Isolation and Functional Characterization of a Floral Repressor, BcMAF1, From Pak-choi (Brassica rapa ssp. Chinensis)

Abstract

MADS-box genes form a large gene family in plants and are involved in multiple biological processes, such as flowering. However, the regulation mechanism of MADS-box genes in flowering remains unresolved, especially under short-term cold conditions. In the present study, we isolated BcMAF1, a Pak-choi (Brassica rapa ssp. Chinensis) MADS AFFECTING FLOWERING (MAF), as a floral repressor and functionally characterized BcMAF1 in Arabidopsis and Pak-choi. Subcellular localization and sequence analysis indicated that BcMAF1 was a nuclear protein and contained a conserved MADS-box domain. Expression analysis revealed that BcMAF1 had higher expression levels in leaves, stems, and petals, and could be induced by short-term cold conditions in Pak-choi. Overexpressing BcMAF1 in Arabidopsis showed that BcMAF1 had a negative function in regulating flowering, which was further confirmed by silencing endogenous BcMAF1 in Pak-choi. In addition, qPCR results showed that AtAP3 expression was reduced and AtMAF2 expression was induced in BcMAF1-overexpressing Arabidopsis. Meanwhile, BcAP3 transcript was up-regulated and BcMAF2 transcript was down-regulated in BcMAF1-silencing Pak-choi. Yeast one-hybrid and dual luciferase transient assays showed that BcMAF1 could bind to the promoters of BcAP3 and BcMAF2. These results indicated that BcAP3 and BcMAF2 might be the targets of BcMAF1. Taken together, our results suggested that BcMAF1 could negatively regulate flowering by directly activating BcMAF2 and repressing BcAP3.

Keywords: BcAP3; BcMAF1; BcMAF2; Pak-choi (Brassica rapa ssp. Chinensis); late flowering.

FIGURE 1

BcMAF1 is a short-term cold response gene encoding an MADS protein. (A) Sequence alignment of BcMAF1, BcMAF2, and AtMAFs. Conserved and similar residues are boxed with red and yellow, respectively. The MADS-box domain is indicated by a straight line. (B) The phylogenic tree of BcMAF1, BcMAF2, and AtMAFs. The gene accession numbers are as follows: AtMAF1 (AT1G77080), AtMAF2 (AT5G65050), AtMAF3 (AT5G65060), AtMAF4 (AT5G65070), AtMAF1 (AT5G65080), BcMAF1 (MG964044), and BcMAF2 (MG964045). (C) The expression of BcMAF1 during the process of vernalization in Pak-choi by qPCR. (D) The expression of BcMAF1 in different tissues of Pak-choi by qPCR. Data shown are means ± SEM of three independent experiments.

FIGURE 2

Subcellular localization of BcMAF1 protein. (A) 35S:GFP and 35S:BcMAF1-GFP construct. (B) Transient expression of 35S:GFP (Scale bars = 50 μm) and 35S:BcMAF1-GFP fusion protein (Scale bars = 20 μm) in tobacco leaves.

FIGURE 3

Overexpression of BcMAF1 in Arabidopsis. (A) Late flowering phenotype of the transgenic plants overexpressed BcMAF1. The 35S:GFP and 35S:BcMAF1-GFP#8, #16, and #23 plants in a chamber (LD, 16 h light/8 h dark photoperiod at 22°C/18°C). Scale bars = 1.5 cm. Rosette leaf number at bolting (B) Days of opening of first flower (C) in the 35S:GFP and 35S:BcMAF1-GFP#8, #16, and #23 plants. Error bars represent standard deviation of the mean number of 30 plants for each line. ^∗∗ indicate significant differences from the control (P < 0.01). (D) Expression analysis of predicted downstream genes in the 35S:GFP and 35S:BcMAF1-GFP#8, #16, and #23 plants.

FIGURE 4

Silencing BcMAF1 in Pak-choi. (A) Expression analysis of BcMAF1 in Pak-choi seedlings bombarded with pTY and pTY-BcMAF1 plasmid. (B) Expression analysis of BcPDS in Pak-choi seedlings bombarded with pTY and pTY-BcPDS plasmid. (C) Early flowering phenotype in Pak-choi seedlings silenced BcMAF1. Scale bars = 2.5 cm. (D) Expression analysis of predicted downstream genes in the pTY, pTY-BcMAF1-1, and pTY-BcMAF1-5 plants.

FIGURE 5

Binding activities of BcMAF1 protein with the promoters of BcMAF2 and BcAP3 detected by yeast one-hybrid assays. The yeast cells were grown on SD/-Leu medium plate supplemented with or without 300 ng/mL AbA.

FIGURE 6

Activation or repression of BcMAF2 or BcAP3 promoter by overexpressing BcMAF1 in the dual luciferase transient assay. (A) Diagram of the effector, reporter, and reference plasmids used in the dual luciferase transient assay. (B) The relative FLUC/RLUC activities of BcMAF2 and BcAP3 promoters activated and repressed by recombinant 35S:BcMAF1-GFP in Arabidopsis protoplasts were detected by Dual-Luciferase^® Reporter Assay System. Bars represent the mean ± SEM. The relative FLUC/RLUC activity of the negative control was set at 1. ^∗∗ indicate a significant difference at P < 0.01.