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Genome-wide identification, molecular evolution and expression analysis of the B-box gene family in mung bean (Vigna radiata L.)

BMC Plant Biology volume 24, Article number: 532 (2024) Cite this article

Abstract

Background

Mung bean (Vigna radiata L.) is an important warm-season grain legume. Adaptation to extreme environmental conditions, supported by evolution, makes mung bean a rich gene pool for stress tolerance traits. The exploration of resistance genes will provide important genetic resources and a theoretical basis for strengthening mung bean breeding. B-box (BBX) proteins play a major role in developmental processes and stress responses. However, the identification and analysis of the mung bean BBX gene family are still lacking.

Results

In this study, 23 VrBBX genes were identified through comprehensive bioinformatics analysis and named based on their physical locations on chromosomes. All the VrBBXs were divided into five groups based on their phylogenetic relationships, the number of B-box they contained and whether there was an additional CONSTANS, CO-like and TOC1 (CCT) domain. Homology and collinearity analysis indicated that the BBX genes in mung bean and other species had undergone a relatively conservative evolution. Gene duplication analysis showed that only chromosomal segmental duplication contributed to the expansion of VrBBX genes and that most of the duplicated gene pairs experienced purifying selection pressure during evolution. Gene structure and motif analysis revealed that VrBBX genes clustered in the same group shared similar structural characteristics. An analysis of cis-acting elements indicated that elements related to stress and hormone responses were prevalent in the promoters of most VrBBXs. The RNA-seq data analysis and qRT-PCR of nine VrBBX genes demonstrated that VrBBX genes may play a role in response to environmental stress. Moreover, VrBBX5, VrBBX10 and VrBBX12 are important candidate genes for plant stress response.

Conclusions

In this study, we systematically analyzed the genomic characteristics and expression patterns of the BBX gene family under ABA, PEG and NaCl treatments. The results will help us better understand the complexity of the BBX gene family and provide valuable information for future functional characteristics of specific genes in this family.

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Background

Zinc finger proteins are among the transcription factors that play important roles in developmental processes and stress responses [1]. The B-box (BBX) gene family belongs to the zinc finger protein. The BBX proteins have one or two B-box domains at the N-terminus of proteins, and some have a conserved C-terminal CONSTANS, CO-like and TOC1 (CCT) domain. Based on the distance between the zinc-binding residues and their consensus sequence, the BBX domain is divided into two categories: B-box1 (B1) and B-box2 (B2) [2]. Both the BBX and CCT domains have important functions. The BBX domain plays an important role in modulating protein‒protein interactions due to the inclusion of conserved cysteine and histidine [3, 4]. The CCT domain is important for transcriptional regulation and nuclear transport [5].

In Arabidopsis, 32 BBX genes are divided into five groups according to the number of B-box domains and whether the protein contains a CCT domain [3]. The first characterized BBX gene in Arabidopsis was CONSTANS (CO)/AtBBX1, which is a key activator of flowering under long inductive days. CO acts as a transcription factor to directly bind the promoter of Flowering Locus T (FT), ultimately triggering the expression of FT and initiating flowering [6]. The CO-FT module is highly conserved in regulating photoperiod flowering in various plants, such as rice, corn, tomato and sunflower [7]. In addition, other members of the BBX family have been reported to mediate flowering, including AtBBX4, AtBBX7, AtBBX21 and AtBBX32 [8,9,10]. Extensive studies have reported that BBX proteins act as key factors in the regulation of early photomorphogenesis. Some BBXs, such as BBX11, BBX21, BBX22, and BBX23, are positive regulators of photomorphogenesis [11,12,13], while others, including BBX19,BBX24, BBX25, BBX28, BBX29, BBX30, BBX31 and BBX32, repress photomorphogenesis in response to a wide range of light signals [7, 14,15,16]. For example, BBX28 negatively regulates photomorphogenesis by inhibiting the activity of transcription factor HY5 and undergoes COP1-mediated degradation [16]. Several BBX genes also have functions in shade avoidance through mediating cell elongation [17].

