Genome-scale identification, expression and evolution analysis of B-box members in Dendrobium huoshanense

Abstract

B-box (BBX) proteins have been recognized as vital determinants in plant development, morphogenesis, and adaptive responses to a myriad of environmental stresses. These zinc-finger proteins play a pivotal role in various biological processes. Their influence spans photomorphogenesis, the regulation of flowering, and imparting resilience to a wide array of challenges, encompassing both biotic and abiotic factors. Chromosome localization, gene structure and conserved motifs, phylogenetic analysis, collinearity analysis, expression profiling, fluorescence quantitative analysis, and tobacco transient transformation methods were used for functional localization and expression pattern analysis of the DhBBX gene. A total of 23 DhBBX members were identified from Dendrobium huoshanense. Subsequent phylogenetic evaluations effectively segregated these genes into five discrete evolutionary subsets. The predictions of subcellular localizations revealed that all these proteins were localized in the nucleus. The genetic composition and patterns showed that the majority of these genes consisted of several exons, with a few variations that could be attributed to transposon insertion. A comprehensive analysis using qRT-PCR was conducted to unravel the expression patterns of these genes in D. huoshanense, with a specific concentration on their responses to various hormone treatments and cold stress. Subcellular localization reveals that DhBBX21 and DhBBX9 are located in the nucleus. Our results provide a deep comprehension of the complex regulatory mechanisms of BBXs in response to various environmental and hormonal stimuli. These discoveries encourage further detailed and focused investigations into the operational dynamics of the BBX gene family in a wider range of plant species.

Highlights

• •Whole-genome duplication and segment duplication events drive the expansion of BBX genes.
• •DhBBX genes exhibit different expression patterns in response to hormone signaling and cold stress.
• •Subcellular localization confirmed that DhBBX21 and DhBBX9 mainly localized in the nucleus.

1. Introduction

Transcription factors (TFs) are pivotal in orchestrating a spectrum of plant biological processes, encompassing developmental regulation, stress adaptation, and the modulation of secondary metabolic pathway [1]. It is possible to categorize TFs into different groups based on their ability to bind cis-acting elements to the promoter regions. One of the most significant groups of transcription factors (TFs) in plants is the zinc finger family. In accordance with the structural and functional characteristics of each of its individual members, zinc finger TFs are divided into a number of subfamilies [2]. Within this group, B-box proteins (BBXs) constitute a subset of zinc-finger transcription factors. Regardless of whether they feature a C-terminal CCT (CO-like, TOC1, and CONSTANS) domain, BBX proteins consistently contain one or more N-terminal B-box domains, which play pivotal roles in transcriptional regulation, protein interactions, and nuclear transport [3,4]. In A. thaliana, the BBX family has undergone a comprehensive identification and classification process, resulting in the categorization of its 32 members into four groups. This classification is based on the number of B-box domains and the presence or absence of a CCT domain [5].

Members of Groups I and II within the AtBBX family are characterized by the presence of two B-box domains, accompanied by a CCT domain. Conversely, Group III members are characterized by a combination of a single B-box domain and a CCT domain. In Group IV, there are two B-box domains, but they lack a CCT domain. Group V, on the other hand, is defined by a single B-box domain [6]. Numerous plants, including Arabidopsis [5], rice [7], pear [8], pepper [9], tomato [10], and wolfberry [11], have identified BBX genes. Segmental duplication and deletion events over evolutionary time have resulted in variations in the consensus sequences of the two B-box domains [12]. The remarkably conserved CCT domain, which spans a range of 42–43 amino acids, plays a critical role in the control of transcriptional functions and the transport of nuclear proteins [13]. Recent genome-wide expression analyses suggest that BBX proteins may play a role in plant hormone signaling responses. Specifically, abscisic acid (ABA), a hormone released by plants in response to stress, has been implicated in modulating the expression of BBX genes [14]. Microarray studies have shown differential expression of BBX genes in response to ABA [15,16]. Furthermore, extensive research has demonstrated the critical involvement of BBX genes in regulating various aspects of plant biology, including growth, development, flowering, and the plant's response to abiotic stress [17,18].

Currently, the scientific community is increasingly intrigued by B-box proteins, recognizing their evolving role in various plant growth processes. Previous studies have cataloged the expression patterns of the BBX family in Oryza sativa, especially in relation to certain hormonal responses [19]. However, there are few studies on the BBX family under abiotic stresses in Dendrobium plants. D. huoshanense, a semi-shaded perennial plant from the Orchidaceae family, is commonly found in various environments, both natural and cultivated, often encounters challenging conditions such as elevated high temperatures, cold stress, drought stress, and additional stressors [[20], [21], [22]]. D. huoshanense is also a valuable Chinese herbal medicine. The polysaccharides in it have significant anti-tumor and immunity-improving effects [23]. Exposure to stress can reduce a plant's ability to tolerate adverse conditions, potentially leading to a significant decrease in productivity. Throughout their life cycle, plants face numerous challenges arising from environmental factors such as low temperatures, drought, salinity, and biotic stressors. These stressors can negatively impact plant growth and development, ultimately affecting overall productivity [24,25]. Such stressful situations result in unfavorable environmental circumstances for growth, development, and productivity. In response to environmental stimuli, plants rely on transcriptional regulation of gene expression for numerous physiological and metabolic functions. This includes reactions to multiple stressors such as salinity, cold, and drought, as well as defensive mechanisms against microbial invasions [26]. These TFs may have the ability to regulate gene expression at the molecular level by maintaining, promoting, and suppressing it. Furthermore, there is limited knowledge regarding the functions of the BBX family in the evolutionary relationship, structural modifications, stress responses, and functional variability of these proteins in D. huoshanense. As a result, the 23 members of the BBX family in D. huoshanense and their patterns of expression under five hormone treatments and cold stress were comprehensively examined in the current work. These results provide valuable insights into the evolutionary history of BBXs in D. huoshanense, shedding light on their potential biological functions related to multiple abiotic stress responses and plant growth regulation.

