Genome-wide identification of BBX gene family and its function in defense of necrotrophic fungus Alternaria sp. in Chrysanthemum

Abstract

BBX genes play an important role in plant growth, development, and stress response. However, systematic analysis of BBX gene family regarding resistance to fungal infections has not been previously conducted in Chrysanthemum. In this study, a systematic analysis of the BBX gene family was performed, and its function in defense of necrotrophic fungus Alternaria sp. has been probed into through publicly available genome and RNA-seq data of Chrysanthemum after Alternaria sp. infection. The systematic analyses included identifying the BBX gene family in Chrysanthemum, their evolutionary relationships, conserved domains, motifs, gene structure, cis-acting elements, and collinearity. Based on the RNA-seq data analyses and expressional pattern, CmBBX32 was selected as a candidate gene for further investigation because it responded continuously to the infection and up-regulated expression when Chrysanthemum was inoculated with Alternaria sp. Gene expression analysis showed the expression of CmBBX32 increased sharply during the infection process, and was highest in flowers. Besides, virus-induced gene silencing (VIGS) of CmBBX32 in Chrysanthemum reduced the resistance to Alternaria sp. infection, as evidenced by phenotypic analysis of infection symptoms, microscopic examination of spore germination and hyphal growth, as well as quantitative analysis of the marker gene associated with the SA and JA defense pathways. Overall, the data generated in this study should form the basis for future functional characterizations of BBX genes in Chrysanthemum, especially regarding the resistance to biological stress in Chrysanthemum.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12864-025-12035-5.

Introduction

Transcription factors (TFs), a class of regulatory proteins in plants, perform a wide range of critical functions throughout plant growth and development by controlling the transcription of their target genes [1, 2]. Zinc finger TFs represent one of the most abundant classes of TFs in plants, and are integral to the regulation of transcription and various biological functions [3–5]. B-Box (BBX) proteins represent a distinct subclass of zinc-finger TFs characterized by the presence of one or two B-box domains (CX2CX8CX7CX2CX4HX8H) in their N-terminus region. Some BBX proteins also possess an additional CCT (CONSTANS, CO-like, and TOC1) conserved domain or VP (valine–proline) motifs located in the C-terminus region [6, 7]. B-box domains are categorized into two distinct types: B-box1 (B1) and B-box2 (B2). Two conserved B-box domains can be recognized based on the spacing between the zinc-binding residues and their consensus sequences [3, 8]. Furthermore, potential segmented duplication and deletion events contribute to distance between the zinc-binding residues and the differences in the consensus sequences in the two B-box domains [3, 8, 9]. Moreover, the CCT domain, consisting of 42 to 43 amino acids, exhibits a high degree of conservation and serves a crucial role in mediating nuclear protein transport and transcriptional regulation [3, 10, 11].

BBX TFs are well-known for regulating flowering time, and it has been reported that several BBX genes function in the regulation of flowering in A. thaliana [1, 3, 12]. For example, CO/AtBBX1, the first discovered BBX gene, serves as a central regulator that promotes flowering under long-day conditions [13, 14]. Then, AtBBX4, AtBBX6, AtBBX7, and AtBBX32 were subsequently identified, and also functionally characterized with roles in the regulation of flowering time [15–17]. Moreover, BBX genes in other plants have also been found to regulate flowering time, including Chinese Cabbage (Brassica rapa L. ssp. pekinensis) [18], sorghum [19], barley (Hordeum vulgare) [20], and rice [21]. BBX TFs also play a significant role in responding to both abiotic and biotic stresses [1]. For instance, AtBBX5 has been shown to enhance salt stress tolerance in an abscisic acid (ABA)-dependent manner [22], while AtBBX18 has been reported to function as a negative regulator of heat tolerance in A. thaliana [23]. Overexpression of apple MdBBX10 in A. thaliana can obviously improve the tolerance to drought and salt stresses [24], and MdBBX37 positively regulates jasmonic acid (JA)-mediated cold-stress resistance in apple [25]. Heterologous overexpression of the GbBBX25 gene from Ginkgo biloba in poplar can enhance the tolerance of transgenic poplar to salt stress [26]. Overexpression of the Chimonanthus praecox CpBBX19 gene in A. thaliana also improved the tolerance to salt and drought stresses [27]. Additionally, in Ipomoea batatas, IbBBX24 plays a crucial role in enhancing resistance to Fusarium oxysporum f. sp batatas, a necrotrophic fungus, by modulating the biosynthesis and signaling of JA [28]. Similarly, CaBBX14 can improve resistance to Phytophthora capsici infection, a biotrophic fungus in pepper (Capsicum annuum L.), by influencing salicylic acid (SA) signaling pathway [29]. Furthermore, more than half of the BBX genes were significantly up-regulated under Arthrinium phaeospermum infection stress in hybrid bamboo [30].

Chrysanthemum, as one of the most renowned ornamental plants, belongs to the Asteraceae family, which is the largest family within the dicotyledons clade, comprising more than 1600 genera and approximately 25,000 species [31]. At present, Chrysanthemum is one of the most economically important floral plants worldwide, and takes up a great proportion of flower industry production [32]. Moreover, considering its nutritional and biologically active components, Chrysanthemum is widely applied in the medical, food, and beverage industries. Furthermore, the genome of Chrysanthemum was sequenced and high-quality assembled with the PacBio RS II platform, which provides a comprehensive research platform for identifying valuable genes and gene families [33]. Additionally, there exists a traditional Chinese medicinal Chrysanthemum variety known as ‘Huaijuhua’ in Jiaozuo City, Henan Province. ‘Huaijuhua’ not only is prized for its high medicinal value but also serves as an ornamental plant and drinking tea [34]. Since ‘Huaijuhua’ is commonly propagated through division propagation and cutting propagation for cultivation, this makes the plant extremely susceptible to infection by pathogens, such as fungi, bacteria, and viruses [34]. Thus ‘Huaijuhua’ is vulnerable to black spot disease, which is caused by the fungus Alternaria sp., a typical necrotrophic fungus. The strain was isolated from infected ‘Huaihuang’ and identified as the pathomycete responsible for causing black spot disease in ‘Huaihuang’ through multiple experimental approaches [34, 35]. This disease can substantially impair the yield and quality of ‘Huaijuhua’ [35]. After six years of rigorous selection and cultivation, a novel disease-resistant cultivar designated as ‘Huaiju 2^#’ (Henan Traditional Chinese Medicine Plant Cultivar identification number: 2016002) has been developed. Furthermore, the transcriptome database of ‘Huaiju 2^#’ post-inoculation with Alternaria sp. has been established and released (PRJNA448499), thereby facilitating the possibility of identifying and characterizing potential resistance-related genes [34, 36].