An increasing number of studies have shown that BBX proteins play critical roles in abiotic stress responses. In Arabidopsis, AtBBX24 was initially isolated as a salt-tolerant protein (STO) conferring salt tolerance in yeast [18]. STO overexpression enhances root growth under high salinity conditions, indicating that STO is involved in the salt-stress response [19]. AtBBX21/STH2 is a homolog of STO. Molecular lesions of STH2 resulted in reduced stomatal aperture and water loss under abscisic acid (ABA) and sodium chloride (NaCl) treatments, indicating that sth2 mutants are sensitive to ABA and salt stress. In addition, BBX21 negatively regulates ABI5 expression by interfering with the binding of HY5 to the ABI5 promoter, contributing to a decelerated ABA response. Therefore, STH2 negatively regulates ABA-mediated dehydration tolerance [20]. The heterologous expression of AtBBX29 in sugarcane enhances drought tolerance and delays senescence under water-deficit conditions by maintaining photosynthesis and enhancing antioxidant and osmolyte capacity [21]. In Chrysanthemum, CmBBX19 interacts with CmABF3 to repress the ABA response, resulting in reduced drought tolerance [22], while CmBBX22 delays drought-induced leaf senescence by negatively regulating the expression of ABF4 and upregulating the expression of ABI3 and ABI5 [23]. CmBBX24 improves freezing and drought stress tolerance by negatively regulating gibberellin biosynthesis and positively regulating genes associated with compatible solutes and carbohydrate metabolism [24]. MdBBX10 overexpression in Arabidopsis significantly enhances tolerance to abiotic stresses, with a higher germination ratio and longer root length. In addition, MdBBX10 overexpression can enhance a plant’s ability to scavenge reactive oxygen species (ROS) under stress, which is correlated with the expression of ROS-scavenging genes such as superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione s-transferase (GST) [25]. MdBBX1 transgenic plants display enhanced tolerance and have a higher survival rate after salt and drought treatments [26]. Overexpression of Ginkgo BBX25 improves salt tolerance in transgenic Populus [27]. CpBBX19 (Chimonanthus praecox) confers salt and drought tolerance in Arabidopsis [28]. Similarly, overexpression of IbBBX24 enhances salt and drought tolerance by scavenging ROS in sweet potato [29]. In addition to their roles in photomorphogenesis, flowering, shade avoidance and stress responses, increased studies have revealed that BBX proteins also play important roles in the circadian rhythm, senescence, and anthocyanin biosynthesis [7].

BBX genes have been systematically identified and analyzed in several species, e.g., there are 30 BBX genes in rice [4], 29 in tomato [30], 25 in pear [31], 64 in apple [32] and 24 in grapevine [33]. However, thus far, BBX genes have not been well studied in mung bean. Mung bean belongs to the subfamily Papilionoid of Fabaceae and is widely cultivated in tropical and subtropical regions. As a functional raw food material, mung bean is rich in nutrients, including high-quality proteins, carbohydrates, dietary fibers and bioactive substances [34]. Because of its nutritional value and broad adaptation, attention has been focused on developing resistance genes to enhance mung bean breeding. With the availability of the mung bean genome sequence [35], the BBX gene family in mung bean could be systematically studied. Here, we analyzed the phylogenetic evolutionary relationships, chromosome localization, motifs, genetic structure, cis-acting elements and expression profiles under polyethylene glycol (PEG), NaCl and ABA treatments. This study provides a new understanding of the evolutionary mechanism of BBX genes and lays a foundation for future investigations of the biological functions of BBX genes in mung bean.

Results

Identification of BBX genes in mung bean

Taking advantage of the availability of the complete genome assembly of mung bean, we initially identified 42 non-redundant BBX genes in mung bean using Arabidopsis BBX proteins as queries. Then, the Hidden Markov Model (HMM) profile of the B-box was used to search for putative BBX genes, and 19 candidate BBX genes were found. The resulting sequences were further confirmed as BBX members by identifying the presence of the B-box domain. As a result, 23 VrBBX genes were confirmed in mung bean and named VrBBX1 to VrBBX23 based on their physical locations on the chromosomes. A total of 17 VrBBX genes were unevenly distributed on 9 of 11 chromosomes, and the other 6 genes (VrBBX18, -19, -20, -21, -22 and -23) have not yet been assembled to any chromosome (Additional file 1). Table 1 shows details on VrBBXs, including gene names, accession number, chromosome location, coding sequence length (CDS), protein length, molecular weight (MW) and theoretical isoelectric point (pI). The divergent lengths of 23 VrBBX proteins resulted in diverse MWs and pIs, indicating that the VrBBXs varied greatly in sequence and physicochemical properties.

Table 1 Detailed information of VrBBX genes in mung bean
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Conserved domain analysis of VrBBXs

Among the 23 VrBBX proteins, eight VrBBXs had two conserved B-box domains and a CCT domain, and nine had two B-box domains. Four VrBBXs consisted of one B-box domain and one CCT domain, while two VrBBXs had only one B-box domain (Fig. 1). The sequence alignment of VrBBX proteins indicated that B-box1, B-box2 and CCT domains had similar conserved amino acid residues and that B-box1 was more conserved than B-box2 (Fig. 2). The conserved B-box1 sequence was CDXCXXXXAXVYCXADXAXLCXXCDXXVHXANXLASRH (where “X” represents any amino acid), and the B-box2 sequence was CDICEXXPAFVXCXXDXXLLCXXCDXXIHXXXXXSXXH. The conserved CCT sequence was REARVLRYREKRKTRKFXKXIRYESRKXXAETRPRIKGRFVK. However, B-box1 and B-box2 retained the same topology, with the form of CX2CX8CX7CX2CX? HX8H (where “?” represents multiple amino acids) (Fig. 2). Figure 3 shows the logos of B-box1, B-box2 and CCT domains.