2. Materials and methods

2.1. Plant sampling and tissue preservation

D. huoshanense seedlings were obtained from the Plant Cell Engineering Center at West Anhui University (Luan, China). All samples utilized for the treatment of plant hormone assays were maintained at room temperature with 60 % relative humidity and subjected to long-day conditions, comprising 12 h of light followed by 12 h of darkness. Plants with uniform growth rates and ages were chosen for the study. Leaves were sprayed with water and 50 mmol/L MeJA solution, respectively. Time intervals between sprays were set at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 16 h. Leaves from both the control and MeJA-treated groups were harvested at distinct time points, immediately frozen in liquid nitrogen, and subsequently stored in a −80 °C freezer for future analysis.

2.2. Identification and characterization of BBX encoding genes in the genome of D. huoshanense

Two distinct approaches were utilized to find the genes that encode BBXs in the D. huoshanense genome. The first method used was retrieving the established sequences of AtBBXs from the TAIR database. Afterwards, we utilized these sequences to conduct a search for putative BBXs in the D. huoshanense genome database using the BLASTP tool, with the E value limit set at 1e⁻³. The second method involved acquiring the hidden Markov model (HMM) profile of the BBX domain (Pfam00643) from the Pfam database. Subsequently, the HMM profile was employed to find all BBXs in the D. huoshanense genome using HMMER 3.0 software, with an E value cutoff of 1e⁻³ [[27], [28], [29], [30]]. The Arabidopsis BBX gene family was obtained from the TAIR 10 database (https://www.arabidopsis.org/browse/genefamily/index.jsp). To assess the completeness of protein sequences and the existence of specific domains, we employed the following online resources: SMART (http://smart.embl-heidelberg.de/) (Schultz et al., 1998), InterPro Scan (http://www.ebi.ac.uk/interpro/), the Conserved Domain Database (CDD) (http://www.ncbi.nlm.nih.gov/cdd/), and ScanProsite (http://prosite.expasy.org/scanprosite/). Through the ExPASy online server (http://web.expasy.org/protparam/), the chemical characteristics of BBX proteins were investigated, including the instability index, protein length, aliphatic index, isoelectric point, molecular weight, and grand average of hydrophobicity [31,32]. The precise position of the DhBBXs gene on the associated chromosomes was depicted using TBtools and MapDraw software. The physical and chemical characteristics of the D. huoshanense B-box protein sequence, including isoelectric point and molecular weight predictions, were examined using the Expasy website (http://expasy.org) [3]. By entering the amino acid sequence of the DhBBX family, the subcellular position was determined using the web program Cell-PLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/).

2.3. Gene structure and motif analysis of DhBBX genes

A multi-gene family's evolution may be facilitated by gene structural variety, according to earlier studies. Gene exon-intron analysis was done to further characterize and comprehend the structural variety of DhBBX genes. The DhBBX genes were investigated and characterized using a systematic approach. The motifs present in each DhBBX gene were meticulously analyzed through the MEME suite (http://meme-suite.org/tools/meme). To visualize the outcomes, the Gene Structure View tool included in the TBtools package was utilized. The motif identifications were set as follows: each motif was expected to have a width ranging from 6 to 200, with a maximum limit of 20 motifs. Furthermore, the gene structures pertaining to DhBBXs were delineated using the essential nucleotide sequence, and this analysis was performed by the online website GSDS software (http://gsds.gao-lab.org/).

2.4. Phylogenetic analysis of DhBBX and AtBBX proteins

Using ClustalW software, the sequence alignment of BBX proteins from D. huoshanense and A. thaliana was employed [33]. The Maximum Likelihood Method (MLM) was used to create the phylogenetic tree using the MEGA X program [34]. One thousand replicates used in the bootstrap analysis by usinig iTOL v6.0 (https://itol.embl.de/itol_account.cgi) were utilized to show the phylogenetic trees [35].

2.5. Gene duplication and collinearity analysis of DhBBX genes

The MCScanX software was utilized to identify BBX gene duplications both intraspecifically and interspecifically. Gene duplication events were determined based on the following parameters: (1) The aligned segment of the two gene sequences extends beyond 80 % of the length of the longer sequence; (2) Among genes in close relation, each participated in WGD or segmental duplication event [36]. By doing an enrichment analysis and applying Fisher's exact test, it was possible to determine the relationship between the number of gene families and a specific genome-wide duplication mechanism [37]. Using a pipeline that has been described, the Ka and Ks values of duplicated gene pairs were determined. The analysis of gene duplication and synteny was executed using the MCscanX software and TBtools software. The exploration of synteny relationships of DhBBX genes among D. huoshanense, O. sativa, A. thaliana, D. nobile, and D. chrysotoxum was undertaken and subsequently visualized employing the MCScanX toolkit.

