Functional characterization of mungbean CONSTANS-LIKE genes reveals a key role for CONSTANS-LIKE 2 in the control of flowering time under short-day conditions

CONSTANS-LIKE (COL) genes play important roles in the regulation of plant growth and development, and they have been analyzed in many plant species. However, few investigations have examined COL genes in mungbean ( Vigna radiata ). In this study, we identified and characterized a total 14 of VrCOL genes from mungbean, which distributed on 7 of the 11 mungbean chromosomes. Based on their conserved domains, VrCOLs were clustered into three groups (I, II and III), which contained 4, 5 and 5 members, respectively. The gene structures and conserved motifs of the VrCOL genes were analyzed, and two duplicated gene pairs, VrCOL2/VrCOL5 and VrCOL6/VrCOL9 , were identified. A total of 82 cis -acting elements were found in the VrCOL promoter regions, and the numbers and types of cis -acting elements in each VrCOL promoter region differed. As a result, VrCOLs showed distinct expression patterns in different tissues. Among these VrCOL genes, VrCOL2 showed a close phylogenetic relationship with Arabidopsis thaliana ( A. thaliana ) CO and displayed daily oscillations in expression under short day conditions but not long day conditions. In addition, overexpression of VrCOL2 accelerated flowering in A. thaliana under short day conditions by activating the expression of flowering time gene FT and TSF . Overall, we identified 14 VrCOL genes from mungbean using genome-wide identification. Characteristics and transcription pattern analysis of VrCOL genes revealed their important roles in plant growth and development, and our results suggested that VrCOL2 regulate flowering time under short day conditions. Our study lays the foundation for further dissection of VrCOL gene functions. cis A. thaliana measured by qRT-PCR. The leaves of transgenic and wild-type plants grown under LD conditions were sampled 5 h after lights-on for qRT-PCR analysis. Gene expression levels were

COL genes belong to the zinc-finger transcription factor family and play central roles in plant growth and development [11,20]. COL proteins are identified based on their conserved structure, which includes one or two BBX (B-Box) domains and one CCT (CONSTANS, CO-like, and TIMING of CAB1) domain [11,20]. The BBX domain can be further divided into two types, B-Box1 and B-Box2, which are recognized by their consensus sequences and the distances between their zinc-binding residues, which are considered to be involved in protein-protein interactions [20]. The CCT domain has important functions in transcriptional regulation and nuclear protein transport [11,[20][21][22]. The COL proteins are classified into three classes based on the number and type of their conserved domains.
Class I and II have two distinct BBX domains and one CCT domain, whereas class III has only one BBX and one CCT domain. Classes I, II, III contain 6, 7 and 4 members in Arabidopsis thaliana (A. thaliana), respectively. In addition, several COL proteins contain valine-proline (VP) motifs in their C termini [11,20].
Among these COL members, CO (BBX1) and its homologs are well-studied in many plant species [11,20,[23][24][25]. CO is expressed in a rhythmic manner and coordinates light pathway and circadian clock signal inputs in A. thaliana [26][27][28][29] [11,20]. The CO protein binds to cis-acting elements in the promoter region of the flowering activator FLOWERING LOCUS T (FT) to active FT expression. Moreover, CO is regulated by many flowering factors, such as GI (GIGANTEA), CDF1 (CYCKLING DOF FACTOR 1) and FKF1 (FLAVIN BINDING, KELCH REPEAT, F-BOX1) [30][31]. Hd1 (Heading date 1), the CO ortholog in rice, accelerates flowering under SD but delays flowering time under LD conditions through the regulation of the FT orthologs Hd3a (Heading date 3a) and RFT1 (RICE FLOWERING LOCUS T1) [32][33][34]. The soybean CO orthologs GmCOL1, GmCOL2, GmCOL3 and GmCOL4 can complement the late flowering phenotype of A. thaliana co mutants [35]. In addition to their functions in flowering time and circadian clock regulation, some COL proteins are also involved in abiotic or biotic stress responses, root development and stomatal opening [11,20].
Mungbean is a diploid legume crop, and its seeds contain proteins and nutrients that are essential for human nutrition [36]. The cultivated mungbean is considered to have been domesticated in India, from which it then spread to other areas [37]. The sequencing of its genome provides genetic resources for the investigation of gene functions in mungbean [38]. Flowering time is an important factor that influences mungbean production, and the investigation of mungbean flowering time genes can therefore provide essential information for further modification of mungbean cultivars to increase yield. In this study, we identified mungbean COL genes and investigated their characteristics, including chromosomal distributions, gene structures, cis-acting elements and gene expression patterns. We also analyzed the functions of VrCOL2 in the regulation of flowering time under SD conditions. Our findings will provide useful information for further characterization of mungbean COL gene functions.