Fig. 1
figure 1

The diagrams of conserved domains for the VrBBX proteins. The length of each protein sequence is represented by the black bar. The colored boxes refer to the conserved domains: red box, B-box1 domain; green box, B-box2 domain; blue box, CCT domain. The sequence length of each protein is represented at the bottom and the scale bar represents 100 amino acids

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Fig. 2
figure 2

Multiple sequence alignments of the B-box1 (a), B-box2 (b) and CCT (c) domains. The identical amino acids, conserved amino acids and similar amino acid residues are shaded in black, charcoal gray and gray, respectively

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Fig. 3
figure 3

The conserved domains in the VrBBX proteins. Logos of the protein alignment of the B-box1 (a), B-box2 (b) and CCT domains (c) are shown. The x-axis represents the conservative sequences of the domains. The height of each letter represents the conservation of each residue across all proteins. The y-axis is the scale of the relative entropy that reflects the conservation rate of each amino acid

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Phylogenetic analysis of the mung bean BBX gene family

To explore the phylogenetic relationships of the BBX family in mung bean, Arabidopsis and soybean, a phylogenetic tree was constructed based on the 97 aligned BBX protein sequences (Additional file 2). All 97 BBXs were divided into five groups (I, II, III, IV and V) (Fig. 4). Members in groups I and II contained two B-boxes and an additional CCT domain, but the amino acid sequence of the B-box2 domain was more conserved in group I than in group II (Additional file 3). Group III BBX members had a B-box1 domain and a CCT domain, and group IV BBX members contained B-box1 and B-box2 domains. Group V BBX members had only a B-box1 domain (Fig. 4). However, some of the BBXs did not fit the presented classification scheme. For example, the members of group II contained two B-boxes and one CCT domain, except for VrBBX18, which only had one B-box domain. VrBBX15 contained the B-box1 and B-box2 domains, suggesting that it belonged to group IV but was clustered in group V in the phylogenetic tree (Figs. 1 and 4). In addition, each group included BBX members from mung bean, Arabidopsis and soybean, indicating that the BBX genes originated before the differentiation of mung bean, Arabidopsis and soybean.

Fig. 4
figure 4

Phylogenetic tree of BBXs. The full-length amino acid sequences of BBX in mung bean, soybean and Arabidopsis are used for Neighbor-joining (NJ) phylogeny reconstruction with 1000 bootstrap replicates and the bootstrap values are indicated at each node. The green circles, red triangles and blue stars represent VrBBXs, AtBBXs and GmBBXs, respectively. The BBX proteins are divided into five groups (I-V) with different colors

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Collinearity analysis of BBX genes in mung bean, rice, Arabidopsis and soybean

To understand the gene duplication mechanisms of BBXs, we constructed three comparative syntenic maps of mung bean associated with three representative species, comprising a monocot (rice) and two dicots (Arabidopsis and soybean). The VrBBX genes exhibited the highest homology with the BBX genes of soybean (24), followed by Arabidopsis (9) and rice (1) (Fig. 5, Additional file 4). Both VrBBX14 and VrBBX19 shared collinearity with only one soybean BBX gene. Some VrBBX genes (VrBBX1, -3, -5, -7, -11, -12, -13, -16 and -17) were associated with two syntenic gene pairs, and VrBBX10 was associated with four syntenic gene pairs. No collinear segments of VrBBX2, -4, -6, -8, -9, -15, -18, -20, -21, -22 and -23 were found in the genomes of mung bean and soybean (Additional file 4). Moreover, VrBBX14 was collinear with the genomes of rice, Arabidopsis and soybean. VrBBX1, -5, -7, -10, -12 and -16 were collinear with the genomes of Arabidopsis and soybean (Fig. 5, Additional file 4).

Fig. 5
figure 5

Synteny analysis of BBX genes between mung bean and monocotyledonous rice (a), monocotyledonous Arabidopsis (b) and soybean (c). V. radiata, O. sativa, A. thaliana and G. max represent Vigna radiata, Oryza sativa, Arabidopsis thaliana and Glycine max, respectively. Gray lines in the background are the duplicated gene pairs between mung bean and other plant genomes, while the red lines indicate the syntenic BBX gene pairs. The colored bars represent chromosomes or scaffolds, and their numbering is displayed at the top or bottom of the colored bars

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Gene duplication of VrBBX genes in mung bean

To better elucidate the expansion patterns of VrBBX genes, synteny was also used to analyze mung bean BBX gene duplication. Six segmental duplication events were identified with ten VrBBX genes, which were located on duplicated segments on chromosomes 1, 2, 3, 6, 7, 8 and 11 and scaffold_364 (Fig. 6). No tandem duplication pairs were observed in our results. Subsequently, the Ka/Ks ratio of the duplicated gene pairs was calculated to determine the selection pressure. All Ka/Ks ratios were less than 1, except for that of gene pair VrBBX1/VrBBX13, indicating that most were under purifying selection during evolution. The divergence time between collinear gene pairs varied from 19.841 to 33.910 million years (Mya) (Table 2).