2.6. Cis-acting element analysis of DhBBX genes

To find out the potential cis-acting elements in the promoter regions of the DhBBX genes, 2000bp DNA sequences from the predicted start of transcription were downloaded from the D. huoshanense genome. These sequences were subsequently analyzed using the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) for the identification of multiple elements in the DhBBX gene promoter [38]. Finally, TBtools software was used for visualization [39].

2.7. Expression profile and transcriptomic analysis of DhBBX genes

To evaluate the expression levels of DhBBX genes, we extracted the fragments per kilobase of exon model per million mapped fragments (FPKM) values from pre-existing transcriptome datasets accessible on the Genome Sequence Archive (GSA) under the accession number CRA006607 (https://ngdc.cncb.ac.cn/gsa/browse/). The computation of these values employed the Salmon tool in conjunction with RNA-seq data [40]. By standardizing the log₂ (FPKM) values for all unigenes, a structured framework was established for hierarchical clustering and the delineation of DhBBX gene expression patterns. To conduct an extensive correlation analysis of DhBBX genes, we employed the SupCorrPlot tool integrated into the TBtools software suite. This allowed us to calculate Spearman correlation coefficients and their corresponding significance values.

2.8. Expression patterns of DhBBXs under various stress conditions via qRT-PCR method

RNA extraction was conducted using the QIAGEN RNA extraction kit from Germany. To ensure the purity of the RNA samples, any remaining genomic DNA was removed using the RNAse-Free DNase Set (QIAGEN, Germany). Subsequently, the quality and integrity of these RNA samples were assessed through the NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, USA) and gel electrophoresis. For the conversion of RNA into complementary DNA, the synthesis of cDNA was employed, and the reverse transcription reaction is performed by using PrimeScriptTM II 1st Strand cDNA Synthesis Kit (TaKaRa, Dalian, China). Quantitative real-time PCR (qRT-PCR) was performed on the 7500 Series Real-Time Fluorescence Quantitative Cycler (Bio-RAD, USA), utilizing the SYBR Premix Ex TaqTM II from TaKaRa (Dalian, China). The Actin gene was selected as the internal control to normalize the results. The qRT-PCR primers were designed using Primer Premier 5.0 software, with detailed information available in Table S1. The expression patterns of the BBX genes under investigation were determined employing the comparative threshold cycle method (2^−ΔΔCt) as described by Ref. [41]. To ensure robustness, each experimental step was meticulously conducted in triplicate.

2.9. Subcellular localization of DhBBX21 and DhBBX9

The homologous recombination primers of the pCAMBIA1305 vector were designed using the homologous recombination method. The homologous primers with XbaI and BamHI sites were presented. The designed homologous primers were used to clone the target fragment. The pCAMBIA1305 empty plasmid was double-digested according to the restriction sites of XbaI and BamHI. The digested carrier fragments were determined by gel electrophoresis. After the homologous recombination reaction between the target fragment and the vector fragment, the recombinant eukaryotic expression plasmid was constructed and successfully obtained. The recombinant vector plasmid was extracted for PCR detection and sequencing. The pCAMBIA1305 plasmid recombined with the target fragment was introduced into Agrobacterium GV3101, and positive single clones were picked out by streaking, and used to infect tobacco after expanding culture. The infection solution used was 10 mmol/L MgCl₂, 200 μmol/L AS (acetosyringone), and 10 nmol/L morpholineethanesulfonic acid (MES) (pH 5.5). The bacterial liquid was centrifuged at 6000 rpm/min for 10 min to collect the bacteria. After mixing the infection solution with the bacteria, the OD600 value was 0.6–0.8, and the cells were cultured in the dark for 3 h. Tobacco leaves were selected for injection on the back side and cultured in the dark for 3 days. The infected parts of the leaves were removed to make slices and observed under a confocal laser microscope.

3. Results

3.1. Identification, physiochemical properties, and chromosomal localization analysis of BBX genes

To identify the BBX genes from the D. huoshanense genome, the HMMER and Pfam softwares were used for the HMM profiles of both the BBox and CCT domains (Finn et al., 2014). The conserved CCT and BBox domains were then verified using SMART, InterPro, and Pfam databases. Finally, 23 BBox genes were identified after removing the split and duplicated DhBBXs (Table 1). Additionally, the online program Expasy Protoparam (http://www.expasy.org/tools/protparam.html) was used to research the physiochemical properties such as the length of the amino acid sequence of BBX members (Table 1). The BBX gene family presumed molecular weights range greatly, from 15992.6 Da (Dhu000014192) to 55543.31 Da (Dhu000010883). The calculated overall hydropathicity, isoelectric point, alphabetic index, and instability index, values ranged from 4.6 to 8.42, 44.43 to 73.99, 51.81 to 96.37, and −0.746 to 0.19, respectively. Subcellular localization prediction shows that all DhBBX proteins are localized in the nucleus.

Table 1.

Physiochemical properties sequence characteristics of 23 B-Box identified in D. huoshanense.

