Brahma is required for proper expression of the floral repressor FLC in Arabidopsis

Abstract

Background: BRAHMA (BRM) is a member of a family of ATPases of the SWI/SNF chromatin remodeling complexes from Arabidopsis. BRM has been previously shown to be crucial for vegetative and reproductive development.

Methodology/principal findings: Here we carry out a detailed analysis of the flowering phenotype of brm mutant plants which reveals that, in addition to repressing the flowering promoting genes CONSTANS (CO), FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), BRM also represses expression of the general flowering repressor FLOWERING LOCUS C (FLC). Thus, in brm mutant plants FLC expression is elevated, and FLC chromatin exhibits increased levels of histone H3 lysine 4 tri-methylation and decreased levels of H3 lysine 27 tri-methylation, indicating that BRM imposes a repressive chromatin configuration at the FLC locus. However, brm mutants display a normal vernalization response, indicating that BRM is not involved in vernalization-mediated FLC repression. Analysis of double mutants suggests that BRM is partially redundant with the autonomous pathway. Analysis of genetic interactions between BRM and the histone H2A.Z deposition machinery demonstrates that brm mutations overcome a requirement of H2A.Z for FLC activation suggesting that in the absence of BRM, a constitutively open chromatin conformation renders H2A.Z dispensable.

Conclusions/significance: BRM is critical for phase transition in Arabidopsis. Thus, BRM represses expression of the flowering promoting genes CO, FT and SOC1 and of the flowering repressor FLC. Our results indicate that BRM controls expression of FLC by creating a repressive chromatin configuration of the locus.

Figure 1. BRM controls expression of CO, FT and SOC1 genes.

A) Analysis of CO and FT expression in wild-type (Col), brm-1 and brm-2 mutant plants by RT-PCR. Total RNA was isolated from seedlings collected 10 h after dawn at 12 days of growth under LD conditions. GAPC transcript levels were also determined as a control for the amount of input cDNA. B) GUS expression patterns of gCO::GUS and pFT::GUS in wild-type and BRM-silenced plants (brm29.1) in whole-mount staining of 6-day-old and 12-day-old seedlings under LD conditions. C) Flowering time of plants grown under LD photoperiod. Data are means and standard deviation of at least 20 plants. Differences between the indicated pairs of data are significant with p<0.05 (*) or p<0.01 (**). D) Analysis of SOC1 expression in wild type (Col), ft-10, ft-10 brm-2 and brm-2 plants by qRT-PCR. Total RNA was isolated from seedlings collected 10 h after dawn at 14 and 15 days of growth under LD conditions. E) Analysis of FT expression in co-10 and co-10 brm-2 mutant plants by qRT-PCR. Total RNA was isolated as in D.

Figure 2. flc-3 mutation enhances the early flowering phenotype of brm mutants.

A) Flowering time of plants grown under LD or SD photoperiod. Data are means and standard deviation of at least 20 plants. Asterisks indicate significant differences between Col and brm-1, brm-2 and flc-3 with p<0.01 (*) for both LD and SD data, or between flc-3 brm-1 and the other background with p<0.001 (**) for SD and p<0.00001 (***) for LD data. B) Analysis of FT expression in wild-type (Col), flc-3, flc-3 brm-1 and brm-1 plants by qRT-PCR. Total RNA was isolated from seedlings collected 10 h after dawn at 14 and 15 days of growth under LD conditions. C) Analysis of SOC1 expression as in C. D) Analysis of FT expression in wild-type (Col), flc-3, flc-3 brm-1 and brm-1 plants by qRT-PCR. Total RNA was isolated from seedlings collected 10 h after dawn at 19 and 20 days of growth under SD conditions. E) Analysis of SOC1 expression as in D. Asterisks indicate significant differences between wt and brm-1 with p<0.005 (*) or p<0.02 (**).

Figure 3. BRM controls expression of FLC.

A) Percentage of flowering under SD (8∶16) conditions of wild-type (Col), brm-1, brm-2, flc-3 and brm-1flc-3 plants. B) Analysis of FLC expression in wild-type (Col) and brm-1 plants by qRT-PCR. Total RNA was isolated from seedlings collected 10 h after dawn at 14 and 15 days of growth under LD conditions and at 19 and 20 days under SD conditions. C) Analysis of the levels of H3K4me3 and H3K27me3 by ChIP-PCR at the FLC promoter in WT and brm-1 mutant plants. A representative experiment of three independent replicates is shown.

Figure 4. BRM is not required for the vernalization response.

A) Flowering behaviour of brm mutants with and without vernalization in long days and short days. Plants were vernalized for 40 days, then transferred either to LD or to SD (as indicated) conditions and flowering time was determined. Data are means and standard deviation of at least 15 plants. Differences between vernalized and not vernalized set of data were significant with p<0.05 (*) or p<0.00001 (**). B) Analysis of FLC expression by RT-PCR of vernalized or not vernalized plants. Total RNA was isolated from non-vernalized seedlings (NV) or 10 days after transferring vernalized plants to LD normal conditions (40VT10).

Figure 5. Interaction of BRM with the autonomous pathway.

A) Analysis of FVE, FCA, FLD, FPA, FY, LD, and FLK expression in wild-type, brm-1 and brm-2 mutant plants by RT-PCR. Total RNA was isolated from seedlings collected 10 h after dawn at 12 days of growth under LD conditions. GAPC transcript levels were also determined as a control for the amount of input cDNA. B) Flowering time of plants grown under LD photoperiod. Data are means and standard deviation of at least 20 plants. Asterisks indicate significant differences between Col and the mutant backgrounds with p<0.00002 (*) or p<0.00001 (**). C) Analysis of FLC expression in wild-type, brm-1, fve-3 and fve-3 brm-1 mutant plants by RT-PCR. Total RNA was isolated as indicated in A. GAPC transcript levels were also determined as a control for the amount of input cDNA.

Figure 6. SEF and PIE1 are not required for expression of FLC in the absence of BRM.

A) Flowering time of plants grown under LD photoperiod. Data are means and standard deviations of at least 20 plants. B) Analysis of FLC expression in wild-type, brm-1, brm-2 sef-2, pie1-5, sef-2 brm-1 and pie1-5 brm-1 mutants by RT-PCR. Total RNA was isolated from seedlings collected 10 h after dawn at 12 days of growth under LD conditions. GAPC transcript levels were also determined as a control for the amount of input cDNA. Col data were significantly different to brm-1 and brm-2 data with p<0.01. Col data were significantly different to sef-2, sef-2 brm-1, pie1-5, pie1-5 brm-2 data with p<0.001. brm-1 and brm-2 data were different to sef-2, sef-2 brm-1, pie1-5, pie1-5 brm-2 data with p<0.01.

Figure 7. Model for the role of BRM in flowering regulation.

A) Under LD conditions in WT plants the photoperiod pathway overcomes the repression mediated by BRM upon CO, FT and SOC1 to promote flowering. In a brm mutant, the high levels of expression of CO, FT and SOC1 leads to an early flowering phenotype in spite of the increase in FLC expression. B) Under SD conditions in WT plants, the photoperiod pathway is not induced and flowering relies on the activation of SOC1 by the GAs pathway; however in brm plants, although FT and SOC1 are still slightly up-regulated in spite of the strong FLC expression, flowering is not induced indicating that there are other pathways (“?” in the scheme) that are also repressed by BRM.