Based on the gene annotation as well as the conserved B-box motif characteristic of the BBX members, a total of 31 SlBBX genes were identified. The detailed information [gene name, gene identifiers, chromosome location, theoretical isoelectric point (pI), molecular weight (MW), genomic, coding sequences (CDS), peptide length, number of exon and intron, instability and aliphatic index, the grand average of hydropathicity (GRAVY) values and subcellular localization) of each SlBBX was presented in Table 1. The lengths of CDS and amino acids (AA) of 31 SlBBXs range from 267 bp and 88 aa (SlBBX18) to 1428 bp and 475 aa (SlBBX27), respectively. Thus, varied MW and pI were observed among SlBBX proteins. The MW of SlBBXs varies from 9.57 (SlBBX18) to 53.14 kDa (SlBBX27). The pI ranged from 4.25 (SlBBX5 and SlBBX7) to 9.28 (SlBBX26), with 74.2% SlBBXs with a pI lower than seven, which indicated that most of the SlBBX proteins were acidic in nature. The pI ranged from 4 to 9 in SlBBX proteins contained one (single) or two (double) B-box domains, while it decreased when plus a CCT domain, especially in the SlBBX proteins with a B-BOX domain plus a CCT domain (Additional file 1: Figure S1), which suggested that the CCT domain in SlBBX proteins may decrease their pI. Majority of SlBBXs were grouped into unstable proteins because their instability index was greater than 40, except for SlBBX6 in this family (Table 1). The predicted aliphatic index ranged from 50.05 to 97.43 in SlBBX proteins. All SlBBX proteins, with the exception of SlBBX18, were predicted to be hydrophilic due to the GRAVY value (< 0). Subcellular localization predicted that most SlBBXs (23 of 31) were localized in the nuclear region, five of them, including SlBBX5, SlBBX 6, SlBBX17, SlBBX25 and SlBBX31 in the chloroplast, while SlBBX16 and SlBBX18 in the cytoplasm, SlBBX19 in the peroxisome (Table 1). In addition, none of the 31 BBX proteins have a transmembrane domain, which indicated that these SlBBX proteins were not located on the cell membrane (Additional file 1: Figure S2).
Protein sequences, phylogenetic analysis and three-dimensional structure of SlBBXs
The domains logos and the sequences of the B-box1, B-box2 and CCT domain of the SlBBX proteins are shown in Fig. 1. Eight members out of the 31 SlBBXs, were characterized by the occurrence of two B-box domains and also a conserved CCT domain, whereas four members of them had a valine-proline (VP) motif (Table 2). Only two B-BOX domains were found in ten SlBBXs, whereas five members had one B-box domain and also a CCT domain, and eight members had only one B-box domain (Table 2). Among the three domains, we found that each tomato B-box motif contained approximately 40 residues with the consensus sequence C-X2-C-X8-C-X2-D-X4-C-X2-C-D-X3-H-X8-H (Fig. 1). The conserved C, C, D and H residues ligated two zinc ions . Additionally, the consensus sequence of the conserved CCT domain was R-X5-R-Y-X-E-K-X3-R-X3-K-X2-R-Y-X2-R-K-X2-A-X2-R-X-R-X-K-G-R-F-X-K (Fig. 1).
To better reveal the evolutionary relationships, we generated a phylogenetic tree based on the 32 AtBBXs and 31 SlBBXs (Fig. 2). All sequences were clustered into five subfamilies according to the phylogenetic analysis and previous article . The BBX genes in clade I (group 1) had two concatenated B-box domains, a CCT domain and a VP motif except for SlBBX1 and SlBBX2, which did not have a VP motif and a CCT domain. The members of group II were characterized by two B-box domains and also a CCT domain with the only exception for SlBBX7, which contained two B-box domains only, and SlBBX8 and SlBBX10, which only had one B-box domain and a CCT domain. In the group III, all the members contained one B-box domain as well as a CCT domain. The group IV and V possessed two and one B-box domain, respectively; nonetheless, SlBBX27 that contained two B-box was also grouped into V. Additionally, BBX proteins from two species showed scattered distribution across the branches of the evolutionary tree, which implies that the duplication events occurred after the lineages diverged.
