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

Background: 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). Results: In this study, we identied 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, VrCOL1/VrCOL2 and VrCOL8/VrCOL9, were identied. 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, the expression patterns of VrCOLs varied in different tissues, and under long day and short day conations throughout the day. 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 owering in A. thaliana under short day conditions by activating the expression of owering time gene AtFT and AtTSF. Conclusion: Overall, we identied 14 VrCOL genes from mungbean using genome-wide identication. Characteristics and transcription pattern analysis of VrCOL genes revealed their important roles in plant growth and development, and our results suggested that VrCOL2 regulate owering time under short day conditions in A. thaliana. Our study lays the foundation for further dissection of VrCOL gene functions. The genomic and CDS sequences of mungbean VrCOL genes were obtained from NCBI and used as inputs to the Gene Structure Display Server (GSDS) to analyze their gene structures [48]. The full-length amino acid sequences of VrCOL proteins were used to analyze the positions of the conserved BBX and CCT domains using the InterPro program [62]. The sequence logos of the conserved BBX1, BBX2 and CCT domains were analyzed using the WebLogo platform [67]. The conserved motifs present in the VrCOL proteins were identied using MEME tools, and the parameters of the optimum motif widths were 11-50 amino acid residues [68]. The cis-acting elements in each VrCOL promoter, 2 kb upstream of the initiation codon, were predicted by PlantCARE [50].

COL genes belong to the zinc-nger transcription factor family and play central roles in plant growth and development [11,20]. COL proteins are identi ed 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 classi ed 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, AtCO (AtBBX1) and its homologs are well-studied in many plant species [11,20,[23][24][25]. AtCO is expressed in a rhythmic manner and coordinates light pathway and circadian clock signal inputs in A. thaliana [26][27][28][29]. Thus, AtCO plays an important role in the regulation of owering time via the photoperiod-dependent pathway. Atco mutants exhibit delayed owering time under long day conditions (LD), but under short day conditions (SD), their owering times are similar to those of wild-type plants. By contrast, AtCO overexpression plants show early owering time under both LD and SD conditions [11,20]. The AtCO protein binds to cis-acting elements in the promoter region of the owering activator FLOWERING LOCUS T (AtFT) to active AtFT expression. Moreover, AtCO is regulated by many owering factors, such as AtGI (GIGANTEA), AtCDF1 (CYCKLING DOF FACTOR 1) and AtFKF1 (FLAVIN BINDING, KELCH REPEAT, F-BOX1) [30][31]. OsHd1 (Heading date 1), the AtCO ortholog in rice, accelerates owering under SD but delays owering time under LD conditions through the regulation of the AtFT orthologs OsHd3a (Heading date 3a) and OsRFT1 (RICE FLOWERING LOCUS T1) [32][33][34]. The soybean AtCO orthologs GmCOL1, GmCOL2, GmCOL3 and GmCOL4 can complement the late owering phenotype of Atco mutants [35]. In addition to their functions in owering 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]. Mungbean is considered to be a SD crop, and the owering time is a critical factor in uencing its production [38][39][40]. Mungbean plants produce a large number of owers, but only a few set pods. Approximately 70-90% of the owers are shed, which mainly occurs in later formed owers of the racemes [41][42]. Thus, it is suggested that the prevention of late owering is an important way to increase mungbean yield [42][43][44]. The sequencing of mungbean genome provides genetic resources for the investigation of gene functions [45], and the investigation of mungbean owering time genes can therefore provide essential information for further modi cation of mungbean cultivars to increase yield. Until now, limited information about gene functions has been identi ed in mungbean owering time regulation. In this study, we identi ed 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 owering time under SD conditions. Our ndings will provide useful information for further characterization of mungbean COL gene functions.

Identi cation of VrCOL genes in mungbean
To search for mungbean VrCOL genes, we used the amino acid sequences of AtCO and AtCOL proteins as blast queries against the mungbean genome database and the NCBI database. The conserved BBX and CCT domains in each candidate gene were con rmed using Pfam and InterPro, and a total of 14 VrCOL members were identi ed 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 in uences gene stability to some degree, ranged from 34.64% to 50.39%, and twelve of the fourteen VrCOL genes had lower than 50% GC content (Table 1).