Sequence ID	Gene Renaming	Number of Amino Acid	Molecular Weight	Theoretical pI	Instability Index	Aliphatic Index	Grand Average of Hydropathicity	Subcellular localization
Dhu000000904	DhBBX3	332	35540.86	5.7	46.56	63.64	−0.234	Nucleus
Dhu000010754	DhBBX2	423	46307.13	8.36	49.48	62.41	−0.39	Nucleus
Dhu000003752	DhBBX21	325	34477.85	6.01	44.93	63.2	−0.258	Nucleus
Dhu000000293	DhBBX22	186	20983.68	6.4	56.19	73.33	−0.507	Nucleus
Dhu000010883	DhBBX17	508	55543.31	7.11	48.29	68.62	−0.412	Nucleus
Dhu000023905	DhBBX9	208	22656.72	6.97	48.15	74.62	−0.256	Nucleus
Dhu000021193	DhBBX8	208	22612.66	6.97	48.15	74.18	−0.255	Nucleus
Dhu000003422	DhBBX18	499	54675.09	7.46	53.79	67.13	−0.519	Nucleus
Dhu000014192	DhBBX5	146	15992.6	8.42	47.37	96.37	0.19	Nucleus
Dhu000023330	DhBBX10	488	53490.13	5.99	44.43	68.83	−0.284	Nucleus
Dhu000002829	DhBBX4	226	24826.28	4.95	73.99	57.57	−0.746	Nucleus
Dhu000010227	DhBBX19	203	22095.56	5.15	61.71	65.96	−0.427	Nucleus
Dhu000001125	DhBBX6	366	40868.9	7.07	60.8	63.99	−0.733	Nucleus
Dhu000013676	DhBBX7	366	40998.11	7.56	62.85	65.85	−0.716	Nucleus
Dhu000000981	DhBBX1	238	25553.79	4.6	65.52	51.81	−0.653	Nucleus
Dhu000000066	DhBBX20	237	25989.73	4.78	57.45	66.75	−0.392	Nucleus
Dhu000010353	DhBBX11	281	31594.37	5.25	62.09	64.23	−0.583	Nucleus
Dhu000023175	DhBBX12	281	31594.37	5.25	62.09	64.23	−0.583	Nucleus
Dhu000022626	DhBBX15	395	42293.61	6.31	53.45	64.63	−0.45	Nucleus
Dhu000018351	DhBBX16	395	42252.43	5.9	55.09	64.63	−0.462	Nucleus
Dhu000024355	DhBBX14	166	18528.03	4.94	49.19	84.04	−0.256	Nucleus
Dhu000020406	DhBBX13	187	21036.62	4.86	58.22	75.61	−0.408	Nucleus
Dhu000003374	DhBBX23	140	16008.51	6.43	69.89	87.79	−0.013	Nucleus

3.2. Phylogeny analysis of DhBBXs and ATBBXs

An evolutionary tree was constructed to depict the relationship between BBX proteins of D. huoshanense and A. thaliana, employing the Maximum Likelihood (ML) method. Based on bootstrap value and sequence homology of the phylogenetic tree, combined with Arabidopsis nomenclature, the categorization of BBX genes into five distinct families: I, II, III, IV and V. BBX proteins can be categorized into four distinct classes based on their domain arrangements: those possessing a single B-box domain, those with dual B-box domains, those combining a B-box with a CCT domain, and those equipped with two B-box domains accompanied by a CCT domain. Homologs from both D. huoshanense and A. thaliana genomes were carefully selected to explore the evolutionary and functional relationships among BBX genes. Their multiple sequence alignment and subsequent phylogenetic tree analysis aimed to unravel insights into the evolutionary dynamics and potential functional distinctions within this gene family. (Fig. 1). Considering the disparities in protein topological architecture, this research delineated the BBX family grouped into five distinct and highly conserved subfamilies, each corroborated by strong bootstrap validation. Based on our investigation, the phylogenetic tree classified the BBX into five groups based on clades. Notably, Groups I, IV, and V were distinguished by a significant number of DhBBX members, while subfamily III comprised a more limited representation with only five members. Subfamilies III had included both domains the BBX and CCT. In addition, the only CCT domain-related genes found in subfamily II and subfamily III. A significant portion of BBX genes possessing two B-box domains fell within a specific subfamily I. Almost all subfamilies belong to BBX, except II and III. In addition, we examined the D. huoshanense BBX genes to investigate their evolutionary connection to A. thaliana BBX members. In this research, we discovered that the A. thaliana BBX genes and D. huoshanense BBX genes were clustered together and showed a strong relationship.

Fig. 1.

Phylogenetic tree of DhBBXs genes from D. huoshanense and A. thaliana. The construction of the phylogenetic tree was facilitated by the MEGA 7.0 software, utilizing full-length protein sequences for the analysis. The Maximum Likelihood method was applied, and the reliability of the tree was assessed using 1,000 bootstrap replicates.

3.3. Microsynteny, gene duplication events and chromosomal localization of DhBBXs

The chromosomal distribution and gene duplication occurrences of DhBBX genes were examined to shed more light on how these genes evolved. There are 17 chromosomes total, and nine of them were discovered to include 23 DhBBX genes (Fig. 2). Four DhBBX genes among them were found on chromosome 16, followed by chromosomes 3, 6, and 8, which each had three BBX genes. Only two genes were embedded on chromosomes 7, 10, 11, and 18, and just one gene on chromosome 1.

Fig. 2.

The chromosomal mapping and distribution of BBX genes in the D. huoshanense genome. Each chromosome is represented by a vertical blue bar, the numerical designation of which is conspicuously displayed on the left-hand side of the bar. These numerical designations serve as a means of identification, aiding in the explication and differentiation of the individual chromosomes.