Identification of VrCOL genes in mungbean
To search for mungbean VrCOL genes, we used the amino acid sequences of A. thaliana CO and COL proteins as blast queries against the mungbean genome database and the NCBI database. The conserved BBX and CCT domains in each candidate gene were confirmed using Pfam and InterPro, and a total of 14 VrCOL members were identified in the mungbean genome. Multiple characteristics of the VrCOL members were analyzed based on their genomic and protein sequences ( Table 1). The genomic lengths of VrCOL genes ranged from 1,506 (XP_014502470) to 14,007 bp (XP_014523701), the CDS lengths ranged from 933 (XP_014502470) to 1,329 bp (XP_022637309), and the amino acid numbers ranged from 310 to 442. The isoelectric points of VrCOL proteins varied from 4.86 (XP_014523701) to 9.22 (XP_014523547), and their molecular weights ranged from 33,756.82 (XP_014502470) to 48,806.9 (XP_022637309) Da. The GC content, which influences gene stability to some degree, ranged from 34.64-50.39%, and twelve of the fourteen VrCOL genes had lower than 50% GC content (Table 1).

Chromosomal distribution of the VrCOL genes
Plant COL genes evolved from several common original genes, and the chromosomal locations of COL genes can represent the alteration of gene distributions during evolution. To visualize the chromosomal locations of the VrCOL genes, we mapped them to their physical positions in the mungbean genome. XP_014523547 was discarded due to a lack of related chromosome information.
Seven of the fourteen VrCOL genes were located on the positive strand, and the VrCOL genes were named from VrCOL1 to VrCOL14 based on their chromosomal locations ( Table 1). Seven of the eleven mungbean chromosomes contained VrCOL genes, with the exception of chromosomes 2, 9, 10 and 11 ( Fig. 1, Table 1). Chromosome 5 contained the most number of VrCOL genes (three), followed by chromosomes 1, 4, 7 and 8, with two genes on each. In addition, most of the VrCOL genes were located on the relatively long chromosomes (1, 5, 6, 7 and 8). Only three members (VrCOL3, VrCOL4 and VrCOL5) were located on the relatively short chromosomes 3 and 4 ( Fig. 1).
Classification and phylogenetic analysis of VrCOL proteins 6 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  (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 wellstudied 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-stressrelated elements, three site-binding-related elements, nine hormone-responsive elements, three promoter-related elements and twenty light-responsive elements ( indicating that VrCOL9 may function in stress response ( Table 2) . 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 FT and TSF accelerate flowering and are regulated by CO in A. thaliana [11,20], and we therefore  (Fig. 10). These results further support the conclusion that VrCOL2 is involved in flowering time regulation under SD conditions.