Phylogenetic and classi cation analysis of VrCOL proteins
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, 16 rice, 18 maize and 14 mungbean CO and COL proteins [11,35,[46][47]. The VrCOL genes were named from VrCOL1 to VrCOL13 based on their phylogenetic relationships with the soybean orthologous genes (Fig. 1, Table 1). Among these VrCOL members, VrCOL1 and VrCOL2 showed close relationships to A. thalianaAtCO, soybean GmCOL1a, GmCOL1b, GmCOL2a, and GmCOL2b and rice OsHd1 (OsCOL-A), all of which have documented roles in the regulation of owering time [11,20,35]. This result suggests that VrCOL1 and VrCOL2 may play critical roles in the owering time regulation of mungbean.
Nine VrCOL proteins contained one BBX1, one BBX2 and one CCT domain, and ve VrCOL proteins contained one BBX1 and one CCT domain (Fig. 3). The VrCOL proteins were further classi ed into three groups based on differences in these conserved domains ( Fig. 1 and 3). 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 VrCOL10, which showed a closer relationship with class II members ( Fig. 1 and 3).

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 [48]. 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 VrCOL10, which contained 6 exons and 6 introns and had a close relationship with group II members ( Fig. 3-4). 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. 4). 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 identi ed, and all VrCOL proteins contained motifs 1 and 2, which appeared to represent the conserved CCT and BBX1 domains, respectively (Fig. 4, Additional le 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 VrCOL10) (Fig. 4).

Chromosomal distribution and duplication analysis of 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. VrCOL7b was discarded due to a lack of related chromosome information. Seven of the fourteen VrCOL genes were located on the positive strand. Seven of the eleven mungbean chromosomes contained VrCOL genes, with the exception of chromosomes 2, 9, 10 and 11 ( Fig. 5, 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 (VrCOL1, VrCOL11 and VrCOL13) were located on the relatively short chromosomes 3 and 4 (Fig. 5).
Mungbean has experienced one round of whole-genome duplication that produced many duplicated gene pairs [45,49]. To investigate the evolutionary relationships among the VrCOLs, we searched for duplicated gene pairs among them. Two interchromosomal duplication events were identi ed in chromosomes 1, 4, 5 and 6, including the duplicated gene pairs VrCOL1/VrCOL2 and VrCOL8/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 VrCOL1 and VrCOL2 showed similar exon-intron organization and similar motifs, as did VrCOL8 and VrCOL9 (Fig. 4), 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 [50]. A total of 82 cis-acting elements were found across the 14 VrCOL promoter regions (2 kb upstream of the initiation codon) (Additional le 3). Forty-ve 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 le 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 VrCOL8 (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 VrCOL8 promoter (nine elements), indicating that VrCOL8 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 le 3), suggesting potential functions of these genes in related signaling pathways.

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, owers, pods and seeds. VrCOL genes showed distinct expression patterns in different tissues (Fig. 7). For example, VrCOL3 was highly expressed in all the tested tissues, whereas VrCOL2 and VrCOL7a showed low expression in most tissues. Some genes were expressed at high levels in speci c tissues, suggesting that they may have critical functions in these tissues. For example, VrCOL6 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 [51][52]. To investigate the conservation and diversity of duplicated genes, we also analyzed their tissue-speci c expression patterns. VrCOL1 and VrCOL2 differed in their expression levels across all the tissues we examined, indicating that they had undergone functional divergence. VrCOL8 and VrCOL9 showed similar expression levels in roots and nodule roots, but they exhibited different expression levels in other tissues (Fig. 7, Additional le 4).

Diurnal rhythm of VrCOL gene expressions
In A. thaliana, the expressions of CO, COL1 and COL2 are regulated by the circadian clock and show diurnal oscillations [11,53]. We therefore investigated whether VrCOL genes exhibited diurnal expression rhythms in mungbean leaves under LD and SD conditions. Gene expression analysis revealed that VrCOL4, VrCOL6, VrCOL12, and VrCOL13 showed daily oscillations under both LD and SD conditions, while VrCOL1, VrCOL2, VrCOL5, VrCOL7a, VrCOL7b, VrCOL10 and VrCOL11 only showed daily oscillations under SD conditions, but not LD conditions (Fig. 8). The duplicated genes VrCOL8 and VrCOL9 exhibited similar expression patterns under both LD and SD conditions, while VrCOL1 and VrCOL2 showed distinct expression patterns under both LD and SD throughout the day (Fig. 8).
Overexpression of VrCOL2 accelerates owering in A. thaliana under SD conditions VrCOL1 and VrCOL2 displayed close phylogenetic relationships with AtCO (Fig. 1), and the amino acid sequences of VrCOL1 and VrCOL2 showed 49.35% and 50.93% similarities with AtCO, respectively, thus VrCOL2 was selected for further analysis. To investigate the potential functions of VrCOL2 in owering 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 le 5). The VrCOL2 transgenic A. thaliana lines showed high levels of VrCOL2 expression (Additional le 6). The VrCOL2 overexpression lines exhibited similar owering time to wild-type plants under LD conditions but exhibited earlier owering time than wild-type plants under SD conditions (Fig. 9), indicating that VrCOL2 regulates owering time under a photoperiod-dependent pathway.
AtFT and AtTSF accelerate owering and are regulated by AtCO in A. thaliana [11,20], and we therefore investigated the expression of AtFT and AtTSF in wild-type and VrCOL2 transgenic plants under LD and SD conditions. AtFT and AtTSF showed similar expression levels in VrCOL2 transgenic and wild-type plants under LD conditions. By contrast, AtFT and AtTSF showed higher expression levels in VrCOL2 transgenic plants than in wild-type plants under SD conditions (Fig. 9). In addition, the expressions of two mungbean FT and TSF homologous genes (XP_014496932, XP_014497364), which contained CORE cisacting elements in their promoter regions, increased in VrCOL2 transgenic hair roots under SD conditions, but not LD condition (Additional le 7). These results further support the conclusion that VrCOL2 is involved in owering 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 owering time regulation, stress response and root development [11,20]. Mungbean is a globally important legume crop, and the mechanisms of its owering time regulation are still largely unknown. In this study, we identi ed and characterized 14 VrCOL genes from the mungbean genome and investigated the function of VrCOL2 in owering time regulation.
The A. thaliana, soybean, Medicago and mungbean genomes contained 17, 26, 11 and 14 CO and COL members, respectively (Fig. 1) [35,54], and their genome sizes are 125 Mb [55], 1100 Mb [56], 500 Mb [57] and 579 Mb [45], respectively. Thus, genome size has no direct relationship with the number of COL genes in plants. Soybean has undergone two rounds of whole-genome duplication, whereas mungbean has experienced only one such duplication [45,56]. As a result, the COL gene number in mungbean is approximately half that of soybean. Seven of the eleven (63.6%) mungbean chromosomes (Fig. 5), seven of the eight (87.5%) Medicago chromosomes and sixteen of the twenty (80.0%) soybean chromosomes contained COL genes [35,54], 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 VrCOL10, which contained one BBX and one CCT domain and showed a close relationship with group II members ( Fig. 1 and 3). VrCOL10 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. 4), suggesting that VrCOL10 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 [51]. Two duplicated gene pairs, VrCOL1/VrCOL2 and VrCOL8/VrCOL9 (Fig. 6), were found among the mungbean VrCOLs. The duplicated genes showed close relationships in the phylogenetic tree and contained similar motifs (Fig. 3-4), 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, VrCOL8 and VrCOL9 shared similar numbers of several cis-acting elements in their promoter regions, including promoter-related elements and site-binding related elements, but differed in the numbers of development-related elements, environmental-stress-related elements, hormoneresponsive elements and light-responsive elements (Table 2, Additional le 3). VrCOL8 and VrCOL9 showed similar expression levels in roots and nodule roots, but their expression differed in owers, pods, leaves, seeds, stems and shoot apices (Fig. 7, Additional le 4). 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 VrCOL6 and VrCOL12) showed tissue-speci c 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, VrCOL7a and VrCOL10 (Fig. 7, Table 2, Additional le 3). Gene expression is in uenced by many factors. For example, many circadian clock and owering time regulation genes are controlled by photoperiod. Their expression changes under different photoperiods and during the day and night [1,4,6,53]. 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 eld-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). CO and CO-homologous genes, such as OsHd1, play critical roles in owering time regulation [11,20]. VrCOL2 showed higher expression levels at the stages of during owering, and after owering, than that at the stage of before owering, indicating that VrCOL2 played an important role in owering time regulation (Additional le 8). VrCOL2 showed close relationships with A. thaliana CO, soybean GmCOL1a, GmCOL1b, GmCOL2a and GmCOL2b and rice OsHd1 (OsCOL-A), and accelerated owering under SD but not LD conditions in transgenic A. thaliana lines (Fig. 9). AtCO regulates AtFT and AtTSF to accelerate owering [26][27][28][29], and the expression of AtFT and AtTSF increased in VrCOL2 transgenic A. thaliana lines under SD but not LD conditions (Fig. 9), indicating that VrCOL2 regulates downstream genes via photoperiod-dependent pathways. In addition, AtCO protein accumulation is also regulated by circadian clock. AtCO mRNA abundance is highly expressed from late afternoon to the dawn, but AtCO protein only accumulates in the late afternoon under long day conditions [27,[58][59][60]. 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 owering time under LD conditions. Overexpression of AtCO accelerates owering under both LD and SD conditions [11,20], while in rice OsHd1 accelerates owering under SD but delays owering time under LD conditions [32][33][34]. Mungbean [38][39] and rice are short day plants, and A. thaliana is a long day plant, which might be the reason why CO homologous genes have different functions in different plant species. These results suggest that CO and its homologs are involved in owering time regulation under photoperiod-dependent pathways and have distinct roles in different plant species. Thus, in summer LD conditions, the expression of VrCOL2 may be low and have little effect on the acceleration of owering. In the autumn, as days become shorter, the expression of VrCOL2 may increase and accelerate mungbean owering. In addition, VrCOL1 and VrCOL2 form a duplicated gene pair and show a close relationship with one another ( Fig. 1 and 6), indicating that VrCOL1 may have similar functions to VrCOL2 in owering time regulation. Much more work is needed to fully elucidate the mechanisms by which VrCOL2 affects owering time and circadian clock regulation in mungbean.

Conclusion
In this study, we identi ed 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, classi cations, phylogenetic relationships, gene structures, conserved motifs, duplicated gene pairs, cis-acting elements, and expression pro les. Among these VrCOL genes, VrCOL2 showed a close relationship with AtCO and the expression of VrCOL2 exhibited daily oscillations under SD conditions. Further investigation revealed that VrCOL2 accelerated owering under SD conditions by activating the expression of AtFT and AtTSF, but not LD conditions.

Plant materials and growth conditions
The reference mungbean genome variety VC1973A, stored at Seoul National University, was identi ed and supplied by Suk-Ha Lee at Seoul National University, Seoul, Korea [45], and the variety was used for all experiments in this study. Mungbean seeds were geminated in tap water for 1 day and then planted in

Identi cation 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

Phylogenetic relationship analysis
The amino acid sequences of CO and COL proteins from A. thaliana, soybean, Medicago, mungbean, rice and maize were aligned using ClustalW2 [63], and the resulting alignment was used to construct a phylogenetic tree in MEGA 7.0 using the Neighbor-Joining method with default parameters [64]. 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 identi ed using OrthoMCL software as described by Jin et al. [19,65]. The duplicated gene pairs were identi ed with amino acid sequences more than 60% similarity, and visualized using Circos software [66].
Analyses of exon-intron organizations, conserved domains, sequence logos, protein motifs and cis-acting 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) to analyze their gene structures [48]. The full-length amino acid sequences of VrCOL proteins were used to analyze the positions of the conserved BBX and CCT domains using the InterPro program [62]. The sequence logos of the conserved BBX1, BBX2 and CCT domains were analyzed using the WebLogo platform [67]. The conserved motifs present in the VrCOL proteins were identi ed using MEME tools, and the parameters of the optimum motif widths were 11-50 amino acid residues [68]. The cis-acting elements in each VrCOL promoter, 2 kb upstream of the initiation codon, were predicted by PlantCARE [50].

Plasmid construction and plant transformation
To investigate the functions of VrCOL2, a 35S: CDS-VrCOL2 plasmid was constructed. The VrCOL2 CDS was ampli ed 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 veri ed by sequencing. It was then introduced into A. thaliana using the oral dip method [69], and successful transformation was con rmed by PCR. In addition, the VrCOL2 overexpression construct was transformed into VC1973A to obtain transgenic root hairs using the method as described by Kereszt et al. [70]. Three-week old transgenic root hairs grown under LD and SD conditions were collected for gene expression analysis, respectively. Each sample was analyzed using three independent root hairs. All primers are listed in Additional le 9.
RNA extraction and transcription analysis RNA isolation and quantitative real-time PCR (qRT-PCR) analysis were carried out as described in Li et al. [49]. 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 le 9.

Declarations
Ethics approval and consent to participate 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. Additional le 8: Expression levels of VrCOL2 in leaves at three growth stages, before owering (S1), during owering (S2), and after owering (S3).
Additional le 9: Primers used in this study.   Sequence logos of the BBX1, BBX2 and CCT domains of VrCOL proteins. The conserved domains were analyzed using the WebLogo platform.    Relative expression levels of VrCOL genes in different tissues analyzed by qRT-PCR. The expression level of VrCOL1 in owers was set as 1, and the others were adjusted accordingly. ***, ** and * are signi cantly different at P < 0.001, P < 0.01 and P < 0.05, respectively, compared with the expression levels in the relative owers.

Tables
Page 27/28 Figure 8 Relative expression of VrCOLs in leaves throughout the day under SD and LD conditions. The SD condition was set as 8:00 am-6:00 pm light, 6:00 pm-8:00 am dark; the LD condition was set as 8:00 am-0:00 am light, 0:00 am-8:00 am dark. ZT, Zeitgeber Time. Expression level of VrCOLs was normalized to an ACTIN gene from mungbean.