In Chrysanthemum, BBX TFs plays a critical role in regulating flowering time and stress responses. For instance, CmBBX8 accelerates the flowering process by directly targeting the CmFTL1 (Flowering Locus T Like 1) gene [37]. In contrast, CmBBX5 interacts with CmBBX8 to negatively regulate CmFTL1, thereby preventing premature flowering [38]. Conversely, CmBBX7 and CmBBX8 synergistically interact to positively regulate CmFTL1 expression, promoting flowering [39]. Furthermore, CmBBX29 has been shown to regulate flowering time by integrating the FLC (Flowering Locus C) and photoperiod pathways [40]. Additionally, CmBBX24 modulates flowering time and abiotic stress tolerance by influencing gibberellins (GA) biosynthesis [41]. Meanwhile, CmBBX22 may function as a regulator mediating drought stress tolerance and delaying leaf senescence [42]. Although BBX gene family has been identified in numerous plant species, such as Solanum lycopersicum [43], Capsicum annuum [1], Iris germanica [6], Prunus avium [44], Vitis Vinifera [45], Artemisia annua [46], and apple [47], there is a paucity of studies about BBX gene family in Chrysanthemum. Furthermore, as previously mentioned, Chrysanthemum is frequently threatened by various biotic and abiotic stresses during its growth and development. Among them, pathogen infection is a very severe biotic stress. Although BBX genes are widely recognized for their critical roles in modulating both abiotic and biotic stress responses, there have been relatively few studies on the role of BBX genes in mediating resistance to pathogens in Chrysanthemum.

Additionally, Virus-induced gene silencing (VIGS) is an siRNA-mediated silencing approach that utilizes viral vectors carrying a fragment of a target gene to produce long double-stranded RNAs, which are processed by Dicer into siRNAs for the silencing of the target gene [48]. This siRNA-mediated VIGS, referred to as “SIR VIGS” hereafter, has been established as an effective and rapid gene knockdown technology and is widely applied to define gene functions [49]. This technique avoids the need for stable plant transformation, and virus-induced silencing phenotypes can be readily observed within a short timeframe [48]. Compared with siRNAs, miRNAs cause more accurate gene silencing, as deduced from genome-wide expression profiling [50]. Furthermore, artificial miRNA (amiRNA) technology has been developed using endogenous miRNA precursors to generate new target-specific miRNAs for gene silencing in plants [51–53]. Thus, a modified CaLCuV(Cabbage leaf curl virus) [54] vector, based on the VIGS vector, has been developed to trigger siRNA-mediated silencing [48, 55]. This system can not only express amiRNAs to induce efficient VIGS using amiRNAs but also express endogenous miRNAs to study their biological functions [48]. Therefore, this technology enables rapid silencing of target genes and facilitates preliminary investigations of their functions.

In this study, we analyzed the BBX gene family in Chrysanthemum genome (http://www.amwayabrc.com), with a focus on gene structure, conserved motifs, phyletic evolution, chromosome distribution, cis-acting elements and collinearity. We identified a candidate CmBBX gene (CmBBX32) in ‘Huaiju 2^#’ through the transcriptome database and investigated their expression patterns across different tissues and following Alternaria sp. (Strain: HQJH10092301) inoculation. Furthermore, we investigated the role of CmBBX32 in resistance to Alternaria sp. through VIGS in ‘Huaiju 2^#’. This study provides insights into the BBX gene family and the function of CmBBXs in controlling and preventing black spot disease caused by Alternaria sp.

Materials and method

Identification of the members of BBX in chrysanthemum

Using the TAIR database [56], we retrieved the nucleotides and amino acids sequences of the 32 AtBBX genes. These amino acid sequences were then employed as queries in a BLASTp [57] search against the Chrysanthemum genome (10.6084/m9.figshare.21655364.v2; NCBI accession number: PRJNA796762 and PRJNA895586) [32, 33] using BioEdit v7.2.6.1 software [58]. Following established protocols for gene family member screening [59], we successfully identified the BBX gene family in Chrysanthemum. The theoretical isoelectric point (pI) and Molecular weights (Mw) of the BBX proteins in Chrysanthemum were calculated using the Compute pI/Mw tool on the ExPASy server [60].

Multiple sequence alignment and phylogenetic analyses

The Conserved Domain Database (CDD) program available through the NCBI website (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) was used to extract conserved sequences of Chrysanthemum BBX proteins. Subsequently, multiple sequence alignment of the conserved amino acid sequences was conducted using the DNAMAN 6.0 program (Lynnon Corporation, San Ramon, CA, USA). Additionally, to investigate the evolutionary relationship among BBX genes in Chrysanthemum, A. annua, A. thaliana and rice, as well as to determine orthologous and paralogous relationships among BBX genes in this plant, we constructed an un-rooted phylogenetic tree using Bayesian inference implemented in MrBayes 3.2.2 [61, 62] with the BBX amino acid sequences of Chrysanthemum, A. annua, A. thaliana, and rice. PhyloSuite v1.2.1 [63] was employed to select the model for phylogenetic tree construction, and the phylogenetic tree was visualized using Treeview1.61 [64].

Gene structure and motif analysis

The gene structure of BBXs in Chrysanthemum was analyzed using the Gene Structure Display Server (GSDS 2.0) [65], by aligning each coding sequence (CDS) with its corresponding gene sequence. Additionally, conserved motifs within the BBX gene family in Chrysanthemum were identified using the online MEME Suite 5.5.8, with the following parameters: the E-values less than 2 × 10⁻³⁰, and any number of repetitions of a motif [66, 67].

Cis‑acting elements in the promoter regions analysis

To gain insights into the regulatory information of gene expression, we investigated the cis-regulatory elements in the promoters of BBX genes in Chrysanthemum. The 2.0 kb upstream sequence of the start codon (ATG) of each BBX gene was extracted from the Chrysanthemum genome database. Then, the cis-regulatory elements in the promoters of each BBX gene were predicted using the online tool PlantCARE [68]. Finally, we drew the distribution map of the cis-regulatory elements of each BBX gene promoter using the cis-regulatory element visualization tool in TBtools-II v2.210 [69].

Chromosomal localization and collinearity analysis

The chromosomal locations of each BBX gene in Chrysanthemum were determined based on the physical location information from the Chrysanthemum genome annotation [32. 33] with the TBtools-II v2.210 [69]. Collinearity analysis was conducted for the Chrysanthemum BBX genes, as well as between Chrysanthemum, and A. thaliana, also using the TBtools-II v2.210 [69].

Excavation of the differentially expressed BBX genes response to necrotrophic fungus Alternaria sp. in Chrysanthemum

The transcriptome sequencing datasets of Chrysanthemum morifolium cv. ‘Huaiju 2^#’ after inoculation with the necrotrophic fungus Alternaria sp. at 3 d and 5 d (designated as A3 and A5) were retrieved from the NCBI Sequence Read Archive (SRA) repository (http://www.ncbi.nlm.nih.gov/sra/, project accession number PRJNA448499) [34]. By screening the transcriptome database, we identified a total of 32 sequences annotated as the BBX gene [34, 36]. The expression patterns of these BBX sequences were examined using FPKM values [70], and an expression heatmap was generated using the pheatmap R package [12, 71]. Based on this analysis, CmBBX genes showing up-regulated expression in response to Alternaria sp. inoculation were selected as potential candidate genes for further investigation into their role in resistance to necrotrophic fungus Alternaria sp.