Several DhBBX genes, including DhBBX17, DhBBX18, DhBBX19, DhBBX20, and DhBBX21 were grouped together on chromosome 16, likely due to tandem duplication events. Tandem and segmental duplication are typically used to expand gene families. We were unable to locate any tandem duplication pairings in the current investigation. The gene pair collinearity in the genome of D. huoshanense was analyzed through the MCScanX software. Dhu000003752, Dhu000000066, Dhu0000010227, Dhu000003422 and Dhu0000010883 showed microsynteny with Dhu000000981, Dhu000010754, Dhu000000904 and Dhu000002829 genes situated on chromosomes 2, 3, and 18. Notably, genes Dhu000000981, Dhu000010754, Dhu000000904 and Dhu000002829 also showed microsynteny with Dhu000020406 and Dhu000024355. Microsynteny studies revealed pronounced collinearity for DhBBX genes situated on chromosomes 2, 3, 6, 7, 8, 11, and 18 (Fig. 3). Additionally, a collinear relationship was identified between Dhu000023905 and Dhu000021193.

Fig. 3.

Microsynteny analysis of BBXs genes within the D. huoshanense genome. (A) Employing a multi-layered visual representation to illuminate the intricacies of gene duplication, chromosomal length, gene density, and syntenic relationships. Duplicated blocks within the chromosomes are delineated by blue lines, offering a visual cue to identify regions of genetic redundancy or repetition. (B) Synteny relationship between BBX genes of D. huoshanense.

We conducted comparative genomics analyses involving D. huoshanense, D. nobile, O. sativa, D. chrysotoxum, and A. thaliana to elucidate the evolutionary and orthologous gene connections between various species, as well as to explore the emergence and numerous functional divergences of BBX homologous members (Fig. 4 and Table S2). We observed more extensive synteny blocks between D. huoshanense and D. nobile, and between D. huoshanense and D. chrysotoxum comprising 20 and 21 pairs, respectively. This pattern was followed by D. huoshanense O. sativa, D. huoshanense and A. thaliana, which exhibited 7 and 6 pairs, respectively. These findings align with the established evolutionary relationships among these species.

Fig. 4.

Comparative genomic analysis performed on D. huoshanense genes between D. huoshanense and A. thaliana, D. huoshanense and O. sativa, D. huoshanense and D. nobile, D. huoshanense and D. chrysotoxum. Black lines specifically highlight the collinear gene pairs that include members of the DhBBXs gene family.

3.4. Gene structure and conserved motif analysis of DhBBXs

The DhBBX genes, based on their chromosomal location and annotation, appear to be sporadically distributed throughout the chromosomes of the examined species. A multi-gene family's evolution may be facilitated by gene structural diversity, according to earlier studies [34]. Gene exon-intron analysis was done in order to characterize and comprehend the structural diversity of the DhBBX genes (Fig. 5). Upon detailed scrutiny of the gene structure, it is apparent that the DhBBXs feature complete open reading frames, including untranslated regions (UTRs), exons, and introns. A majority of the DhBBX genes were characterized by the presence of multiple exons and introns (Fig. 5). The phylogeny analysis revealed that DhBBX genes with significant similarity shared comparable exon and intron configurations. Similarly, when examining conserved motifs, it was evident that DhBBX genes originating from the same branch displayed similar motif arrangements. The number of exons ranged from 1 to 5, and Dhu000023330 has the most exons and introns of all the DhBBX. On the other hand, Dhu000014192 has one exon (Fig. 5).

Fig. 5.

Phylogeny, conserved motifs and gene structural elements within the DhBBXs.

In addition, DhBBX genes were found to be grouped in one clade with a strikingly similar exon-intron structure. For instance, Dhu000010883 and Dhu000003422 had 4 exons. These findings demonstrated exon loss or gain during the evolution of the DhBBX gene family, which led to functional divergence among the closely related DhBBX (Fig. 5). Utilizing the MEME Suite tool, we identified twenty representative motifs within DhBBX. Motifs 1, 2, and 6 were prevalent in the majority of DhBBX genes, representing the standard structures associated with BBX and zinc-finger domains. Additionally, unique motifs were discovered in specific DhBBX genes. Notably, the highest motifs were present in Dhu000010883 and Dhu000003422. As well as the lowest motifs were identified in Dhu000003374 and Dhu000014192 (Fig. 5). These distinctive patterns were associated with the diverse functionalities of the BBX genes. According to these findings, the DhBBX gene family had undergone exon loss or exon gain during its evolution, which caused the functional divergence among the closely related DhBBXs.

3.5. Cis-elements analysis of DhBBXs genes

To unravel regulatory elements and functions, we conducted an analysis of the cis-acting elements present in the promoter regions of DhBBX genes. These promoter regions in DhBBX were found to encompass a wide array of regulatory elements associated with processes related to multiple hormones, growth, and development, secondary metabolism, as well as abiotic stress responses (Fig. 6A). We identified a total of 67 distinct cis-acting elements, which were classified into different types (Table S3). Notably, a significant number of these elements were associated with processes such as ABA response (ABRE motif), anaerobic induction (ARE motif), and light response (including the G-box motif and Box 4 motif), respectively. Hormonal response elements, such as ABRE (48) related ABA response element, CGTCA-motif (32) related to the MejA response element), and GARE-motif (5) related to the gibberellin response (Fig. 6B). Light-responsive elements are G-box element (40). The meristematic expression-related elements are TGACG-motif (32) plays a key role in DhBBXs gene regulation of plant meristem and organogenesis. Stress-related elements, such as MBS (11) as well as some other additional regulatory variables like GC motif (2) and circadian (5) have major functions in anoxic, zein metabolism regulation. Nonetheless, the configurations of cis-acting elements among the majority of closely related DhBBX members exhibited variations, showcasing a range of cis-elements. This observation highlights the concept that despite originating from a common ancestor through duplication of genes, paralogous genes maintain unique and closer functions and are subject to varying degrees of regulatory control.

