Classification and phylogenetic analysis of VrCOL proteins
The COL proteins were classified into three groups based on differences in the numbers and types of conserved BBX and CCT domains [11, 20]. We analyzed the VrCOL BBX and CCT domains and found two distinct BBX domains (BBX1 and BBX2) and one CCT domain (Additional file 1). The sequence logos of the BBX1 (CX2CX8CX4AXLCX2CDX3HX8HXR), BBX2 (CX2CX4AX3CX7CX2CDX3HX8H) and CCT (RYX2KX3RX3KX2RYX2RKX2AX2RXR) domains were determined using WebLogo (Fig. 2, Additional file 1). Nine VrCOL proteins contained one BBX1, one BBX2 and one CCT domain, and five VrCOL proteins contained one BBX1 and one CCT domain (Fig. 3). The VrCOL proteins were further classified into three groups based on differences in these conserved domains. Classes I, II and III contained 4, 5 and 5 VrCOL members, respectively. The BBX1 and BBX2 domains were located close to one another in the class I and II genes (Fig. 3). Most of the VrCOL genes from the same class were clustered into the same clade in the phylogenetic tree, with the exception of class III member VrCOL8, which showed a closer relationship with class II members (Fig. 3).
To analyze the evolutionary relationships among the VrCOL genes and obtain information from well-studied CO homologs in other species, a phylogenetic tree was constructed using 17 A. thaliana, 26 soybean, 11 Medicago and 14 mungbean CO and COL proteins [11, 35]. The proteins from each group were clustered together in the phylogenetic tree (Fig. 4). VrCOL2 and VrCOL5 showed close relationships to A. thaliana CO and soybean GmCOL1a, GmCOL1b, GmCOL2a, and GmCOL2b, all of which have documented roles in the regulation of flowering time [11, 20, 35]. This result suggests that VrCOL2 and VrCOL5 may play critical roles in the flowering time regulation of mungbean.
Gene structures and conserved motifs of the VrCOL genes
To investigate the gene structures of the VrCOL genes, we downloaded their genomic and CDS sequences from the NCBI and analyzed them using the GSDS program [39]. All the VrCOL members contained 5’ UTR and 3’ UTR regions. Their exon numbers ranged from 2 to 6, and their intron numbers ranged from 1 to 6. Most group I and III VrCOL members contained two exons and one intron, suggesting conserved functions of the genes within each group. An exception was class III member VrCOL8, which contained 6 exons and 6 introns and had a close relationship with group II members (Fig. 5). By contrast, group II members contained various numbers of exons (3 to 5) and introns (2 to 5), suggesting potential functional diversity among these genes (Fig. 5). To further investigate the conservation and diversity of VrCOL protein structures, we analyzed putative protein motifs in the VrCOLs. A total of 17 distinct motifs were identified, and all VrCOL proteins contained motifs 1 and 2, which appeared to represent the conserved CCT and BBX1 domains, respectively (Fig. 5, Additional file 2). Most members of the same group shared some conserved motifs. For example, group I proteins shared motifs 1, 2, 3, 9 and 16, group II members shared motifs 1, 2, 3, and 5, and most group III members shared motifs 1, 2, 4, 8, 12, and 13 (except for VrCOL8) (Fig. 5).
Duplication analysis of VrCOL genes
Mungbean has experienced one round of whole-genome duplication that produced many duplicated gene pairs [38, 40]. To investigate the evolutionary relationships among the VrCOLs, we searched for duplicated gene pairs among them. Two interchromosomal duplication events were identified in chromosomes 1, 4, 5 and 6, including the duplicated gene pairs VrCOL2/VrCOL5 and VrCOL6/VrCOL9 (Fig. 6). The duplicated genes were clustered together in the phylogenetic tree (Fig. 3). All the duplicated genes contained one BBX1, one BBX2 and one CCT domain and belonged to groups I and II, no duplicated gene pairs were found in group III. The duplicated genes VrCOL2 and VrCOL5 showed similar exon-intron organization and similar motifs, as did VrCOL6 and VrCOL9 (Fig. 5), indicating that the duplicates may share similar functions.
Cis -acting element analysis of the VrCOL promoter regions
To predict the potential expression response of VrCOL genes, we investigated the cis-acting elements in their promoters using PantCARE [41]. A total of 82 cis-acting elements were found across the 14 VrCOL promoter regions (2 kb upstream of the initiation codon) (Additional file 3). Forty-five of them had predicted functions, including six development-related elements, four environmental-stress-related elements, three site-binding-related elements, nine hormone-responsive elements, three promoter-related elements and twenty light-responsive elements (Table 2, Additional file 3). The various VrCOL promoter regions had different numbers and types of cis-acting elements, highlighting the functional diversity of these genes. All VrCOL promoters contained hormone-responsive elements, light-responsive elements and promoter related elements, and light-responsive elements were the most abundant element in each VrCOL promoter, with the exception of VrCOL9 (Table 2), indicating that VrCOL genes may play critical roles in light-dependent signaling pathways. Environmental-stress-related elements were the most abundant element in the VrCOL9 promoter (nine elements), indicating that VrCOL9 may function in stress response (Table 2). All the VrCOL genes contained the promoter-related elements CAAT-Box and TATA-Box, which are basic promoter components. Thirteen of the 14 VrCOLs contained the hormone-responsive elements CGTCA-motif and TGACG-motif and the light-responsive element Box 4 (Additional file 3), suggesting potential functions of these genes in related signaling pathways.