Plant materials and pathogenic strains

Based on the previously described culture methods [34], ‘Huaiju 2^#’ used in this study were cultivated in 13 cm diameter pots containing a peat-vermiculite mixture (v/v = 1:1). These plants were grown in a controlled greenhouse under the following conditions: 20–25°C, 40–60% relative humidity, and a 14 h light/10 h dark cycle with a light intensity 60 mE·s⁻¹·m⁻² [34]. Alternaria sp. (GenBank accession number: KF688111) was used as the pathogenic strain to inoculate the‘Huaiju 2^#’, which was maintained for experimental using at the Engineering Technology Research Center of Nursing and Utilization of Genuine Chinese Crude Drugs in Henan Province, Henan Normal University, Xinxiang, China [34].

Isolation and qRT‑PCR analyses of candidate gene (CmBBX32)

To further characterize the candidate gene (CmBBX32) screened from the transcriptome sequencing data mentioned above, we performed gene cloning and qRT-PCR expression analysis. Total RNA was extracted from the leaves of 35-day-old ‘Huaiju 2^#’ plants using the TRIzol method [72]. The RNA quantity was determined by a NanoDrop 2000 C Spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Subsequently, cDNA was synthesized with HiScript^® II Q RT SuperMix (Vazyme Biotech, Nanjing, China) following the manufacturer’s protocol. Gene-specific primers CmBBX32-F and CmBBX32-R (Supplementary Table 1) were designed with Premier 5.0 (Premier, Canada) based on the unigenes identified in the transcriptome database [34]. Then, we amplified the CmBBX32 with the PCR reaction system (50 µL) consisting of 25 µl 2×Phanta^® Max Buffer; 1µL of template cDNA (1:100), 2 µL each oligo of primer (10 µM); 1 µL dNTP Mix (10 mM each); 1 µL Phanta^® Max Super-Fidelity DNA Polymerase (Vazyme Biotech, Nanjing, China), and 18 µL of ddH₂O. The PCR program was conducted as follows: 3 min at 95 °C; 40 cycles of 95 °C for 15 s, 49 °C for 15 s and 72 °C for 60 s; followed by a final extension at 72 °C for 5 min. The PCR products were purified using a FastPure^® Gel Extraction Mini Kit (Vazyme Biotech, Nanjing, China), cloned into the pCE2 TA/Blunt-Zero (Vazyme Biotech, Nanjing, China), and sequenced at Sangon Biotech (Shanghai, China). Furthermore, we employed the SMART tool (http://smart.embl-heidelberg.de/) to identify the conserved domains in CmBBX32 amino acid sequence. To investigate the evolutionary relationship of CmBBX32 with the BBX genes in A. thaliana, we constructed a Neighbor-joining (NJ) phylogenetic tree using the full-length amino acid sequences of CmBBX32 and AtBBXs with MEGA 6.0 software [73].

The tissue-specific expression patterns of CmBBX32 were examined in tissues of ‘Huaiju 2^#’, including roots (R), stems (S), leaves (L) of 30-day-old tissue culture-derived plantlets, and flowers (F) at full bloom with qRT-PCR. Samples were immediately frozen in liquid nitrogen and stored at −80 °C. The procedures for total RNA extraction, RNA quality determination, and first-strand cDNA synthesis according to the methods mentioned above. qRT-PCR was conducted on a Light Cycler 96 Instrument (Roche, Basel, Switzerland). Each reaction contained 20 ng of first-strand cDNA, 10 µL of 2 x AceQ qPCR SYBR Green Master Mix (Vazyme, Nanjing, China), and 500 nM each primer. The thermal cycling protocol included 30 s of polymerase activation at 95 °C, followed by 45 cycles of 95 °C for 10 s and 60 °C for 30 s. The Ubiquitin gene (CmUBI) was selected as the reference gene [36]. Gene-specific primers were designed with Premier 5.0 software, and their sequences are listed in Supplementary Table 1. All qRT-PCR experiments included three biological replicates and three technical replicates. Relative expression levels were calculated using the 2^–ΔΔCt method [74], and were presented as means ± standard deviation (SD). The amplicon sizes ranged from 100 to 250 bp.

Alternaria sp. inoculation and sampling

Thirty-day-old plantlets of ‘Huaiju 2^#’ were transplanted in vermiculite that had been saturated with water for 7 days. Subsequently, they were transplanted into the greenhouse under the cultivation condition as described above. After 14 days, they were inoculated with Alternaria sp. following the previously described inoculation method [34, 75]. Briefly, activated Alternaria sp. spores were diluted in sterile distilled water (SDW) to achieve a density of 10⁷ spores per milliliter. Three leaves from each plant were randomly selected for inoculation, and five sites on each selected leaf were punctured with a needle (approximately 0.41 mm diameter). Then, each punctured site was inoculated with 10 µL of the spore suspension. As a control, 10 µL of SDW was applied to each puncture wound on the leaves of mock-inoculated plants.

After inoculation, all inoculated plants were placed in a dark incubation chamber under environmental conditions as previously described [34, 75]. Forty-eight hours later, the plants were transferred to the greenhouse under the cultivation conditions described above. Leaves were harvested from plants at 0 d (prior to inoculation), 1, 3, 5, 7, 15, and 21 d post-inoculation (dpi) with Alternaria sp. and SDW (mock), respectively. Harvested samples were immediately frozen in liquid nitrogen and then stored at −80 °C for qRT-PCR testing. The procedures for total RNA extraction, RNA quantification, and first-strand cDNA synthesis adhered to the methods outlined above.

CaLCuV-BBX32 vector construction

To investigate the function of CmBBX32, we constructed a virus-induced instantaneous gene silencing (VIGS) vector. The modified vector of CaLCuV based on the VIGS mentioned above was generously provided by Bo Hong professor of China Agricultural University. Detailed protocols for designing and obtaining the amiRNA sequences were consistent with the methods described in previous study [48, 51, 76, 77]. Briefly, candidate 21-mer amiRNA sequences were designed using the Web MicroRNA Designer (http://wmd3.weigelworld.org/cgi-bin/webapp.cgi). Subsequently, WMD Oligo (http://wmd3.weigelworld.org/cgi-bin/webapp.cgi? page=Oligo; project=stdwmd) was employed to design four primers I, II, III, and IV (Supplementary Table 1) for overlap amplification of individual amiRNA sequence, along with two common primers oligo A and oligo B (Supplementary Table 1) with the A. thaliana endogenous miR319a precursor as backbone (At4g23713). Finally, the PCR products were digested with KpnI and XbaI and cloned into CaLCuV to generate CaLCuV-BBX32. All cloned amiRNA sequences were confirmed through DNA sequencing.