Fig. 6.

Prediction of cis-acting elements in the promoter regions of DhBBXs genes. (A) The cis-elements are predicted using a 2000bp sequence of promoter region of DhBBX genes and are visually represented as colored ellipses. (B) Composition and number of cis-acting elements in the promoter of DhBBX genes.

3.6. Functional annotation of DhBBX genes

The gene ontology enrichment analysis unveiled associations with cellular components, molecular and biological processes, with an additional sixteen groups identified as pivotal players in plant molecular functions. When examining the biological processes governed by DhBBXs participate in the regulation of flower development, regulation of photomorphogenesis, regulation of flower development, red light signaling pathway, regulation of photomorphogenesis, regulation of DNA-templated transcription, photomorphogenesis, and regulation of DNA-templated transcription (Fig. 7). DhBBXs regulate a variety of biological processes, including zinc ion binding, transcription cis-regulatory region binding, protein binding, DNA-binding transcription factor activity, and zinc ion binding. In addition to their roles in biological processes, DhBBX proteins were identified as being engaged in the regulation of various molecular functions. Additionally, the GO (gene ontology) enrichment analysis predicted the involvement of DhBBXs protein in processes related to the nucleus, chloroplast, and cytoplasmic (∼5 %) compartments, as detailed in Table S4. Predictions for subcellular localization showed that the majority of DhBBX proteins were found in the nucleus and chloroplast, with only one localized in the Cytoplasm.

Fig. 7.

Analysis of the Gene Ontology (GO) annotations for DhBBXs proteins. GO annotation was conducted across three distinct categories: biological process, cellular component, and molecular function. The dot in the graphical representation displays the proportion of predicted proteins in terms of percentages for each category.

3.7. Expression profiles of DhBBX genes

To get a full picture of the effects of methyl jasmonate (MeJA) treatment, we did a comparative transcriptomic analysis that covered 16 h of exposure. We carefully looked at the expression profiles of 15 DhBBXs genes using sequences from clean reads that had been carefully filtered (Table S5). After subjecting these genes to heatmap clustering analysis, we discovered distinct patterns of the genes with consistently high expression levels at the Time6-vs-Time7 intervals, such as Dhu000023330, Dhu000018351, Dhu000021193, Dhu000023905, and Dhu000003422 (Fig. 8).

Fig. 8.

The heatmap of expression profile of DhBBXs genes at different time with MeJA treatment. Expression changes are quantified using fold changes, which have been normalized using the logarithm base 2 function of the ratio of FPKM values between two comparative groups.

Notably, the influence of MeJA treatment was profound, as evidenced by the robust induction of Dhu000010227, Dhu000020406, Dhu000024355, and Dhu000013676, whereas Dhu000000904, Dhu000010883, and Dhu000000066, Dhu000010353, Dhu000022626, and Dhu000003374 exhibited no discernible response to MeJA treatment. Furthermore, the expression profile of DhBBX genes were employed at various time points (Fig. 8). The majority of DhBBX genes exhibited upregulated expression at 0.25 h (Time 2) after treatment, with the highest expression observed at 4 h (Time 6). Remarkably, Dhu000021193 showed the most expression of fold change (highlighted by the red block) when revealed after 8 h of MeJA treatment (Time 7), signifying a significant upregulation in its expression. Conversely, Dhu000024355 displayed the most pronounced fold change after just 2 h of treatment (Time 3), implying a potent inhibitory effect of MeJA on its expression. Our findings suggest that DhBBX genes might reveal its special behavior to JA signaling.

3.8. Expression analysis of DhBBX genes under abiotic stresses

A comprehensive investigation was undertaken to analyze the expression patterns of DhBBX genes through qRT-PCR. This meticulous assessment was conducted following the application of distinct exogenous hormones (ABA, IAA, MeJA, SA, GA) and exposure to cold stress in D. huoshanense at different time intervals. This time point was strategically chosen due to its correlation with the peak hormonal activity in D. huoshanense. The overarching objective of this investigation was to gain deeper insights into the dynamic expression levels of DhBBX genes when exposed to stress-related hormones. It is worth noting that the response dynamics were meticulously monitored at multiple time intervals, including 2 and 8 h, but ABA hormone on 2 and 4 days (Fig. 9). The outcomes of this endeavor unveiled a notable alteration in the expression profiles of DhBBX members across the spectrum of multiple hormones and time points. This intricate interplay between genes and hormonal stimuli provides valuable insights into the molecular responses of D. huoshanense under the influence of these stress-related hormones. We observed that the DhBBX genes exhibited unique and nuanced responses to each hormone intervention. Under ABA treatment, Dhu000000293 and Dhu000023330 showed a substantial surge in expression on the 2nd, while Dhu000000066, Dhu000000904, Dhu000003752, and Dhu000010227 exhibited little expression on different levels (Fig. 9A). On the other hand, IAA treatment had no significant expression (Fig. 9B). GA treatment led to the heightened expression of Dhu000000981, and Dhu000001125 on the 16 h, while Dhu000013676 showed the highest expression on 16 h as compared to other genes (Fig. 9C). Dhu000002829 showed the highest gene expression at 2 h. Meanwhile, the application of MeJA resulted in the significant upregulation of Dhu000000981 on the 2 h, while the Dhu000003422 gene continued to exhibit heightened expression on the 2 h and 16 h of treatment (Fig. 9D). Under the influence of SA, Dhu000000981 initially elevated in expression at the 2 h and 16 h but rebounded with the heightened expression in the 8 h (Fig. 9E). The cold stress conditions revealed that all genes had little expression as compared to the control (Fig. 9F). These empirical findings serve to substantiate the broad-ranging involvement of DhBBX genes in the intricate process of development, underscoring their pivotal role in responding to abiotic stressors.