Table 2
Numbers and types of cis-acting elements in each VrCOL promoter region
Gene name | Development related elements | Environmental stress related elements | Hormone-responsive elements | Light-responsive elements | Promoter related elements | Site-binding related elements | Others |
VrCOL1 | 1 | 3 | 3 | 6 | 2 | 0 | 17 |
VrCOL2 | 1 | 3 | 4 | 6 | 2 | 1 | 18 |
VrCOL3 | 0 | 2 | 5 | 6 | 3 | 0 | 20 |
VrCOL4 | 4 | 1 | 4 | 6 | 2 | 2 | 15 |
VrCOL5 | 0 | 3 | 4 | 11 | 2 | 0 | 18 |
VrCOL6 | 0 | 1 | 5 | 7 | 2 | 0 | 14 |
VrCOL7 | 2 | 1 | 4 | 8 | 2 | 0 | 19 |
VrCOL8 | 1 | 2 | 4 | 6 | 2 | 1 | 14 |
VrCOL9 | 1 | 9 | 4 | 8 | 2 | 0 | 13 |
VrCOL10 | 1 | 0 | 4 | 11 | 2 | 2 | 17 |
VrCOL11 | 1 | 0 | 4 | 11 | 2 | 2 | 17 |
VrCOL12 | 0 | 3 | 4 | 7 | 2 | 1 | 16 |
VrCOL13 | 1 | 1 | 5 | 8 | 2 | 2 | 17 |
VrCOL14 | 0 | 0 | 4 | 7 | 2 | 0 | 14 |
Transcription patterns of VrCOL genes in different tissues
To shed light on the potential functions of VrCOL genes during plant development, we analyzed the expression of VrCOL genes in different tissues, including roots, nodule roots, shoot apices, stems, leaves, flowers, pods and seeds. VrCOL genes showed distinct expression patterns in different tissues (Fig. 7). For example, VrCOL7 was highly expressed in all the tested tissues, whereas VrCOL2 and VrCOL8 showed low expression in most tissues. Some genes were expressed at high levels in specific tissues, suggesting that they may have critical functions in these tissues. For example, VrCOL11 showed high expression in leaves but low expression in nodule roots and roots.
Duplicated genes may retain some common functions and evolve some new functions [42–43]. To investigate the conservation and diversity of duplicated genes, we also analyzed their tissue-specific expression patterns. VrCOL2 and VrCOL5 differed in their expression levels across all the tissues we examined, indicating that they had undergone functional divergence. VrCOL6 and VrCOL9 showed similar expression levels in roots and nodule roots, but they exhibited different expression levels in other tissues (Fig. 7).
Diurnal rhythm of VrCOL2 expression
In A. thaliana, the expressions of CO, COL1 and COL2 are regulated by the circadian clock and show diurnal oscillations [11, 44]. VrCOL2 displayed a close phylogenetic relationship with CO, COL1 and COL2 (Fig. 4). We therefore investigated whether VrCOL2 exhibited diurnal expression rhythms in mungbean leaves under LD and SD conditions. The expression of VrCOL2 showed daily oscillations under SD conditions, reaching a peak at ZT 16. However, under LD conditions, VrCOL2 showed no daily oscillations (Fig. 8). In addition, the expression of VrCOL2 was higher under SD than under LD conditions at ZT 16, suggesting that VrCOL2 is a daily oscillation gene and works under a photoperiod-dependent pathway.
Overexpression of VrCOL2 accelerates flowering in A. thaliana under SD conditions
To investigate the potential functions of VrCOL2 in flowering time regulation, VrCOL2 was transformed into A. thaliana under the control of the 35S promoter. The empty vector was also transformed into A. thaliana, and the transgenic plants showed no difference with wild type under both LD and SD conditions (Additional file 4). The VrCOL2 transgenic A. thaliana lines showed high levels of VrCOL2 expression (Additional file 5). The VrCOL2 overexpression lines exhibited similar flowering time to wild-type plants under LD conditions but exhibited earlier flowering time than wild-type plants under SD conditions (Fig. 9), indicating that VrCOL2 regulates flowering time under a photoperiod-dependent pathway.
FT and TSF accelerate flowering and are regulated by CO in A. thaliana [11, 20], and we therefore investigated the expression of FT and TSF in wild-type and VrCOL2 transgenic plants under LD and SD conditions. FT and TSF showed similar expression levels in VrCOL2 transgenic and wild-type plants under LD conditions. By contrast, FT and TSF showed higher expression levels in VrCOL2 transgenic plants than in wild-type plants under SD conditions (Fig. 10). These results further support the conclusion that VrCOL2 is involved in flowering time regulation under SD conditions.