VIGS of CmBBX32 in ‘Huaiju 2^#’ and phenotypic observation of transgenic plants to Alternaria sp. inoculation

The recombinant CaLCuV-BBX32 vector was introduced into the Agrobacterium tumefaciens (GV3101) strain (Sangon Biotech, Shanghai, China). Subsequently, the A. tumefaciens strain harboring the recombinant vector was transformed in to the ‘Huaiju 2^#’ via the negative-pressure vacuum filtration method [78, 79]. Briefly, the A. tumefaciens were suspended in an infiltration buffer (10 mM MES, 10 mM MgCl₂ and 200 mM acetosyringone pH 5.6) to a final OD600 of 1.0 [79]. The suspension was incubated in the dark at 28 °C for 3–4 h. Tissue culture plantlets of ‘Huaiju 2^#’, which had been cultured for 40 days, were carefully removed from the rooting medium. These plantlets were immersed in the infiltration buffer, vacuum-infiltrated at −0.7 MPa for 3 min, and subsequently washed with deionized water. Following inoculation, the plants were incubated in the dark at 8 °C for 72 h. Thereafter, all plants were transferred to pots containing a peat-vermiculite mixture (v/v = 1:1) in a controlled greenhouse, under the cultivation conditions described above.

Following an 8-day growth period for the transgenic plants, their leaves were promptly immersed in liquid nitrogen and subsequently stored at −80 °C for qRT-PCR analysis of gene silencing efficiency. The procedures for total RNA extraction, RNA quantification, and first-strand cDNA synthesis adhered to the methods mentioned above. Subsequently, one week later, the transgenic plants were inoculated with Alternaria sp. employing the protocol outlined earlier [34]. We utilized photography to document the phenotypic alterations in ‘Huaiju 2^#’ and control plants, both prior to and following inoculation with Alternaria sp. at 7, 15, and 21 d.

Alternaria sp. resistance assay of transgenic plants

To further faster investigate whether instantaneous silencing of CmBBX32 reduces resistance to Alternaria sp. infection in Chrysanthemum, we re-transformed the‘Huaiju 2^#’ with the CaLCuV empty vector (CaLCuV::00) and recombinant CaLCuV-BBX32 vector with the method mentioned above. Following the generation of transgenic seedlings, we re-infected those transgenic plants with Alternaria sp. at a concentration 10 times higher than the initial inoculum concentration using the same infection protocol as above.

Evans blue staining was utilized to examine spore germination and hyphal growth at 3 and 5 days post-inoculation (dpi) with Alternaria sp., following the methodology previously described [36, 80]. Briefly, leaf samples were submerged in a 0.5% (w/v) Evans blue solution in the dark at room temperature for 24 h. Following this, the leaves were rinsed three times with SDW, blotted gently to remove excess surface moisture, and then transferred to 95% ethanol for decolorization. The ethanol was replaced every 2 h until the leaves lost all pigmentation. The decolorized leaves were subsequently examined and photographed under a stereomicroscope. Additionally, the spore germination and hyphal growth of the black spot pathogen Alternaria sp. on the inoculated leaves were monitored and documented using a light microscope.

Furthermore, to evaluate the resistance of transgenic plants to Alternaria sp., qRT-PCR was conducted to analyze the expression of marker genes involved in the SA and JA defense pathways. The marker genes of SA defense pathway were CmPR1 and CmPR5, while the JA defense marker gene was CmPDF1.2 [34]. The procedures for total RNA extraction, RNA quantification, and first-strand cDNA synthesis were performed according to the methods described above. Gene-specific primers sequences are listed in Supplementary Table 1.

Results

Sequence feature of BBX genes in Chrysanthemum

A total of 26 members of Chrysanthemum BBX genes were identified (Supplementary Table 2). The lengths of these BBX genes in Chrysanthemum vary from 672 bp (CsG_LG8.g09866) to 4664 bp (CsG_LG3.g31983). The protein lengths range from 189 aa (CsG_LG7.g69629 and CsG_LG6.g04593) to 456 aa (CsG_LG3.g34272) (Supplementary Table 2). The protein molecular weights range from 20.95 kDa (CsG_LG6.g04593) to 51.93 kDa (CsG_LG3.g34272) and the theoretical isoelectric points range from 4.24 (CsG_LG6.g04593) to 9.18 (CsG_LG8.g12827) (Supplementary Table 2).

Multiple sequence alignment and phylogenetic analysis

Using the Conserved Domain tool and DNAMAN 6.0 software, all conserved amino acid sequences of BBX proteins in Chrysanthemum were aligned to identify the conserved regions. Results revealed that all BBX family members in Chrysanthemum harbor the conserved B-box domain (Supplementary Fig. 1). To investigate the phylogenetic relationships and divergence among Chrysanthemum BBX proteins, an un-rooted tree was constructed under the model of JTT + I + G in MrBayes program with BBX amino acid sequences from Chrysanthemum, A. annua, A. thaliana, and rice. Consistent with prior phylogenetic analyses of the BBX gene family in A. thaliana [81], the 26 Chrysanthemum BBX proteins were clearly clustered into five distinct groups based on their functional annotation and sequence identity (Fig. 1).

Fig. 1.

The phylogenetic tree for the BBX gene family in Chrysanthemum, A. annua, A. thaliana and rice. The tree was constructed using Bayesian inference implemented in MrBayes based on the amino acid sequences of the CmBBXs, AaBBXs, AtBBXs and OsBBXs under the model of JTT + I + G. Groups I, II, III, IV and V are marked with red, pink, blue, black, and green respectively

Among these CmBBX proteins, five were classified into subclass I, with four members typically containing two B-box domains and one CCT domain, while CsG_LG1.g02520 uniquely possessed one B-box and one CCT domain (Fig. 2). Subclass II also contained five members, four characterized by two B-box domains and one CCT domain, with the exception of CsG_LG3.g31983 possessed one B-box and one CCT (Fig. 2). In subclass III, two BBX proteins (CsG_LG3.g34272 and CsG_LG8.g31201) were found featured one B-box domain and one CCT domain. Four BBX proteins were grouped together in subclass V, each containing only one B-box domain (Fig. 2). Subclass IV represented the largest group, comprising ten BBX proteins, eight typically containing two B-box domains, except for CsG_LG9.g71760 and CsG_LG5.g16911 each harboring only one B-box domain (Fig. 1).

Fig. 2.