Discussion
In recent decades, the investigation of CO and COL genes in many plant species has greatly increased our knowledge about the molecular mechanisms of flowering time regulation, stress response and root development [11,20]. Mungbean is a globally important legume crop, and the mechanisms of its flowering time regulation are still largely unknown. In this study, we identified and characterized 14 VrCOL genes from the mungbean genome and investigated the function of VrCOL2 in flowering time regulation. whereas mungbean has experienced only one such duplication [38,47]. As a result, the COL gene number in mungbean is approximately half that of soybean. Seven of the eleven (63.6%) mungbean chromosomes ( Fig. 1), seven of the eight (87.5%) Medicago chromosomes and sixteen of the twenty (80.0%) soybean chromosomes contained COL genes [35,45], indicating that the distribution of COL genes has changed much during evolution in legumes. The COL genes were clustered into three groups based on their conserved domains, and most of VrCOL genes in each group were clustered into the same clade in the phylogenetic tree, with the exception of the group III member VrCOL8, which contained one BBX and one CCT domain and showed a close relationship with group II members ( Fig. 3-4). VrCOL8 protein lacked motifs 4, 8, 12, and 13, which were found in all other VrCOL group III members, but it did contain motifs 3 and 5, which were found in all VrCOL group II members (Fig. 5), suggesting that VrCOL8 may derive from a group II ancestor and that one BBX domain may have been lost during evolution.
Plant genome evolution produces many duplicated gene pairs and provides resources for new gene functions [42]. Two duplicated gene pairs, VrCOL2/VrCOL5 and VrCOL6/VrCOL9 (Fig. 6), were found among the mungbean VrCOLs. The duplicated genes showed close relationships in the phylogenetic tree and contained similar motifs (Fig. 3-5), indicating that they evolved from the same origin and likely shared similar functions. However, the duplicated gene pairs contained different numbers and types of cis-acting elements in their promoter regions and exhibited different expression levels in some tissues (Fig. 7, Table 2), suggesting that they might have evolved novel functions compared with their original gene. For example, VrCOL6 and VrCOL9 shared similar numbers of several cisacting elements in their promoter regions, including promoter-related elements and site-binding related elements, but differed in the numbers of development-related elements, environmentalstress-related elements, hormone-responsive elements and light-responsive elements ( Table 2, Additional file 3). VrCOL6 and VrCOL9 showed similar expression levels in roots and nodule roots, but their expression differed in flowers, pods, leaves, seeds, stems and shoot apices (Fig. 7). This result suggests that they may have retained some common functions from the original gene in roots and nodule roots but evolved novel functions in other tissues.
The expression of VrCOL genes in different tissues provides clues to their potential functions, and many VrCOL genes (such as VrCOL11 and VrCOL12) showed tissue-specific expression patterns (Fig. 7). However, several VrCOL genes showed low expression levels in all tissues tested, despite the fact that their promoter regions contained many cis-acting elements, including VrCOL2, VrCOL8 and VrCOL13 (Fig. 7, Table 2 [1,4,6,44]. For example, VrCOL2 appeared to be a daily oscillation gene whose expression changed during the day under SD conditions but showed low expression throughout the day under LD conditions (Fig. 8). The different field-grown mungbean tissues were collected in the afternoon under relatively LD conditions in July, and that may explain why VrCOL2 showed low expression levels in the tissue expression analysis (Fig. 7). VrCOL8 and VrCOL13 may exhibit higher expression levels at other time points or under other photoperiod conditions.
CO and CO-homologous genes, such as OsHd1, play critical roles in flowering time regulation [11,20].
VrCOL2 showed close relationships with A. thaliana CO and soybean GmCOL1a, GmCOL1b, GmCOL2a and GmCOL2b, and accelerated flowering under SD but not LD conditions in transgenic A. thaliana lines (Fig. 9). A. thaliana CO regulates FT and TSF to accelerate flowering [26][27][28][29], and the expression of FT and TSF increased in VrCOL2 transgenic A. thaliana lines under SD but not LD conditions ( Fig. 10), indicating that VrCOL2 regulates downstream genes via photoperiod-dependent pathways.
In addition, CO protein accumulation is also regulated by circadian clock. CO mRNA abundance is highly expressed from late afternoon to the dawn, but CO protein only accumulates in the late afternoon under long day conditions [27,[49][50][51]. Although VrCOL2 is controlled by 35S promoter and can be expressed under both LD and SD conditions, the accumulation of VrCOL2 proteins might be low under LD condition, which might be the reason why VrCOL2 had no effect on flowering time under LD conditions. Overexpression of A. thaliana CO accelerates flowering under both LD and SD conditions [11,20], while in rice OsHd1 accelerates flowering under SD but delays flowering time under LD conditions [32][33][34]. Mungbean [52][53]

Conclusion
In this study, we identified and characterized 14 VrCOL genes from mungbean genome using genomewide analysis, and many characteristics of these VrCOL genes were investigated, including chromosomal distributions, sequence logos, classifications, phylogenetic relationships, gene structures, conserved motifs, duplicated gene pairs, cis-acting elements, and expression profiles.
Among these VrCOL genes, VrCOL2 showed a close relationship with A. thaliana CO and the expression of VrCOL2 exhibited daily oscillations under SD conditions. Further investigation revealed that VrCOL2 accelerated flowering under SD conditions by activating the expression of FT and TSF, but not LD conditions.