Fig. 9.

The expression levels of DhBBXs genes in D. huoshanense under six distinct types of abiotic stressors (ABA, GA, IAA, MeJA, SA, and cold stress). The quantification of gene expression is achieved through quantitative reverse transcription polymerase chain reaction (qRT-PCR), and the actin gene serves as the normalization control. (A) The seedlings are exposed to abscisic acid (ABA) treatments, with the time frames of exposure categorized as CK (control), 2 days, and 4 days. (B) Delineates the gene expression patterns under indole-3-acetic acid (IAA) treatments at distinct intervals labeled as CK, 2 H, and 8 H. (C) The effects of gibberellic acid (GA) treatments on DhBBXs gene expression, with observations taken at CK, 2 H, and 8 H. (D) Explores the impact of Methyl Jasmonate (MeJA) treatments, with temporal markers set at CK, 2 H, and 8 H. (E), The seedlings undergo salicylic acid (SA) treatments, and gene expression is recorded at CK, 2 H, and 8 H. (F) The DhBBXs gene expression changes under cold stress conditions, specifically at 4 °C, with time points at CK, 2 H, and 8 H. Statistical rigor is maintained through the inclusion of error bars, which are calculated based on three biological replicates for each treatment. Significance levels are denoted by asterisks placed atop the error bars, providing a visual cue for statistical validity. A single asterisk (*) indicates a significance level where P < 0.05; two asterisks (**) signify P < 0.01; and three asterisks (***) represent an even more stringent significance level with P < 0.001.

3.9. Subcellular localization of DhBBX21 and DhBBX9

To figure out the functional location of the DhBBX proteins in cells, two target genes were connected to the pCAMBIA1305-GFP subcellular localization vector. The results showed that the empty fluorescent signal was distributed in the nucleus, cytoplasm, and cell membrane. The fluorescent signal of pCAMBIA1305-DhBBX21-GFP was localized on the nucleus and cytoplasm, which indicated that the DhBBX21 protein mainly functions on the nucleus and cytoplasm. The fluorescent signal of pCAMBIA1305-DhBBX9-GFP also localized on the nucleus, which suggested that the DhBBX9 protein mainly functions on the nucleus (Fig. 10).

Fig. 10.

Subcellular localization analysis of DhBBX proteins. Subcellular localization of pCAMBIA1305-DhBBX21-GFP and pCAMBIA1305-DhBBX9-GFP. GFP: green fluorescence signal, Bright Field: bright field, Merge: superposition field. The scale bar is 10 μm.

4. Discussion

In recent years, the scientific community has shown a growing fascination with the BBX gene family. Previous research has already delved into the identification of BBX genes throughout the entire genome of rice [42] as well as other significant plants such as potato, Arabidopsis, apple, tomato, and pear [[43], [44], [45], [46], [47]]. The comprehensive genome-wide identification of BBX genes in D. huoshanense was conducted, with a specific focus on their responses to various hormone treatments (ABA, GA, IAA, MeJA, and SA). Additionally, we analyzed their expression responses under cold stress conditions. Notably, genome duplication events emerged as the prevailing mechanism, potentially playing a pivotal role in the proliferation of DhBBXs. Microsynteny analysis involving D. huoshanense, A. thaliana, O. sativa, D. nobile, and D. chrysotoxum revealed that duplicated regions of DhBBXs remained highly conserved and experienced purifying selection throughout their evolutionary journey. Moreover, an in-depth examination of their physicochemical characteristics, gene structures, and motifs revealed diversification in terms of quantity, functional divergence, and structural attributes.

The number of BBX genes exhibits some variation across various crops, with rice, Arabidopsis, tomato, and potato having 30, 32, 29, and 30 members, respectively [48,49]. There is virtually no variation in BBX genes between the different plants. But there are 67 BBX genes altogether in the apple [50]. Due to the heterozygous nature and large size of the apple genome, there could be differences in the count of BBX genes when compared to both tree and crop plants. In our analysis, we identified 23 BBX gene members within the D. huoshanense genome. Based on domain architecture in tomato and Arabidopsis, previous investigations classified BBX proteins into four distinct categories [51,52].