Gene structure, protein domain diagram and motif phases of CmBBX genes mapped on phylogenetic tree. a. The phylogenetic tree. The groups I, II, III, IV, and V are marked with red, pink, blue, black, and green respectively. b. The exon and intron phases of CmBBX genes. The exons were represented by green rectangles. The black lines connecting 2 exons represented introns. c. The conserved domain distribution of CmBBX proteins. d. The conserved motifs on the CmBBX proteins

Gene structure, protein domains and motif analysis of BBX in Chrysanthemum

Through gene structure analysis, we observed that the number of exons in CmBBX genes ranged from 1 to 5, and the average number of exons in the groups being either three or four. With the exception of CsG_LG8.g09866, which lacks introns, the average number of introns in the other genes ranges from 1 to 4. Furthermore, the correlation between gene structure and phylogenetic tree analysis revealed that CmBBX genes in subgroups II and IV generally possessed a higher number of exons and introns (Fig. 2a, b).

To elucidate the arrangement of protein domains, we conducted a domain analysis among BBX proteins of Chrysanthemum. A total of eight CmBBX proteins possess two B-box domains and one CCT domain, with six of them sharing identical B-box domains, and the other two possess distinct B-box domains. Additionally, four BBX proteins contain one B-box domain and one CCT domain, while one BBX protein (CsG_LG8.g12827) possesses one B-box domain and one PTZ00072 superfamily domain. Moreover, eight CmBBX proteins were found to possess only two identical B-box domains, and four BBX proteins contain only one B-box domain. Notably, only one BBX protein (CsG_LG9.g71760) was found to possess one B-box superfamily domain (Fig. 2c). Furthermore, the domain distribution also varied among different subgroups: CmBBX proteins in subgroups I and II all have a CCT domain, whereas CmBBX proteins with one B-box domain are primarily located in subgroups III and V. Moreover, the majority of members in subgroup IV possess two B-box domains (Fig. 2a, c).

Using the online MEME suite, we identified five motifs within the conserved amino acid sequences of Chrysanthemum BBX proteins. The consensus sequences of these five motifs are detailed in Supplementary Table 3. Subsequent analysis revealed that motif frequencies ranged from 2 to 26 occurrences (Supplementary Table 3), with individual motif lengths varying from 11 to 41 amino acids. Furthermore, the number of motifs present in each BBX conserved protein ranged from 1 to 6 (Fig. 2d, Supplementary Table 3). Notably, motifs 1, 2 and 3 were broadly distributed across BBX conserved proteins (Fig. 2d). Phylogenetic combined with motif analysis demonstrated that BBX proteins within the same subgroup exhibited similar motif orders and compositions (Fig. 2a, d), suggesting that genes clustered within the same subgroup may share similar functional roles.

Cis-regulatory elements of promoter analysis in CmBBX genes

A total of 39 cis-elements were identified and categorized into three primary classes, including abiotic and biotic stresses, phytohormone responsive, and plant growth and development (Fig. 3, Supplementary Table 4) [3].

Fig. 3.

Cis-elements of BBX genes in Chrysanthemum. The middle panel is the heat map showing the levels of cis-acting element enrichment. The chart on the right is a proportional map showing the abundance of cis-elements: the green rectangle, Abiotic and biotic stresses; the blue rectangle, Plant growth and development; the red rectangle, Phytohormone responsive

The majority of cis-regulatory elements identified within the Plant growth and development class were light-responsive elements, such as Box 4, G-box, I-box, and GT1-motif, which are widespread in plants and collectively accounted for 60.0% of the elements in this group (Fig. 3, Supplementary Table 4). The abscisic acid-responsive elements (ABREs) were the most abundant cis-regulatory elements in the phytohormone responsive group, occurring with a frequency of 88 times across the 26 CmBBX genes. Other notable elements included the CGTCA-motif and TGACG-motif, which are associated with methyl jasmonate (MeJA) responsiveness, the as-1 element linked to SA and oxidative stress responsiveness, and the ERE element corresponding to ethylene responsiveness (Fig. 3, Supplementary Table 4). These findings highlight that the regulation of CmBBX genes is involve in multiple hormonal pathways [22, 28, 29]. The group of abiotic and biotic stresses-related elements was associated with various stress responses. All BBX genes in Chrysanthemum harbored the cis-acting regulatory element responsible for anaerobic (ARE) inductions, suggesting that these genes are potentially induced under low oxygen conditions.

Furthermore, MYB and MYC elements were identified in all 26 CmBBX genes. This result suggests the potential involvement of CmBBX genes in modulating the defense response of Chrysanthemum to pathogens. The MYB transcription factor is known to integrate multiple resistance genes and defense signaling pathways, playing a pivotal role in plant defense against pathogens [82, 83]. Meanwhile, the MYC protein is involved in co-regulating the aromatic amino acid defense mechanism with jasmonate ZIM-domain (JAZ) proteins, thereby increasing resistance to necrotrophic pathogens [84, 85]. Additionally, a range of other stress-related elements, including the W box (involved in wounding and pathogen responsiveness), the STRE (stress-related element associated with heat shock proteins), and the MBS (MYB binding site involved in drought inducibility), were also present in this group (Fig. 3, Supplementary Table 4). These findings further highlight the potential role of BBX genes in mediating responses to both abiotic and biotic stresses in Chrysanthemum [41, 42].

Chromosomal distribution and collinearity analysis among CmBBX genes

Based on the annotated genomic locations, the 26 CmBBX genes exhibit a widespread distribution pattern across the Chrysanthemum chromosomes. Chromosome 5 harbors the largest number of CmBBX genes (five), followed by Chromosomes 7 and 8, each containing four CmBBX genes, while Chromosomes 3 and 6 each possess three CmBBX genes. Meanwhile, Chromosomes 1, 4 and 9 each possess two CmBBX genes, and Chromosome 2 contains a single CmBBX gene (Fig. 4).

Fig. 4.

Chromosome distribution of Chrysanthemum BBX genes. Chromosomal mapping was based on the physical position in 9 Chrysanthemum chromosomes. The scale on the left is in megabases (Mb). The chromosome numbers are indicated at the side of each bar. The gene names on the right side of each chromosome correspond to the approximate locations of each BBX gene

Collinearity analysis revealed five segmental duplication events: CsG_LG6.g07241 to CsG_LG7.g66670, CsG_LG6.g06424 to CsG_LG7.g64575, CsG_LG5.g61487 to CsG_LG7.g30264, CsG_LG3.g34272 to CsG_LG8.g31201, and CsG_LG2.g45063 to CsG_LG4.g39072 (Fig. 5, Supplementary Table 5). Notably, no tandem duplications have occurred during the evolution of Chrysanthemum BBX genes, consistent with previous findings [86, 87], and indicating that segmental duplications have primarily contributed to their evolutionary expansion. To further investigate the gene expansion mechanisms, we conducted a genomic collinearity analysis between CmBBX and AtBBX genes. The results identified a total of 18 gene pairs, comprising 14 CmBBXs and 14 AtBBXs (Fig. 6, Supplementary Table 6). The CmBBX genes exhibited one-to-one (10 pairs) and two-to-one (4 pairs) relationships with AtBBX genes. These findings suggested that both gene loss and expansion events have occurred in Chrysanthemum when compared with A. thaliana.