Fig. 6
figure 6

Synteny analysis of VrBBX genes on mung bean chromosomes. The light purple lines in the background indicate all synteny blocks in the mung bean genome between each chromosome, and the thick red lines indicate duplicate VrBBX gene pairs. The colored boxes indicate the different chromosomes or scaffolds and their numbering is shown on the outside of boxes. The scale bar marked on the chromosome is the length of the chromosome (Mb)

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Table 2 Ka/Ks analysis for duplicated gene pairs of BBXs in mung bean
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Gene structure and conserved motif analysis of the BBX gene family in mung bean

We mapped the exon‒intron map of VrBBX genes, and the number of exons ranged from 1 to 7. The VrBBX genes of groups I and III had two exons, except for VrBBX1 (five exons) and VrBBX2 (three exons). In groups II and IV, the majority of BBX genes contained three or four exons, with the exceptions of VrBBX8 and VrBBX23 (five exons). In group V, VrBBX19 contained the fewest exons (one), and VrBBX15 had the most exons (seven). (Fig. 7a). In addition, the intron patterns formed by relative position and phase in each group were highly conserved (Fig. 7a).

Fig. 7
figure 7

Phylogenetic tree, exon–intron structure and motif analysis of the BBX gene family in mung bean. a Structure analysis of VrBBX genes. Exons and introns are represented by yellow boxes and black lines, respectively. The untranslated regions (UTRs) are represented by blue boxes. The numbers (0, 1 and 2) indicate the intron phase. b Conserved motif analysis of VrBBX proteins. Each motif is represented by a number in a colored box

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To further explore the potential function of VrBBXs, we analyzed the conserved motifs and detected seven novel motifs (motifs 1–7) in addition to the B1, B2 and CCT domains (Fig. 7b, Additional file 5). Some conserved motifs were restricted to specific groups. For instance, motifs 5 and 7 were detected only in group III, and motifs 1, 2 and 6 were found only in group IV (Fig. 7b), indicating a functional difference between groups III and IV. The results also showed different motifs within the same group, suggesting that VrBBXs within the same group performed different functions. For example, VrBBX1 in group III lacked motif 7, whereas the other three VrBBXs (VrBBX13, -16 and -20) in group III contained motif 7 (Fig. 7b). This phenomenon was also observed in the other groups. VrBBX19 in group V did not contain these seven motifs.

Cis-acting element analysis in the promoters of VrBBX genes

In the present study, we analyzed the cis-acting elements, including stress response and hormone-related elements, in the − 1.5 kb promoter region of the VrBBX genes. Anaerobic induction-responsive element, methyl jasmonate (MeJA)-responsive element and abscisic acid-responsive element were abundant in the promoter regions of VrBBX genes (Fig. 8, Additional file 6). Drought inducibility-responsive element was identified in the promoter of VrBBX1, -2, -3, -4, -5, -10, -17, -19 and -22, and the wound-responsive element was identified in the VrBBX1, -5 and -22 promoter regions. Low temperature-responsive element was found in the VrBBX2, -9, -11, -17, and -19 promoter regions, and defense- and stress-responsive elements were found in the VrBBX1, -4, -9, -13, -14, -17, -19 and -22 promoter regions (Fig. 8, Additional file 6). These results indicate that some VrBBX genes might respond to various stresses and react collectively under the same stress. In addition, the promoter regions of VrBBX genes from the same group had similar cis-acting elements. For example, most of the group I VrBBX promoters had ABA- and MeJA- responsive elements, and most of the group III members contained anaerobic induction-responsive element and MeJA-responsive element (Fig. 8, Additional file 6). These results indicate that the same group of VrBBX genes may have similar functional mechanisms. In group III, only VrBBX20 contained an auxin-responsive element, and only VrBBX1 contained a wound-responsive element (Fig. 8, Additional file 6). This phenomenon was also found in other groups, indicating that the same group of VrBBX genes might have different mechanisms of action.

Fig. 8
figure 8

Predicted cis-elements in the promoter regions of VrBBX genes. The scale bar at the bottom indicates the length of promoter sequence (− 1500 bp). The cis-acting elements were represented by colored boxes

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Expression analysis ofVrBBXgenes.

For a better understanding of BBX genes involvement in abiotic stress, we analyzed the available RNA sequence data on the SRA database to get the expression of VrBBX genes. In the project, leave samples were taken after the drought stress treatment for 3 days and 6 days. Clustering analysis revealed clusters of genes (i) upregulated in response to drought stress (VrBBX1, -8, -10, -12, -20, -21 and -22), (ii) no significant expression changes in response to drought stress (VrBBX2, -3, -5, -6, -7, -9, -11, -14, -15, -16, -17, -18 and -23), (iii) downregulated in response to drought stress (VrBBX4, -13 and -19) (Fig. 9).