Plant materials and growth conditions
The reference genome variety VC1973A (named Zhonglu in China) supplied by Suk-Ha Lee at Seoul National University, Seoul, Korea, was used for all experiments [38]. Mungbean seeds were geminated in tap water for 1 day and then planted in soil-filled pots. Seedlings were grown in growth chambers

Identification of mungbean VrCOL members
The amino acid sequences of A. thaliana CO and COLs were used as blast queries against the National Center for Biotechnology Information (NCBI) and mungbean genome database (http://plantgenomics.snu.ac.kr/mediawiki-1.21.3/index.php/Main_Page) [38] to search for mungbean VrCOL proteins. The presence of conserved BBX and CCT domains in candidate genes were confirmed using the Pfam database [54] and InterPro program with default parameters [55].

Phylogenetic relationship analysis
The amino acid sequences of CO and COL proteins from A. thaliana, soybean, Medicago and mungbean were aligned using ClustalW2 [56], and the resulting alignment was used to construct a phylogenetic tree in MEGA 7.0 using the Neighbor-Joining method with default parameters [57]. In addition, VrCOL proteins were aligned separately in ClustalW2 and used to construct a phylogenetic tree in MEGA7.0 with the Neighbor-Joining method.

Chromosomal distribution and duplication analyses
The physical positions of VrCOL genes were obtained from NCBI, and a chromosomal location map was constructed using MapInspect software (Mike Lischke, Berlin, Germany). Duplicated gene pairs were identified using OrthoMCL software as described by Jin et al. [19,58]. The duplicated gene pairs were identified with amino acid sequences more than 60% similarity, and visualized using Circos software [59].
Analyses of exon-intron organizations, conserved domains, sequence logos, protein motifs and cisacting elements The genomic and CDS sequences of mungbean VrCOL genes were obtained from NCBI and used as inputs to the Gene Structure Display Server (GSDS) [39] to analyze their gene structures. The fulllength amino acid sequences of VrCOL proteins were used to analyze the positions of the conserved BBX and CCT domains using the InterPro program [55]. The sequence logos of the conserved BBX1, BBX2 and CCT domains were analyzed using the WebLogo platform [60]. The conserved motifs present in the VrCOL proteins were identified using MEME tools, and the parameters of the optimum motif widths were 11-50 amino acid residues [61]. The cis-acting elements in each VrCOL promoter, 2 kb upstream of the initiation codon, were predicted by PlantCARE [41].

Plasmid construction and plant transformation
To investigate the functions of VrCOL2, a 35S: CDS-VrCOL2 plasmid was constructed. The VrCOL2 CDS was amplified from the cDNA of the sequenced mungbean variety VC1973A using primers with XhoI and XbaI digestion site sequences. The resulting PCR fragment was digested by the restriction endonucleases XhoI and XbaI to generate cohesive ends. The pRTL2 vector was digested with XhoI and XbaI to generate a linearized plasmid. Then the VrCOL2 and pRTL2 fragments were ligated using T4 DNA ligase (Promega). The constructed plasmid was verified by sequencing. It was then introduced into A. thaliana using the floral dip method [62], and successful transformation was confirmed by PCR.
All primers are listed in Additional file 6.
RNA isolation and quantitative real-time PCR (qRT-PCR) analysis were carried out as described in Li et al. [40]. Gene expression levels were normalized to an Actin gene from mungbean (Vradi03g00210).
Each sample was analyzed using three biological replicates. All primers are listed in Additional file 6.
For RNA-seq analysis, total RNA was extracted from different tissues of mungbean variety VC1973A, including flowers, pods, leaves, seeds, nodule roots, stems, roots and shoot apices, and the RNA purity and RNA integrity were checked using the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA) and the RNA Nano 6000 Assay Kit of the

Availability of data and materials
The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests. Roots, nodule roots, shoot apices, stems, leaves, flowers, pods and seeds were used for analysis. Figure 1 Chromosomal locations of the VrCOL genes. Chromosome number and length are indicated.       Relative expression of VrCOL2 in leaves throughout the day under SD and LD conditions.