Fig. 5.

Distribution and intraspecific collinearity analysis of CmBBX genes on Chrysanthemum chromosomes. The grey lines indicate duplicated blocks and red lines indicate duplicated gene pairs of BBXs

Fig. 6.

Distribution and interspecific collinearity analysis of BBX genes between Chrysanthemum and A.thaliana. The grey lines indicate duplicated blocks and red lines indicate duplicated gene pairs of BBXs

Excavation of differentially expressed BBX genes response to Alternaria sp. in Chrysanthemum

Based on the transcriptome datasets of ‘Huaiju 2^#’ after inoculation with Alternaria sp. at 3 dpi and 5 dpi [34, 36], we assessed the expression profiles of 32 annotated BBX genes using FPKM values [70] according to the established criteria for selecting differentially expressed genes [88]. The results demonstrated that six CmBBX genes exhibited continuous up-regulation in response to Alternaria sp. inoculation at both 3 dpi and 5 dpi (Supplementary Fig. 2). Among these genes, the gene with transcript ID C81653_g1 showed the most significant increase, with its expression levels increasing by an approximately 3.7-fold at 3 dpi and a 5.12-fold at 5 dpi (Supplementary Fig. 2). Due to its remarkable up-regulation pattern, C81653_g1 was selected for further functional characterization.

Colon and expression analysis of CmBBX32

The full-length complementary DNA (cDNA) of C81653_g1 was obtained through PCR amplification and submitted to GenBank under the accession number PV002721. This cDNA fragment encompasses a 681-bp open reading frame (ORF), which encodes a predicted polypeptide consisting of 226 amino acids, with an isoelectric point of 8.37 and a molecular weight of 25.2 kDa (Supplementary Fig. 3a). Phylogenetic analysis with AtBBX gene family revealed that C81653_g1 clusters with AtBBX32 (Supplementary Fig. 3b). Based on this analysis, we propose renaming C81653_g1 to CmBBX32. Additionally, a single B-box domain is located at the N-terminus of CmBBX32 (Supplementary Fig. 3c).

Furthermore, qRT-PCR was performed to characterize the expression profiles of CmBBX32 at 0 d (prior to inoculation), and 1, 3, 5, 7, 15, and 21 dpi with Alternaria sp., using plants treated with SDW as controls. The results demonstrated a significant up-regulation of CmBBX32 expression in response to Alternaria sp. infection, with expression levels reaching 5.11 times the control level at 1 dpi. This up-regulation continued from 3 to 5 dpi, peaking at 5 dpi with an expression level of 23.95 times the control level. Subsequently, the expression gradually decreased but remained elevated compared to the control group throughout the experiment duration (Fig. 7a). Additionally, tissue-specific expression analysis revealed that CmBBX32 exhibited higher expression in flowers at full bloom. In tissue culture plantlets, CmBBX32 showed higher expression in leaves and stems, whereas its expression was relatively low in roots (Fig. 7b).

Fig. 7.

Expression profiles of CmBBX32. a: Expression of CmBBX32 in ‘Huaiju 2^#’ after inoculation with Alternaria sp. and SDW (mock) for 0 d (before inoculation), 1, 3, 5, 7, 15, and 21 dpi. b: Tissue-specific expression of CmBBX32 in root (R), stem (S), leaf (L) and flower (F) in ‘Huaiju 2^#’. (Student’s t-test, **p < 0.01, *p < 0.05)

Phenotypic observation of transgenic plants to Alternaria sp. inoculation

To further verify the function of CmBBX32 is associated with the resistance of ‘Huaiju 2^#’ to Alternaria sp. inoculation, we employed VIGS to instantaneous silence CmBBX32 in ‘Huaiju 2^#’. Following selection using rifampicin resistance, we obtained 15 positive transgenic lines. Subsequent verification via qRT-PCR revealed that the expression level of CmBBX32 was reduced in 13 transgenic lines compared to wild-type (WT). Among these, 10 lines exhibited a significant reduction in expression (Supplementary Fig. 4).

Furthermore, based on the expression levels of the transgene, three transformants were selected to evaluate their resistance to Alternaria sp. infection: CaLCuV::CmBBX32 #7 (with the highest degree of silencing), CaLCuV::CmBBX32 #5 (with moderate silencing) and CaLCuV::CmBBX32 #6 (with the lowest degree of silencing). The results indicated that all three lines displayed weakened resistance to Alternaria sp. infection, as evidenced by increased black spot development. Specifically, the CaLCuV::CmBBX32 #7 line exhibited the most severe symptoms, suggesting that lower expression levels of CmBBX32 correlate with poorer resistance to infection (Fig. 8).

Fig. 8.

Changes in phenotype after ‘Huaiju 2^#’ was inoculated with Alternaria sp. on leaves of WT and CaLCuV::CmBBX32 after 7, 15, 21 dpi. The inserted white box in the top right corner is a magnified image of the leaf selected from the bottom of the plants highlighted by a white frame. The numbers represent the transformants strains (Bar = 1 cm)

Instantaneous silencing of CmBBX32 reduces resistance to Alternaria sp. infection in Chrysanthemum

Using the screening method described above, we re-identified five transgenic lines that showed significantly reduced expression compared to WT and CaLCuV::00 (Supplementary Fig. 5). Among those transgenic lines, the CaLCuV::CmBBX32 #3 and CaLCuV::CmBBX32 #7 were selected to evaluate their resistance to Alternaria sp. infection. These transgenic plants were subsequently inoculated with Alternaria sp. at a concentration 10 times higher than the initial inoculum concentration.

Phenotypic analysis of infection symptoms demonstrated a more rapid expansion of black spot lesions in transgenic plants compared to both WT and CaLCuV::00 plants at 3 and 5 dpi (Fig. 9a). Evans blue staining was used to assess infection development, revealing that spore germination was observed in plants as early as 3 dpi. Furthermore, spore density in transgenic plants (1.65 × 10³ pcs/mm² in CaLCuV::CmBBX32 #7 and 2.05 × 10³ pcs/mm² in CaLCuV::CmBBX32 #3) were significant higher than WT (6.87 × 10² pcs/mm²) and CaLCuV::00 (5.49 × 10² pcs/mm²) plants (Fig. 9b, Supplementary Table 7). Additionally, evident hyphae growth was observed at 5 dpi, and the growth rate of hyphae was significantly faster in transgenic plants compared to WT and CaLCuV::00 plants (Fig. 9b).

Fig. 9.

Changes in phenotype (a) (Bar = 1 cm), and microscopic observation spore germination and hyphae development (b) (Bar = 200 μm) after ‘Huaiju 2^#’ was inoculated with Alternaria sp. on leaves of WT, CaLCuV::00, CaLCuV::CmBBX32 #3 and CaLCuV::CmBBX32 #7 after 3 and 5 dpi

In particular, the CaLCuV::CmBBX32 #3 line exhibited the most severe symptoms (Fig. 9), suggesting that reduced CmBBX32 expression correlates with decreased resistance. To further investigate this, we conducted qRT-PCR to analyze the expression levels of marker genes in the SA and JA defense pathways in transgenic plants. The results showed that the expression levels of the SA defense pathway marker genes, such as CmPR1 and CmPR5, were consistently lower in CaLCuV::CmBBX32 #3 compared to WT and CaLCuV::00 plants at 3 and 5 dpi with Alternaria sp. (Fig. 10; Table 1).

Fig. 10.

Expression profiles of two genes (CmPR1 and CmPR5) of the SA response pathway and one gene (CmPDF1.2) of the JA response pathway in WT CaLCuV::00, CaLCuV::CmBBX32 #3 plants after inoculation for 3 or 5 days. (Student’s t-test, **p < 0.01, *p < 0.05)

Table 1.

Effect of Alternaria sp. treatment on the expression of CmPR1, CmPR5 and CmPDF1.2 in VIGS Transgenic ‘Huaiju 2^#’ lines

Lines	Expression level
		3 dpi			5 dpi
		CmPR1	CmPR5	CmPDF1.2	CmPR1	CmPR5	CmPDF1.2
WT	Relative expressional level	1.60	6.63	1.64	11.93	25.05	3.36
CaLCuV:: 00		1.62	6.54	1.53	11.91	24.99	3.45
CaLCuV:: CmBBX32#3		1.09	2.29	3.13	1.77	3.51	1.56
CaLCuV:: CmBBX32#3	Reduction rate compared to WT (%)	31.88	65.45	−91.46	85.21	86.01	53.49
CaLCuV:: CmBBX32#3	Reduction rate compared to CaLCuV:: 00 (%)	32.51	64.96	−104.14	85.17	85.97	54.75

The expression level of the CmPDF1.2, the JA defense pathway marker gene, displayed higher levels at 3 dpi and lower levels at 5 dpi in CaLCuV::CmBBX32 #3 compared to WT and CaLCuV::00 plants (Fig. 10; Table 1). These findings demonstrate that the silencing of CmBBX32 in ‘Huaiju 2^#’ impaired the activity of the SA defense pathway, which is associated with resistance to Alternaria sp. infection in ‘Huaiju 2^#’. Meanwhile, the JA defense pathway exhibited an initial negative response followed by a subsequent positive response to Alternaria sp. infection. However, its responsiveness was significantly weaker compared to the SA defense pathway (Fig. 10; Table 1). These findings align with our previous studies [34].

Discussion

In plants, transcription factors are central regulators in responses to various forms of biotic stress. For example, heterologous overexpression of PsWRKY7, a WRKY transcription factor originating from Paeonia suffruticosa, significantly enhances resistance to Alternaria alternate in transgenic Populus davidiana × P. bolleana [89]. In Solanum torvum, StoERF109, an Apetala2/ethylene-responsive factor (AP2/ERF) transcription factor, positively functions against Verticillium dahliae infection [90]. Additionally, 12 R2R3-MYB transcription factors identified from Chinese flowering cabbage, potentially play a role in responding to Pectobacterium carotovorum infection [82]. Furthermore, CmC3H24 and CmC3H33, which belong to the Zinc Finger Protein (ZFP) transcription factor family, are potentially involved in modulating resistance to Fusarium wilt infection in Cucumis melo [89, 91]. BBX as a member of the Zinc Finger transcription factor family plays an important role in biochemical and physiological processes, such as response to biotic and abiotic stress, regulation of flowering time, photomorphogenesis, and secondary metabolism [3, 92–96]. With the advancement of high-throughput sequencing technologies, an increasing number of BBX gene family members have been identified across different plant species. However, there is limited information available regarding the BBX gene family in Chrysanthemum, which is one of the most renowned ornamental plants.

The number of BBX genes varies across different plant species, for instance, the largest number of BBX members identified was 64 in apple [47], while the smallest was 19 in millet [87, 97]. Additionally, among the 14 species of higher plants in which the BBX gene family has been identified, their genomes generally contain approximately 30 BBX gene members on average [1], which suggests that the number of BBX genes in plants is relatively stable [97]. In the present study, we identified 26 BBX genes in the Chrysanthemum genome, which also demonstrates a relatively stable number of BBX genes in this specie [97]. Previous studies have shown that the number of BBX gene family members does not correlate directly with plant genome size [1, 3]. The genome size of the Chrysanthemum used in this study is 2.53 Gb [33], while the genome size of A. thaliana is significantly smaller at 125 Mb [1, 98]. Based on these findings, we further propose that species-specific duplications or deletions during evolution may contribute to significant variations in gene family members among plants [1, 87, 97].

Phylogenetic analyses have previously categorized the majority of plant BBX genes into five distinct subgroups. In this study, a phylogenetic tree was constructed using BBX protein sequences from A. thaliana, rice, A. annua, and Chrysanthemum, which also supported their classification into five subfamilies. This finding is consistent with previous studies [3, 9, 81, 99, 100] (Fig. 1). In Chrysanthemum, the numbers of BBX members with one B-box, two B-boxes, one B-box plus one CCT domain, and two B-boxes plus one CCT domain were 5, 8, 4, and 8, respectively. Similar distribution patterns were observed in other dicotyledonous species, with corresponding counts of 7, 8, 4, and 13 in A. thaliana [81], 6, 9, 4, and 7 in pear [100], and 6, 10, 5 and 8 in tomato [43]. In contrast, monocotyledonous species exhibited distinct variation, with counts of 2, 8, 9, and 5 in Sorghum [101], 4, 17, 10, and 5 in Maize [102], 1, 8, 5, and 5 in Millet [101], and 3, 10, 10, and 7 in in rice [99]. These results suggest that BBX genes share a common ancestor and have undergone independent expansions after the divergence between monocotyledonous and dicotyledonous species [3, 100].

Although the number of members in each subgroup varies among different plants, the conserved domain composition of BBX proteins within the same subgroups is highly similar across plant species. This similarity suggests that BBX proteins within the same subgroups share similar functional roles. Additionally, the majority of CmBBX proteins within the same phylogenetic clade exhibited similar motif distributions, which also suggests that BBX proteins in the same group may share similar functions. Specifically, motif 4 was found exclusively in clade I and II, while motif 5 was present only in clade I. These distinct motif distributions suggest that BBX proteins in different groups have likely undergone functional divergence (Fig. 2d).