Fig. 9
figure 9

Heatmap expression profiles of VrBBX genes under drought stress. Red color indicates up-regulated gene expression, yellow color indicates no significant change in gene expression, and blue color indicates down-regulated gene expression under drought stress

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In silico expression analysis and cis-regulatory element analysis show that BBX genes may be involved in response to different abiotic stresses. Therefore, to predict the potential role of the BBX genes in mung bean, qRT-PCR was performed to estimate the transcript abundance of the BBX genes in leaf tissues under ABA, PEG and NaCl treatments. According to the homology of reported abiotic stress-related BBX genes and the presence of “defense and stress responsive element”, “drought-inducibility responsive element” or “abscisic acid-responsive element” in the promoters of VrBBX genes, nine VrBBX genes (VrBBX12 from group I, VrBBX5 from group II, VrBBX1 and VrBBX16 from group III, VrBBX3, VrBBX10, VrBBX21 and VrBBX22 from group IV, and VrBBX19 from group V) were selected to study their expression profiles when treated with ABA, PEG and NaCl at 4, 12 and 24 h. Among them, VrBBX3 and VrBBX21 were orthologous to AtBBX24, and VrBBX10 was orthologous to AtBBX21. AtBBX24 was involved in salt-stress response, and AtBBX21 mutants were sensitive to ABA and salt stress [19, 20]. The promoters of VrBBX1, -19 and -22 contained “defense and stress responsive elements.” All nine VrBBX gene promoters, except for VrBBX12, -16 and -21, contained “drought-inducibility responsive element.” All nine VrBBX gene promoters had an “abscisic acid-responsive element”. The results showed that most of VrBBX genes were responsive to ABA, PEG and NaCl treatments. The expression profiles showed that VrBBX5, -10 and -12 were upregulated under all three treatments (Fig. 10). Among the upregulated genes, the relative expression level of VrBBX10 showed the maximum fold change at 24 h under ABA treatment, and VrBBX5 showed the maximum fold change at 12 h under NaCl stress. The relative expression levels of VrBBX5 and VrBBX12 were highest under PEG stress (Fig. 10). In case of ABA treatment, VrBBX19 was found downregulated, while VrBBX1, VrBBX5, VrBBX10, VrBBX12 and VrBBX16 were found upregulated. There were no significant changes found in VrBBX3, VrBBX21 and VrBBX22 (Fig. 10). In case of drought stress, VrBBX19 was found downregulated, while VrBBX1, VrBBX3, VrBBX5, VrBBX10, VrBBX12, VrBBX16, VrBBX21 and VrBBX22 were found upregulated (Fig. 10). In case of NaCl stress, VrBBX1 and VrBBX16 were found downregulated, while VrBBX3, VrBBX5, VrBBX10, VrBBX12, VrBBX21 and VrBBX12 were found upregulated. There was no significant change found in VrBBX19 (Fig. 10).

Fig. 10
figure 10

Expression profile of nine selected VrBBX genes in response to ABA (a), PEG (b) and NaCl (c) treatments. The mean value was calculated from three independent replicates. Vertical bars indicate the standard deviation. Asterisks indicate corresponding genes that were significantly upregulated or downregulated compared with the control (* p < 0.05; ** p < 0.01)

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Discussion

BBX proteins are important regulators in mediating developmental processes and stress responses [36]. In the present study, we identified 23 VrBBX genes by performing a whole genome-wide analysis in mung bean. The number of BBX genes in mung bean was less than their orthologues in other species, such as Arabidopsis (32) [3] and rice (30) [4]. The genome size of mung bean is approximately 494–579 Mb, and 22,368 genes have been predicted [35]. Notably, the mung bean genome size is larger than those of rice (403 Mb) [37] and Arabidopsis (125 Mb) [38]. These results indicate that the BBX family members might not be directly related to the genome sizes of different plants [31]. Based on the phylogenetic relationships, the number of B-box they contain and whether there was an additional CCT, VrBBX genes were divided into five groups and clustered with the BBXs from other plant species, suggesting that they might have undergone similar evolutionary diversification [33]. The number of groups varied among plant species. There were 13, 4, 8 and 7 BBXs in Arabidopsis [3]; 7, 10, 10 and 3 BBXs in rice; and 9, 4, 8 and 2 BBXs in mung bean in groups I/II, III, IV and V, respectively [4]. These results suggest that BBX family members had a common ancestor and were independently expanded during speciation.

Previous research has shown that the amino acid sequence of B-box1 is different from that of B-box2, while B-box1 and B-box2 have the same topology [4, 31]. Our results showed that the B-box1 domain was more conserved than the B-box2 domain (Fig. 2). These results suggest that the early BBX proteins in green plants likely initially had one B-box domain and that the other B-box domain evolved by duplication events [2]. This assumption is supported by the evidence that most green algae have only a single B-box domain, and the first B-box duplication event occurred in CrBBX1 of the green alga Chlamydomonas before the colonization of land plants [39]. The BBX proteins of groups I and II contained two B-boxes and an additional CCT domain. However, the amino acid sequence of the B-box2 domain in group I was more conserved than that in group II (Additional file 3), indicating that the evolutionary mechanism of B-box2 in groups I and II might be different. Previous research predicted that the BBX proteins of group II (B1 + B2 + CCT) were generated by adding a CCT domain at the C-terminus of group IV BBX proteins (B1 + B2). Then, the deletion of the B2 domain generated BBX proteins with a single B-box and CCT domain (group III). The duplication events of the B1 domain in group III BBX proteins may produce the B1 + B2 + CCT domain (group I). The early BBX proteins of group IV (B1 + B2) may be the ancestors of BBX members in group V (B1) after B2 domain deletion [2]. Therefore, the B-box2 domain in groups I and II differed in amino acid sequence, which is consistent with our research result. This indicates that the evolution of VrBBX in mung bean is consistent with the evolutionary model of previous studies.

