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. 2021 Feb 4:12:608603.
doi: 10.3389/fpls.2021.608603. eCollection 2021.

Characterization of Mungbean CONSTANS-LIKE Genes and Functional Analysis of CONSTANS-LIKE 2 in the Regulation of Flowering Time in Arabidopsis

Chenyang Liu  1 , Qianqian Zhang  1 , Hong Zhu  2 , Chunmei Cai  1 , Shuai Li  1
Affiliations

Characterization of Mungbean CONSTANS-LIKE Genes and Functional Analysis of CONSTANS-LIKE 2 in the Regulation of Flowering Time in Arabidopsis

Chenyang Liu et al. Front Plant Sci. .

Abstract

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 studies have examined COL genes in mungbean (Vigna radiata). In this study, we identified and characterized 31 mungbean genes whose proteins contained B-Box domains. Fourteen were designated as VrCOL genes and were distributed on 7 of the 11 mungbean chromosomes. Based on their phylogenetic relationships, VrCOLs were clustered into three groups (I, II, and III), which contained 4, 6, and 4 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 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, the expression patterns of VrCOLs varied in different tissues and throughout the day under long-day and short-day conditions. Among these VrCOL genes, VrCOL2 showed a close phylogenetic relationship with Arabidopsis 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 Arabidopsis under short-day conditions by affecting the expression of the flowering time genes AtFT and AtTSF. Our study lays the foundation for further investigation of VrCOL gene functions.

Keywords: CONSTANS; VrCOL2; flowering time; genome wide; mungbean.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Phylogenetic relationships and conserved domain analyses of the VrBBX proteins. (A) Phylogenetic relationship analysis of 31 VrBBX proteins. The amino acid sequences of mungbean proteins containing BBX domains were used to construct a phylogenetic tree with the neighbor-joining method. (B) The positions of conserved BBX1, BBX2, and CCT domains in the VrBBX proteins. The blue, purple and orange boxes in each VrBBX protein indicate the BBX1, BBX2, and CCT domains, respectively. (C) The positions of the conserved BBX1, BBX2, and CCT domains in each VrBBX protein.
FIGURE 2
FIGURE 2
Sequence logos of the BBX1, BBX2, and CCT domains of VrCOL proteins. The conserved domains were analyzed using the WebLogo platform. The amino acid sequences of each conserved domain are presented on the x-axis, and the height of each letter corresponds to the conservation of each residue.
FIGURE 3
FIGURE 3
Evolutionary relationships among VrCOL proteins and COL proteins from other species. The amino acid sequences of COL proteins from Arabidopsis, soybean, Medicago, mungbean, rice and maize were used to construct a phylogenetic tree in MEGA 7.0 with the neighbor-joining method. VrCOL proteins are grouped into three classes and indicated with different colors.
FIGURE 4
FIGURE 4
Gene structures and conserved motifs of the VrCOL proteins. (A) Exon-intron organization of the VrCOL genes. The length of each VrCOL gene is indicated, and the blue boxes, pink boxes and black lines indicate UTRs, exons and introns, respectively. (B) Conserved motifs of the VrCOL proteins. Conserved motifs were analyzed using MEME tools, and different motifs are indicated by different colored boxes.
FIGURE 5
FIGURE 5
Chromosomal locations of the VrCOL genes. Chromosome number, chromosome length, and the positions of VrCOLs on the chromosome are indicated.
FIGURE 6
FIGURE 6
Duplication analyses of VrCOL proteins. The positions of VrCOL genes on the chromosomes are presented, and duplicated gene pairs are connected by colored lines.
FIGURE 7
FIGURE 7
Relative expression levels of VrCOL genes in different tissues. The expression levels of VrCOL genes were analyzed by qRT–PCR. The expression level of VrCOL1 in flowers was set to 1, and other values were adjusted accordingly. The gene expression results were visualized using a heatmap generated with Multiple Experiment Viewer 4.9.0 (Saeed et al., 2003). Different colors in the heatmap indicate different expression levels.
FIGURE 8
FIGURE 8
Relative expressions of VrCOLs in mungbean 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. The gray and red lines indicate VrCOL expression levels under LD and SD conditions, respectively. Each sample was analyzed using three biological replicates.
FIGURE 9
FIGURE 9
Overexpression of VrCOL2 accelerates flowering under SD conditions in Arabidopsis. Flowering time phenotypes of three VrCOL2 overexpression transgenic lines and wild-type Arabidopsis (Col) plants grown under LD (A) and SD conditions (B). Bar = 4 cm. The rosette leaf numbers of VrCOL2 overexpression transgenic lines and wild-type plants grown under LD (C) and SD conditions (D) as shown in panels (A,B). The expression levels of AtFT and AtTSF throughout the day in VrCOL2 transgenic lines and wild-type plants under LD (E,G) and SD (F,H) conditions. Leaves of 2-week-old VrCOL2 overexpression transgenic lines and wild-type plants were sampled every 4 h after lights-on. Expression levels of AtFT and AtTSF were normalized to an Actin gene from Arabidopsis. 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.
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