Protein structural features are crucial for understanding the biological properties as well as the evolutionary origins of proteins. Here, we found that most members of SlBBX proteins in a subfamily had a similar three-dimensional structure (Fig. 3). In addition, we found physical connections in each protein sector in the tertiary structure. Moreover, a distinct functional role, and an independent mode of sequence divergence in the protein family, reflected the evolutionary histories of the conserved biological properties of BBX proteins.
Gene structure, conserved motifs, chromosomal localization and synteny analysis of SlBBXs
The evolution of multigene families can be driven by gene structural diversity. Examination of the genomic DNA sequences revealed that most SlBBXs contained one to five introns, while SlBBX16, SlBBX17 and SlBBX30 had no introns (Fig. 4b and Table 1; Additional file 1: Figure S3). Among them, nine SlBBXs had one intron, followed by ten SlBBXs with two introns, five SlBBXs with three exons, four SlBBXs with four exons, and one SlBBXs with five introns. Generally, members of each subclass, which are most closely related, exhibited analogous exon-intron structures. For instance, the members in group I and V had one to two, and zero to one intron, respectively (Fig. 4a and 4b; Additional file 1: Figure S3). However, a few SlBBX genes showed dissimilar exon-intron arrangements. For instance, SlBBX18 and SlBBX19 had high sequence similarity, but SlBBX18 and SlBBX19 contained two and five introns, respectively (Fig. 4a and 4b; Additional file 1: Figure S3). These divergences indicated that both the gain and loss of introns during evolution, may better explain the functional diversity of SlBBX homologous genes.
To further examine the structural features of SlBBXs, the conserved motif distributions were analyzed. Twenty conserved motifs were predicted (Fig. 4c), while multilevel consensus sequences and the E-value of them were shown in Additional file 2: Table S1. The results showed that motif 17 was the largest motif depending on the width, followed by motif 8 and motif 2 (Additional file 2: Table S1). Motif 1 was found in all the SlBBXs (Fig. 4c). Notably, 74.2% and 70.1% SlBBXs contained motif 4 and motif 3, respectively. Motif 2 was unique to the group I, II and III of SlBBXs, while motif 5 was unique to group II except for the SlBBX27. Motif 10 was found only in group III of SlBBXs. Our results showed that members that are most closely related in the phylogenetic tree contained common motifs on the basis of alignment and position, which indicated that they may have a similar biological function.
Chromosomal locations showed that 31 SlBBX genes were unevenly distributed on the 12 chromosomes (Fig. 5a). A maximum number of SlBBX genes were found on chromosome 12 (Chr12), comprising of six genes. Five genes were located on Chr2 and Chr7. Four and three SlBBX genes were located on Chr5 and Chr4, respectively. Both Chr1 and Chr6 contained two members of SlBBX genes, whereas only one gene was detected on Chr3, 8, 9 and 10. Additionally, no SlBBX genes were found on Chr11.
To examine the duplication of SlBBX genes, the MCScanX program was used. Thirty-six pairs of SlBBXs were identified as segmental duplication in the tomato genome (Fig. 5b). Chr2, 7 and 12 had more duplication regions, which partially explain the greater numbers of SlBBX genes that were located on these three chromosomes. Although SlBBX1 and SlBBX3 were located on the same chromosome (Fig. 5a), and their sequence identity was 83% (Additional file 1: Figure S4), they were not tandem duplication. To further examine the evolutionary relationships between SlBBXs and AtBBXs, a synteny analysis was performed with MCScanX software. A total of 16 of SlBBX-AtBBX orthologous pairs were identified (Fig. 5c), which indicated the existence of numerous SlBBX genes prior to the divergence of Arabidopsis and tomato. Some members of SlBBXs were not localized in the syntenic block, suggesting that these genes might have certain specificity due to their evolution time.