To further clarify the evolutionary history of the BBX gene family, the synteny relationships of BBXs from monocots (rice) and dicots (mung bean, Arabidopsis and soybean) were systematically analyzed. VrBBX14 had homologous gene pairs with BBX in Oryza sativa (1) (Fig. 5), implying that BBX family members had a common ancestor and that large-scale expansion of BBXs might have occurred after the division of monocots and dicots. Seven VrBBX genes (VrBBX1, -5, -7, -10, -12, -14 and -16) had syntenic gene pairs in both Arabidopsis and soybean (Fig. 5), indicating that these VrBBX genes originated before the differentiation of mung bean, Arabidopsis and soybean. Five VrBBX genes (VrBBX3, -11, -13, -17 and -19) might have originated before legume differentiation because of their collinear relationship between mung bean and soybean, while some VrBBX genes had no collinear segments (Fig. 5). Gene duplication plays a key role in generating novel genes during the process of plant evolution. There are two main patterns of gene replication in plants, segmental duplication and tandem duplication, which have been demonstrated to play an important role in the expansion of the gene family member [40]. Therefore, we investigated the gene duplication of BBX genes in mung bean. In the present study, no tandem duplications of VrBBX genes were found. Six VrBBX gene pairs with segmental duplication were identified in mung bean chromosomes (Fig. 6). These results indicate that segmental duplications were the main expansion pattern of mung bean BBX gene family members. The same potential mechanism of gene family evolution has also been identified in the NAC gene family in mung bean [41]. Previous studies have reported that tandem duplication often occurs in large and rapidly evolving gene families, while segmental duplication usually occurs in slowly evolving gene families [42]. The present results indicated that the mung bean BBX gene family had slow evolutionary characteristics. Furthermore, the Ka/Ks ratios of all duplicated VrBBX gene pairs were less than 1, except for VrBBX1/VrBBX13, indicating that most of the duplicated VrBBXs experienced purifying selective pressure (Table 2). Since purifying selection limits gene differentiation, duplicated VrBBX genes might retain similar functions after replication [43].

The intron-exon pattern bears the imprint of gene family evolution [44]. In the present research, the exon number of VrBBX genes ranged from 1 to 7, which showed similar genetic diversity to other species, such as tomato [30] and pear [31]. The different splicing patterns of exons and introns might be meaningful for the evolution of the VrBBX gene. Additionally, we investigated the conserved motifs of VrBBXs. Each group had several common motifs, while some groups had special motifs (Fig. 7b). The different motif compositions in each group might indicate the functional diversity of VrBBXs, and the same motif in the same group might indicate the functional synergy of VrBBXs [45]. In conclusion, the gene structures and motif distributions of VrBBXs were highly conserved in each group, supporting the close evolutionary relationships and group classifications (Fig. 7).

Transcriptional regulation of stress-responsive genes is an important part of plant stress responses. Transcription factors are potential activators or repressors that control the expression of gene clusters by binding the cis-acting element in the promoter regions of target genes [32]. In this study, the analysis of VrBBX promoter regions revealed a series of frequent occurrences of cis-acting elements corresponding to abiotic stress (Fig. 8), indicating their potential functions in abiotic stress. For example, the promoters of VrBBX5 and VrBBX10 contained drought inducibility-responsive element and the relative expression levels of VrBBX5 and VrBBX10 were nearly 8-fold higher than that of the control (0 h) under PEG stress (Figs. 8 and 10). To further verify the involvement of the VrBBX genes in the regulation of abiotic stress, the expression analysis based on the publicly available RNA sequence data and qRT-PCR were conducted. The present results suggest that the majority of VrBBX genes were induced or repressed to varying degrees depending on stress treatments. According to their expression changes under stress, VrBBX10, VrBBX5 and VrBBX12 can be further studied as candidate genes in response to ABA, NaCl and PEG, respectively. The expression of VrBBX1 and VrBBX16 was upregulated by ABA and PEG treatments but downregulated under NaCl treatment, suggesting that VrBBX1 and VrBBX16 might have different mechanisms to maintain protection against various abiotic signals [33]. In particular, the expression of VrBBX5, -10 and -12 was upregulated under three abiotic stresses (Fig. 10), indicating that these VrBBX genes may play a key role in response to multiple abiotic stress networks [33]. Previous studies have shown that the BBX genes in the same group might perform similar functions. For example, the Arabidopsis BBX genes in group I (such as AtCO and AtCOL) were mostly associated with photoperiod or photoperiod-regulated flowering [46, 47], while the majority of the BBX genes in group IV, including AtBBX18, AtBBX19, AtBBX21, AtBBX22, AtBBX24 and AtBBX25, were related to photomorphogenesis [13, 48, 49]. The present study showed that VrBBX1 and VrBBX16 in group III, and VrBBX3 and VrBBX21 in group II showed similar expression profiles, indicating that they may have similar functions in responding to abiotic stress. The expression characterization of VrBBX genes in response to abiotic stress will greatly improve our understanding of the functions of abiotic stress signaling pathways.

Conclusions

In this study, 23 VrBBX genes were identified in mung bean, and systematic and comprehensive analyses of the VrBBX gene family were performed, including conserved domain, phylogenetic tree, duplication event and expansion pattern, gene structure, conserved motif, chromosome location, cis-acting elements in promoters and expression patterns. The VrBBX genes were divided into five groups, namely I (three genes), II (six genes), III (four genes), IV (eight genes), V (two genes), which were supported by conserved domain analysis. Gene duplication analysis suggested that segmental duplication was the main expansion pattern of mung bean BBX genes. Numerous cis-acting elements were found in the VrBBX promoters, indicating that VrBBX genes are involved in complex regulatory networks that control development and responses to abiotic stress. The expression profiles of VrBBX genes showed that most VrBBX genes were responsive to ABA, PEG and NaCl treatments. Therefore, genome-wide analysis of the VrBBX gene family will provide a solid basis for functional analyses of VrBBX genes, and further study of several VrBBX genes is currently underway to understand their biological functions. Detailed knowledge of stress-responsive VrBBXs in mung bean will be a valuable resource for future molecular breeding in legumes.

Methods

Identification of BBX gene family members in mung bean

Two methods were used to identify the BBX genes in mung bean. First, we used the Basic Local Alignment Search Tool (BLAST) to search for potential BBXs in the V. radiata genome using Arabidopsis BBX protein sequences as queries, and the cutoff E-value was 0.001. Second, the HMM of the B-box (pfam00643) was used to query the V. radiata whole-genome protein database [50]. The V. radiata genome database (genome assembly: Vradiata_ver6) was downloaded from EnsemblPlants (http://plants.ensembl.org/index.html). Each potential candidate sequence was verified using the SMART (http://smart.embl.de/) [51], InterProscan (http://www.ebi.ac.uk/Tools/pfa/iprscan/) [52] and Pfam (http://pfam.xfam.org/) databases for the presence of a B-box. The MW and pI of the identified VrBBXs were calculated using online ProtParam (http://web.expasy.org/protparam/) [53].

Phylogenetic analysis and sequence alignment

The ClustalW program was used to align BBX protein sequences [54], and the phylogenetic tree was built using the MEGA 7 program with the neighbor-joining method and 1000 replicate iterations [55]. Domains were identified with SMART, InterProScan and pfam programs. The WebLogo website (http://weblogo.berkeley.edu/logo.cgi) was used to construct sequence logos of conserved domains [56]. The conserved motifs of VrBBX proteins were identified using the MEME program (http://memesuite.org/tools/meme).

Gene structure, chromosomal location, and duplication analysis

The exon and intron locations of the VrBBX genes were obtained according to the mung bean genome annotation file, and a map of the VrBBX gene structure was generated using the GSDS website (http://gsds.cbi.pku.edu.cn/) [57]. The chromosome location image was generated with MapInspect software according to the physical positions of the VrBBX genes on the chromosomes or scaffolds. MCScanX software (http://chibba.pgml.uga.edu/mcscan2/) [58] was used to identify duplications of VrBBX genes with default parameters, and the relationship was plotted with Circos software [59]. Calculator 2.0 software [60] was used to estimate the Ka and Ks of the gene duplication pairs, and the selective pressure was calculated using the Ka/Ks ratio. The approximate date of the duplication event that occurred in the mung bean was estimated using the Formula: T = Ks/2λ × 10–6 Mya (λ = 6.5 × 10− 9) [61].

Cis-regulatory elements and expression pattern of transcriptome analysis

The genomic DNA sequence approximately 1.5 kb upstream of the transcriptional start sites was used to analyze the cis-acting elements. The PLACE website (http://www.dna.affrc.go.jp/PLACE/signalscan.html) [62] was used to identify the cis-acting elements involved in abiotic stress and hormone responses. To gain insight into the expression patterns of VrBBXs genes under drought stress, RNA-Seq data (BioProject: PRJNA764584) was obtained from SRA-NCBI (https://www.ncbi.nlm.nih.gov/sra) [63]. Paired-end clean reads were downloaded and aligned to the reference genome using bowtie2. Differential expression fold change of genes was calculated by comparing the treatment with the control. The genes with false discovery rate (FDR)<0.05, Log2FC>1 and Log2FC<-1 were considered to be significantly differential expression. The heatmaps were made with the mean value of Log2FC in expression of VrBBX-regulated transcript.

Plant materials

Ten mung bean seeds (VC1973A) were sown in a pot (diameter = 22 cm) filled with nutrient soil and cultured in a growth chamber at 24 °C with a 16 h/8 h light/dark cycle. After the first trifoliolate leaf was fully expanded, 20% PEG-6000, 100 mM NaCl and 100 µM ABA solution were poured onto seedling. Leaves were collected after treatment at 0, 4, 12 and 24 h. Three biological replications were conducted for each sample, and each replicate included three to four plants. All collected samples were stored at − 80 °C for RNA extraction and gene expression analysis.

Expression profile analysis of VrBBX genes under ABA, PEG and NaCl treatments

The total RNA from leaves was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and the RNA concentration and quality were measured using NanoDrop Spectrophotometer (Thermo Fisher Scientifc, Waltham, MA, USA), with 260/280 ≥ 1.8 and 260/230 ≥ 2.0. First-strand cDNA was synthesized using a SuperScript™ III Reverse Transcriptase Kit (Invitrogen) according to the manufacturer’s instructions. The resulting cDNA was used for qRT-PCR analysis using SYBR Green PCR mix (Takara, Tokyo, Japan) with the ABI 7500 Real-Time PCR System. The PCR conditions were as follows: 95 °C for 2 min followed by 45 cycles of 95 °C for 10 s and 60 °C for 1 min. Additional file 7 lists the primer sequences used for qRT-PCR. The relative expression levels of VrBBX genes were determined using the 2–ΔΔCT method with the VrACTIN3 gene (Vradi03g00210) serving as an endogenous control. The expression patterns of the VrBBX genes were identified by three independent biological replicates.

Data availability

The Arabidopsis BBX protein sequences were collected from the Arabidopsis information source database (http://www.arabidopsis.org). The BBX protein sequences of mung bean and soybean were obtained from EnsemblPlants (http://plants.ensembl.org/index.html). The genome sequences of mung bean, Arabidopsis, soybean and rice were downloaded from EnsemblPlants. The datasets used and analyzed during the study are available from the corresponding author on reasonable request.

Abbreviations

BBX:

B-box

CCT:

CONSTANS, CO-like and TOC1

ABA:

Abscisic acid

PEG:

Polyethylene glycol

NaCl:

Sodium chloride

B1:

B-box1

B2:

B-box2

CO:

CONSTANS

FT:

Flowering Locus T

ROS:

Reactive oxygen species

SOD:

Superoxide dismutase, APX: Ascorbate peroxidase

GST:

Glutathione s-transferase

Mya:

Million years

HMM:

Hidden Markov Model

CDS:

Coding sequence length, MW: Molecular weight

pI:

Isoelectric point

UTR:

Untranslated regions

MeJA:

Methyl jasmonate

BLAST:

Basic Local Alignment Search Tool

SMART:

Simple Modular Architecture Research Tool

MEGA:

Molecular Evolutionary Genetics Analysis

NJ:

Neighbor-joining

MEME:

Multiple EM for Motif Elicitation

Ka:

Non-synonymous substitution rate

Ks:

synonymous substitution rate

qRT‒PCR:

Quantitative real-time PCR

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Acknowledgements

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Funding

This study was financially supported by the National Natural Science Foundation of China (Grant No. 32102340), the Research Project of Industry-Education Integration of Shanxi Datong University (2021CXZ8) and Doctoral Scientific Research Foundation of Shanxi Datong University (2018-B-07). The funding bodies only provided the financial support and had no role in the design of the experiment, data collection and analysis, and drafting the manuscript.

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Authors and Affiliations

  1. College of Agronomy and Life Sciences, Shanxi Datong University, Datong, 037009, People’s Republic of China

    Lili Yin, Ruilan An, Yaxin Feng & Yaqi Qiu

  2. School of Landscape and Ecological Engineering, Hebei University of Engineering, Handan, 056038, People’s Republic of China

    Ruigang Wu

  3. Institute of Forestry and Pomology, Beijing Academy of Agriculture and Forestry Sciences, Beijing, 100093, People’s Republic of China

    Meiling Zhang

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  1. Lili Yin
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Contributions

LY and MZ conceived and designed the study. LY, RW, RA, YF and YQ conducted the experiments and analyzed the data. LY and RW wrote the manuscript. All authors reviewed the manuscript.

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Correspondence to Meiling Zhang.

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The authors confirm that the mung bean cultivar ‘VC1973A’ used is available and public. The experimental research and field studies on plants complied with relevant institutional, national, and international guidelines and legislation. The mung bean cultivar ‘VC1973A’ was cultivated under controlled conditions and do not pose any risk to other plant species, and no specific permission was required to collect the sample material used in this study.

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Yin, L., Wu, R., An, R. et al. Genome-wide identification, molecular evolution and expression analysis of the B-box gene family in mung bean (Vigna radiata L.). BMC Plant Biol 24, 532 (2024). https://doi.org/10.1186/s12870-024-05236-9

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  • DOI: https://doi.org/10.1186/s12870-024-05236-9

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BMC Plant Biology

ISSN: 1471-2229

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