BBX proteins exhibit a diverse classification into five distinct groups based on their structural features. These include proteins with a single B-box domain, those possessing two B-box domains, proteins with a B-box coupled with additional CCT domains, and those presenting two B-boxes accompanied by supplementary CCT domains. However, we did observe a slight divergence in the composition of one category of BBX proteins across various species. Therefore, it is suggested that during their evolutionary relationship, members of the subfamily BBX had similar gene structures and various functional characteristics. It has also been previously shown that during rice evolution, members of the ferric reduction oxidase family had a similar gene structure and functional features [53]. Furthermore, it has already been noted that the domain of CCT is highly conserved [54]. Still, there is a theory that during evolution, a process called deletion happens, which leads to the creation of a new group of BBX genes that can be identified by their single B-box domain [55]. We have formulated a hypothesis suggesting that a deletion event within the B-box2 domain might lead to the emergence of the B-box1 domain. This hypothesis stems from a comprehensive sequence alignment of the two B-box domains (B-box1 and B-box2), which revealed a higher level of conservation in the B-box1 domain within rice BBX genes. Throughout the course of evolution, these gene family members undergo amplification in the genome through both tandem duplication processes and large-scale duplication [56]. On chromosomes, tandem duplications of the genes were also found, which most likely aided in the production and functionalization of paralogous genes. To reveal orthologous genes and explore the evolutionary connections between diverse species, as well as to examine the origins and functional differentiation of BBX homologous genes, we conducted comprehensive comparative genomics analyses that included D. huoshanense, A. thaliana, O. sativa, D. nobile, and D. chrysotoxum (Fig. 4). At closer distances, orthologous gene pairs were more commonly observed. These findings support the idea that gene duplication expedited species development and provided the basis for the creation and neo-functionalization of genes [57].

The majority of plants are vulnerable to biotic and abiotic stress factors, which affect their output and survivability rate during the course of their lives [48]. In response to stress, plants have evolved tolerance mechanisms that alter the biochemistry and physiology of their cells by altering gene expression [50]. A BBX family of transcriptional regulators has 29 members in tomatoes and 32 in Arabidopsis [36,47]. BBXs play an important role in Arabidopsis regulatory networks that affect growth and development processes such as seedling photomorphogenesis, flowering regulation, and avoidance of shade (Khanna et al., 2009; Gangappa & Botto, 2014; Xu et al., 2018). The role of some BBXs in plants' reactions to diverse abiotic stressors has been in-depth investigated in recent years. For instance, AtBBX5 and AtBBX21 were positive regulators that regulated Arabidopsis' ability to tolerate salt and drought [58]. Abscisic acid (ABA), a significant phytohormone, is crucial to how plants react to diverse stimuli. Plants always activate the ABA signal transduction pathway to activate the production of stress defense genes in response to abiotic stressors [59]. The transgenic plants developed a tolerance for abiotic stress as a result of the overexpression of several transcription factor genes [60].

Under ABA treatment, some genes (Dhu000000293 and Dhu000023330) showed a significant increase in expression after 2 h, while others (Dhu000000066, Dhu000000904, Dhu000003752, and Dhu000010227) exhibited minimal expression changes. MeJA treatment led to a notable upregulation of Dhu000000981 at 2 h, and Dhu000003422 continued to show heightened expression at 2 h and 8 h. Under SA treatment, Dhu000000981 initially decreased in expression at 2 h but rebounded with higher expression at 8 h. GA treatment resulted in increased expression of Dhu000000981 and Dhu000001125 at 8 h, with Dhu000013676 showing the highest expression at 8 h compared to other genes, and Dhu000002829 exhibiting the highest gene expression at 2 h. Nevertheless, when exposed to cold stress conditions, all genes exhibited barely any alterations in expression levels in comparison to the control. This result could likely be attributed to the existence of the LTR motif in the promoters. These findings emphasize the diverse responses of genes to different treatments, highlighting the complex role of these genes in development and their importance in response to abiotic stressors.

5. Conclusion

In-depth analysis of B-box (BBX) genes in D. huoshanense illuminated their multifaceted roles in plant physiology. The study focused on analyzing the functional localization and expression patterns of the DhBBX gene in D. huoshanense. Various methods such as chromosome localization, gene structure analysis, conserved motif identification, phylogenetic analysis, collinearity analysis, expression profiling, fluorescence quantitative analysis, and tobacco transient transformation were used. A total of 23 DhBBX members were identified, and they were categorized into five evolutionary subsets based on phylogenetic analysis. The genes were found to be localized in the nucleus. The majority of the genes had multiple exons, with some variations due to transposon insertion. The expression patterns of these genes were analyzed using qRT-PCR, with a focus on their response to hormone treatments and cold stress. DhBBX21 was found to be located in the nucleus and cytoplasm. The insights gained from their gene structures, coupled with their expression dynamics under varied hormonal and stress conditions, enhance our understanding of their pivotal roles in plant growth, morphogenesis, and stress adaptability. This study not only adds to the current knowledge on BBX genes but also outlines a promising path for future studies, which could lead to new biotechnological applications in plant breeding and molecular genetics in Dendrobium plants.

Funding

This work was supported by National Key R&D Program of China (2023YFC3503804), Startup fund for high-level talents of West Anhui University (WGKQ2022025), the Open Fund of Anhui Engineering Research Center for Eco-agriculture of Traditional Chinese Medicine (WXZR202318), and Demonstration Experiment Training Center of Anhui Provincial Department of Education (2022sysx033).

Data availability statement

The raw datasets of transcriptome used in this study was obtained from the NGDC GSA database with accession code CRA006607. All genome sequences used in this study can be downloaded and queried in the NCBI database.

CRediT authorship contribution statement

Hui Deng: Writing – original draft, Visualization, Validation, Software, Methodology, Investigation, Formal analysis. Yingyu Zhang: Writing – original draft, Visualization, Validation, Software, Methodology, Investigation, Formal analysis. Muhammad Aamir Manzoor: Writing – review & editing, Validation, Software, Investigation. Irfan Ali Sabir: Writing – review & editing, Validation, Methodology, Investigation, Formal analysis. Bangxing Han: Writing – review & editing, Resources, Project administration, Funding acquisition. Cheng Song: Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Methodology, Funding acquisition, Data curation, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the Supplementary data to this article: