids to regulate gene expression and promote stem elongation. Cell. 2002; 109(2): 181-191. https://doi.org/10.1016/s0092-8674(02)00721-3.\u003c/li\u003e\n \u003cli\u003eSu M, Huang G, Zhang Q, Wang X, Li C, Tao Y, Zhang S, Lai J, Yang C,Wang Y. The LEA protein, ABR, is regulated by ABI5 and involved in dark-induced leaf senescence in Arabidopsis thaliana. Plant Sci. 2016; 247: 93-103. https://doi.org/10.1016/j.plantsci.2016.03.009.\u003c/li\u003e\n \u003cli\u003eKuang Q, Li L, Peng J, Sun S,Wang X. Transcriptome Analysis of Gerbera hybrida Ray Florets: Putative Genes Associated with Gibberellin Metabolism and Signal Transduction. PLoS ONE. 2013; 8(3): e57715. https://doi.org/10.1371/journal.pone.0057715.\u003c/li\u003e\n \u003cli\u003eLivak K,Schmittgen T. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;CT\u003c/sup\u003e Method. Methods. 2001; 25(4): 402-408. https://doi.org/10.1006/meth.2001.1262.\u003c/li\u003e\n \u003cli\u003eRyu H, Kim K, Cho H, Park J, Choe S,Hwang I. Nucleocytoplasmic Shuttling of BZR1 Mediated by Phosphorylation Is Essential inArabidopsisBrassinosteroid Signaling. Plant Cell. 2007; 19(9): 2749-2762. https://doi.org/10.1105/tpc.107.053728.\u003c/li\u003e\n \u003cli\u003eHuang G, Han M, Yao W,Wang Y. Transcriptome analysis reveals the regulation of brassinosteroids on petal growth in Gerbera hybrida. PeerJ. 2017; 5: e3382. https://doi.org/10.7717/peerj.3382.\u003c/li\u003e\n \u003cli\u003eHelariutta Y, Elomaa P, Mika K, Sepp\u0026auml;nen P,H. Teeri T. Cloning of cDNA coding for dihydroflavonol-4-reductase (DFR) and characterization of dfr expression in the corollas of Gerbera hybrida var. Regina (Compositae). Plant Mol. Biol. 1993; 22: 183-193. https://doi.org/10.1007/bf00014927.\u003c/li\u003e\n \u003cli\u003eSaha G, Park J, Jung H, Ahmed N, Kayum M, Kang J,Nou I. Molecular characterization of BZR transcription factor family and abiotic stress induced expression profiling in Brassica rapa. Plant Physiol Biochem. 2015; 92: 92-104. https://doi.org/10.1016/j.plaphy.2015.04.013.\u003c/li\u003e\n \u003cli\u003eSun F, Yu H, Qu J, Cao Y, Ding L, Feng W, Khalid M, Li W,Fu F. Maize ZmBES1/BZR1-5 Decreases ABA Sensitivity and Confers Tolerance to Osmotic Stress in Transgenic Arabidopsis. Int J Mol Sci. 2020; 21(3): 996. https://doi.org/10.3390/ijms21030996.\u003c/li\u003e\n \u003cli\u003eYin Y, Qin K, Song X, Zhang Q, Zhou Y, Xia X,Yu J. BZR1 Transcription Factor Regulates Heat Stress Tolerance Through FERONIA Receptor-Like Kinase-Mediated Reactive Oxygen Species Signaling in Tomato. Plant Cell Physiol. 2018; 59(11): 2239\u0026ndash;2254 https://doi.org/10.1093/pcp/pcy146.\u003c/li\u003e\n \u003cli\u003eTsang T, Hsueh Y, Wei E, Lundy D, Cheng B, Chen Y, Wang S,Hsieh P. Subcellular Localization of Survivin Determines Its Function in Cardiomyocytes. Theranostics. 2017; 7(18): 4577-4590. https://doi.org/10.7150/thno.20005.\u003c/li\u003e\n \u003cli\u003ePlotnikov A, Zehorai E, Procaccia S,Seger R. The MAPK cascades: Signaling components, nuclear roles and mechanisms of nuclear translocation. Acta Bioch Bioph Sin. 2011; 1813(9): 1619-1633. https://doi.org/10.1016/j.bbamcr.2010.12.012.\u003c/li\u003e\n \u003cli\u003eZhao Q, Luo Z, Chen J, Jia H, Ai P, Chen A, Li Y,Xu G. Characterization of two cis-acting elements, P1BS and W-box, in the regulation of OsPT6 responsive to phosphors deficiency. Plant Growth Regul. 2021; 93(3): 303-310. https://doi.org/10.1007/s10725-020-00688-z.\u003c/li\u003e\n \u003cli\u003eHe G, Liu J, Dong H,Sun J. The Blue-Light Receptor CRY1 Interacts with BZR1 and BIN2 to Modulate the Phosphorylation and Nuclear Function of BZR1 in Repressing BR Signaling in Arabidopsis. Mol Plant. 2019; 12(5): 689-703. https://doi.org/10.1016/j.molp.2019.02.001.\u003c/li\u003e\n \u003cli\u003eGalstyan A,Nemhauser J L. Auxin promotion of seedling growth via ARF5 is dependent on the brassinosteroid‐regulated transcription factors BES1 and BEH4. Plant Direct. 2019; 3(9). https://doi.org/10.1002/pld3.166.\u003c/li\u003e\n \u003cli\u003eZhang L, Li L, Wu J, Peng J, Zhang L,Wang X. Cell expansion and microtubule behavior in ray floret petals of Gerbera hybrida: Responses to light and gibberellic acid. Photochem Photobiol Sci. 2012; 11(2): 279-288. https://doi.org/10.1039/c1pp05218g.\u003c/li\u003e\n \u003cli\u003eXie Z, Nolan T, Jiang H, Tang B, Zhang M, Li Z,Yin Y. The AP2/ERF Transcription Factor TINY Modulates Brassinosteroid-Regulated Plant Growth and Drought Responses in Arabidopsis. Plant Cell. 2019; 31(8): 1788-1806. https://doi.org/10.1105/tpc.18.00918.