Gene structure analyses revealed that CmBBX genes with a similar exon-intron structure tended to cluster together in the phylogenetic tree (Fig. 2a, b). This finding is consistent with previous studies suggesting that exon-intron structure can serve as a reliable indicator for establishing phylogenetic relationships within a gene family [87, 103]. Furthermore, we observed that two gene pairs (CsG_LG5.g61487/CsG_LG7.g30264 and CsG_LG3.g34272/CsG_LG8.g31201) that possess identical exon numbers and highly similar exon lengths (Fig. 2b). These finding suggest that these gene pairs may have originated from segmental duplication events, providing further evidence to support our collinearity analysis (Fig. 5). Additionally, we also observed that certain paralogous genes within the CmBBX gene family exhibit exon loss (CsG_LG2.g45063/CsG_LG4.g39072 and CsG_LG5.g52909/CsG_LG7.g69629) (Fig. 2b). Those results are consistent with previous research, which has shown that exon-intron structural variations, including exon gain or loss, are key mechanisms driving the expansion and diversification of gene families [87, 104, 105].

Segmental duplications and tandem duplications play crucial roles in the evolutionary expansion of gene families [87, 106]. In the present study, five segmentally duplicated gene pairs were identified on chromosomes 2, 3, 4, 6, 7, and 8 of Chrysanthemum, while no tandem duplications were detected (Figs. 4 and 5, and Supplementary Table 5). This finding is consistent with previous studies examining the evolution of the BBX gene family in different plant species, including Poaceae plants [101], grapevine [87], tomato [43] and pear [100]. Additionally, the segmentally duplicated gene pairs (CsG_LG5.g61487 to CsG_LG7.g30264) were found to cluster within the same subgroup, sharing highly conserved gene structures, exon-intron architecture, and motifs (Figs. 1 and 2). This observation is in agreement with prior studies demonstrating that genes originating from segmental duplication events typically exhibit similar structural and functional annotations, often falling within the same grouping due to shared evolutionary origins [87, 99]. Collectively, these results highlight the contribution of segmental duplication to the expansion of the BBX gene family in Chrysanthemum. Moreover, we identified 18 orthologous BBX gene pairs that derived from segmental duplications between Chrysanthemum and A. thaliana (Fig. 6, Supplementary Table 6), suggesting that these genes may share a common evolutionary origin and potentially similar functional roles.

Diverse cis-regulatory elements located within promoter regions play a crucial role in functional diversity [107], and these elements mediate transcriptional regulation through specific interactions with transcription factors [108]. By investigating the multitude of cis-regulatory elements in the promoter regions of CmBBX genes, we could gain preliminary insights into the diverse functions of CmBBX genes in Chrysanthemum growth and development, phytohormone responsiveness, and resistance to abiotic and biotic stresses [43, 97, 109]. In the present study, we found that cis-regulatory elements associated with light responses, hormonal regulation, and responses to both abiotic and biotic stresses were significantly represented among the CmBBX genes. These findings indicated that the BBX gene family in Chrysanthemum may play an active role in regulating various physiological processes and stress responses. This discovery further elucidates the functional role of the BBX gene family in modulating flowering time and conferring tolerance to abiotic or biotic stresses in Chrysanthemum [41, 42].

Through transcriptomic data annotation, we identified 32 BBX sequences in the ‘Huaiju 2^#’. Among these, CmBBX32 was selected as a candidate gene for further investigation due to its continuous up-regulation in response to Alternaria sp. infection. Subsequent qRT-PCR analysis revealed that CmBBX32 expression in ‘Huaiju 2^#’ demonstrated a significant increase following Alternaria sp. inoculation, peaking at 5 dpi. This observation is consistent with the FPKM analysis of transcriptomic data, confirming the strong response of CmBBX32 to Alternaria sp. inoculation.

Furthermore, based on qualitative phenotypic analysis, the results indicated that the instantaneous silencing of CmBBX32 in ‘Huaiju 2^#’ significantly reduced the resistance of transgenic plants to Alternaria sp. infection compared to control (Fig. 8). To further confirm and investigate this, we inoculated those transgenic plants with Alternaria sp. at a 10-fold higher concentration than the initial inoculum. At 3 days post-inoculation, spore emergence was observed on the transgenic plants, exhibiting a notably accelerated growth rate and extensive hyphal spread compared to the control (Fig. 9). Additionally, quantitative analysis of two marker genes (CmPR1 and CmPR5) in the SA defense pathway revealed a significant reduction in their expression levels compared to the control (Fig. 10). Meanwhile, the SA defense pathway plays a crucial role in the defense mechanisms of Chrysanthemum against the necrotrophic fungus Alternaria sp [34, 36]. Silencing the CmBBX32 in ‘Huaiju 2^#’ thereby affects the activity of the SA defense pathway, ultimately leading to decrease in resistance to Alternaria sp. infection, consistent with our previous findings [34]. All those results demonstrate that CmBBX32 plays a crucial role in defense against Alternaria sp. infection in ‘Huaiju 2^#’.

Conclusion

In this study, we conducted a comprehensive genome-wide analysis of the BBX gene family in Chrysanthemum, identifying a total of 26 CmBBX genes. The CmBBX genes are categorized into five major groups that are characterized by gene structure, conserved domains, motifs, cis-elements, and collinearity. Our RNA-seq data combining with real-time quantitative expression analysis indicate that CmBBX32 is a functional BBX gene involved in resistance to Alternaria sp. inoculation in ‘Huaiju 2^#’. The instantaneous silencing of CmBBX32 in ‘Huaiju 2^#’ significantly compromised the resistance to Alternaria sp. infection. Collectively, this research forms the basis for future functonal characterizations of BBX gene family, particularly in relation to resistance against Alternaria sp. inoculation in Chrysanthemum.

Authors’ contributions

X.Z. contributed to supervision, project administration, writing review and validation, T.W. contributed to the experimental design and supervision, writing review and editing. Y.L. and X.H. cultivated sterile tissue Chrysanthemum seedlings. B.W., W.L. and H.G. carried out mainly experiments. B.W. and W.L. finished the draft the manuscript and comprehensively analyzed data from all experimental results. All authors have read and approved the final manuscript.

Funding

This research was supported by the Natural Science Foundation of China (No. 32372746 and No. U1704120), Doctoral Research Initiation Foundation of Henan Normal University (No. 5101049170907).

Data availability

The Chrysanthemum genome raw datasets generated and analyzed during the current study have been deposited in the NCBI under accession number PRJNA796762 and PRJNA895586. The RNA-seq data for Chrysanthemum morifolium cv. ‘Huaiju 2^#’ after inoculation with the necrotrophic fungus Alternaria sp. at 3 and 5 dpi are available in the NCBI Sequence Read Archive (SRA) repository (http://www.ncbi.nlm.nih.gov/sra/; accession number PRJNA448499). The full-length cDNA sequence of CmBBX32 has been deposited in NCBI GenBank under the accession number PV002721 (https://www.ncbi.nlm.nih.gov/nuccore/PV002721.1/).

Declarations

Ethics approval and consent to participate

Not applicable. This study was conducted in accordance with the People’s Republic of China and international authorities’ relevant guidelines and legislation, including the official website of the Committee on Publication Ethics (http://www.publicationethics.org) and the European Association of Science Editors (EASE) and other institutions’ publishing ethics standards.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.