Analysis of cis-elements in the promoter region of SlBBXs
Transcription factors directly bind the cis-elements in regulatory networks controlling gene expression; therefore, analysis of the putative cis-elements is critical to examine the expression of SlBBX genes. A total of 61 major cis-elements were predicted from the PlantCARE database (Fig. 6a), including 22 light responsive, 12 hormone responsive, 11 stress responsive and 16 development. The number of light responsive cis-elements was the largest in the promoters of 31 SlBBX genes (Fig. 6b). The number of cis-elements in the promoters of SlBBX17 and SlBBX2 was the largest and least, respectively. The major light responsive elements contained box4 (21%), G-box (17.9%) and CMA1a/2a/2b (14.3%), which were located on 87.1% (27/31), 83.9% (26/31) and 96.8% (30/31) of SlBBXs promoters, respectively (Fig. 6c). The most common motif were the JA-responsive elements (MYC), abscisic acid (ABA)-responsive element (ABRE), and ethylene-responsive element (ERE), accounting for 24.8%, 21.5% and 17.2% of the scanned hormone responsive motifs, respectively. The stress responsive elements MYB, STRE (stress-related elements) and WUN were located on 96.8% (30/31), 90.3% (28/31) and 77.4% (24/31) of 31 SlBBX genes promoters, respectively. In the development category, various growth and development related elements, such as AT-rich element (19.2%), O2-site for zein metabolism regulation (13.7%), CAT-box for meristem expression (12.3%), GCN4_motif required for endosperm expression (9.6%), were found. Our findings suggest that the promoter regions of SlBBX genes that contained the cis-elements played a critical role in the light and stress responses.
SlBBX genes expression in response to different light quality
To assess whether light signaling regulates SlBBXs, we investigated the gene expression of SlBBXs in tomato plants grown at dark (D), white (W) and different light quality [purple (P), blue (B), green (G), yellow (Y), red (R), and far-red (FR)] conditions. In comparison with D, light decreased the transcripts of SlBBX1, SlBBX8, SlBBX10 and SlBBX12, while it increased the transcripts of SlBBX7, SlBBX13 and SlBBX15 (Fig. 7). Plants grown at R light conditions showed higher expression of SlBBX4, SlBBX14, SlBBX23, SlBBX24 and SlBBX29 than those grown at other light qualities. Whereas FR light significantly up-regulated the transcripts of SlBBX7, SlBBX13, SlBBX15, SlBBX21, SlBBX25, SlBBX26 and SlBBX27, it obviously down-regulated the transcripts of SlBBX14, SlBBX16, SlBBX18, SlBBX24, SlBBX28, SlBBX30 and SlBBX31 (Fig. 7). Transcripts of SlBBX16, SlBBX17, SlBBX18, SlBBX30 and SlBBX31 were induced, while transcripts of SlBBX5, SlBBX6, SlBBX19, and SlBBX20 were inhibited in plants when grown at B light conditions. SlBBX15 was induced by G light irradiation at 6 h, whereas SlBBX9 and SlBBX28 were repressed (Fig. 7). Y light led to an obvious reduction in expression of SlBBX9 and SlBBX31. Obviously, the P light increased the expression of SlBBX3, SlBBX5, SlBBX6, SlBBX15, SlBBX19, SlBBX20, SlBBX21, SlBBX26 and SlBBX27, but decreased the expression of SlBBX10 and SlBBX16. Interestingly, SlBBX4, SlBBX23 and SlBBX29 were only responsive to R light, while SlBBX7, SlBBX13 and SlBBX25 were induced just in response to FR light. Meanwhile, R light induced the expression of SlBBX14 and SlBBX24, but FR light inhibited their expression (Fig. 7). In general, the results showed that SlBBX genes might act a critical role in response to light quality signaling.
Expression pattern of the SlBBX genes in response to chilling stress
To investigate whether SlBBX genes participated in chilling stress, we analyzed the transcriptome data of tomato plants after chilling stress . Results revealed that the expression levels of ten members of SlBBX family genes, including SlBBX3, SlBBX16, SlBBX17, SlBBX19, SlBBX24, SlBBX26, SlBBX28, SlBBX29, SlBBX30 and SlBBX31, were higher in tomato plants after chilling stress than those grown at optimal temperature conditions (Fig. 8). Furthermore, we found the transcripts of SlBBX1, SlBBX7, SlBBX9, SlBBX12, SlBBX13, SlBBX15, SlBBX18, SlBBX21, and SlBBX27 were significantly decreased after chilling stress. These findings suggest that SlBBX genes might have an important role in response to chilling stress, whereas further studies are essencial to investigate the mechanism.