\u003c/li\u003e\n \u003cli\u003eYang Y, Chen B, Dang X, Zhu L, Rao J, Ren H, Lin C, Qin Y,Lin D. Arabidopsis IPGA1 is a microtubule-associated protein essential for cell expansion during petal morphogenesis. J Exp Bot. 2019; 70(19): 5231-5243. https://doi.org/10.1093/jxb/erz284.\u003c/li\u003e\n \u003cli\u003eWang F, Bai M, Deng Z, Oses-Prieto J, Burlingame A, Lu T, Chong K,Wang Z. Proteomic Study Identifies Proteins Involved in Brassinosteroid Regulation of Rice Growth. J Integr Plant Biol. 2010; 52(12): 1075-1085. https://doi.org/10.1111/j.1744-7909.2010.00992.x.\u003c/li\u003e\n \u003cli\u003eChen W, Lv M, Wang Y, Wang P, Cui Y, Li M, Wang R, Gou X,Li J. BES1 is activated by EMS1-TPD1-SERK1/2-mediated signaling to control tapetum development in Arabidopsis thaliana. Nat Commun. 2019; 10(1): 4164. https://doi.org/10.1038/s41467-019-12118-4.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"BZR transcription factor, Expression pattern, Genome-wide analysis, Petal growth","lastPublishedDoi":"10.21203/rs.3.rs-5257183/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5257183/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground: The BZR family genes encode plant-specific transcription factors that serve as pivotal regulators of plant BR signaling pathway, critically influencing plant growth and development.\u003c/p\u003e\n\u003cp\u003eResults: In this study, we performed a genome-wide investigation of \u003cem\u003eBZR\u003c/em\u003e family gene in gerbera in order to identify the key components of BR pathway that may function in petal growth. The identified \u003cem\u003eBZR\u003c/em\u003e genes, named \u003cem\u003eGhBEH1-7 \u003c/em\u003e(\u003cem\u003eGhBEH1\u003c/em\u003e, \u003cem\u003eGhBEH2\u003c/em\u003e, \u003cem\u003eGhBEH3\u003c/em\u003e,\u003cem\u003e GhBEH4\u003c/em\u003e,\u003cem\u003e GhBEH5\u003c/em\u003e,\u003cem\u003e GhBEH6\u003c/em\u003e,\u003cem\u003e GhBEH7\u003c/em\u003e), are distributed across chromosomes 3, 5, 10, 11, 12, 14 and 15. These genes exhibit similar exon-intron structures and possess typical BZR family structure. Phylogenetic analysis clustered these genes into two distinct subgroups. Analysis of cis-acting elementsrevealed their involvement in hormone response, stress response and growth regulation. Subcellular localization analysis indicated nuclear localization for GhBEH1 and GhBEH2, while the remainingfive genes exhibited dual localization in the nucleus and cytoplasm. Transactivation assay indicated GhBEH1 and GhBEH2 may function as transcriptional repressors, contrasting with the transcriptional activation observed for the other five genes. Notably, seven \u003cem\u003eGhBEHs\u003c/em\u003e exhibits various expression patterns under different growth stages of ray florets and BR treatment conditions. While, \u003cem\u003eGhBEH1\u003c/em\u003e and \u003cem\u003eGhBEH2\u003c/em\u003eshowed pronounced responsiveness to BR stimulation.\u003c/p\u003e\n\u003cp\u003eConclusion: Our work explains genome-wide identification, characterization, and expression analysis of BZR transcription factor family in gerbera, and hinted these seven \u003cem\u003eGhBEHs\u003c/em\u003e is involved in regulating petal growth and development. These findings provide a basis for further studies on further research on the biological function of the \u003cem\u003eBZR\u003c/em\u003e gene family in petal growth and a theoretical basis for future horticultural application in gerbera.\u003c/p\u003e","manuscriptTitle":"Genome-wide identification, characterization, and expression analysis of BZR transcription factor family in Gerbera hybrida","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-08 13:21:27","doi":"10.21203/rs.3.rs-5257183/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-29T11:45:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-28T10:56:21+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-28T10:56:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2024-10-14T01:00:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b621cb95-e022-46e0-8bd5-72079896a1d2","owner":[],"postedDate":"November 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-02-10T15:59:05+00:00","versionOfRecord":{"articleIdentity":"rs-5257183","link":"https://doi.org/10.1186/s12870-025-06177-7","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2025-02-04 15:56:58","publishedOnDateReadable":"February 4th, 2025"},"versionCreatedAt":"2024-11-08 13:21:27","video":"","vorDoi":"10.1186/s12870-025-06177-7","vorDoiUrl":"https://doi.org/10.1186/s12870-025-06177-7","workflowStages":[]},"version":"v1","identity":"rs-5257183","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5257183","identity":"rs-5257183","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}