BRAHMA ATPase of the SWI/SNF chromatin remodeling complex acts as a positive regulator of gibberellin-mediated responses in arabidopsis

Abstract

SWI/SNF chromatin remodeling complexes perform a pivotal function in the regulation of eukaryotic gene expression. Arabidopsis (Arabidopsis thaliana) mutants in major SWI/SNF subunits display embryo-lethal or dwarf phenotypes, indicating their critical role in molecular pathways controlling development and growth. As gibberellins (GA) are major positive regulators of plant growth, we wanted to establish whether there is a link between SWI/SNF and GA signaling in Arabidopsis. This study revealed that in brm-1 plants, depleted in SWI/SNF BRAHMA (BRM) ATPase, a number of GA-related phenotypic traits are GA-sensitive and that the loss of BRM results in markedly decreased level of endogenous bioactive GA. Transcriptional profiling of brm-1 and the GA biosynthesis mutant ga1-3, as well as the ga1-3/brm-1 double mutant demonstrated that BRM affects the expression of a large set of GA-responsive genes including genes responsible for GA biosynthesis and signaling. Furthermore, we found that BRM acts as an activator and directly associates with promoters of GA3ox1, a GA biosynthetic gene, and SCL3, implicated in positive regulation of the GA pathway. Many GA-responsive gene expression alterations in the brm-1 mutant are likely due to depleted levels of active GAs. However, the analysis of genetic interactions between BRM and the DELLA GA pathway repressors, revealed that BRM also acts on GA-responsive genes independently of its effect on GA level. Given the central position occupied by SWI/SNF complexes within regulatory networks controlling fundamental biological processes, the identification of diverse functional intersections of BRM with GA-dependent processes in this study suggests a role for SWI/SNF in facilitating crosstalk between GA-mediated regulation and other cellular pathways.

Figure 1. brm mutants show GA-related phenotypic traits and increased sensitivity to paclobutrazol.

(A), Comparison of brm-1 and ga1-3 mutants grown on ½ MS medium for 18 days under long-day conditions. (B), Germination of the brm-1 mutant is abolished in the presence of 10 µM PAC and rescued upon addition of exogenous gibberellin. The progeny of brm-1/BRM plants were analyzed 14 days after sowing. (C), Phenotype of brm-1 plants grown for 25 days on 10 µM PAC after incubation of seeds with exogenous GA. (D), Germination assay of wild type, brm-3 and 3xdella (rga/rgl1/rgl2) lines. Seed coat rupture after 14 days was scored as germination. (E), Root elongation assay of wild type and brm-3 plants grown for 12 days on PAC-containing medium. Bars in A, C and E = 5 mm.

Figure 2. GA responses of the brm-1 mutant.

(A, B), Elongation of brm-1 hypocotyls and roots in response to 1 µM GA₄. Plants were grown on ½ MS medium for 8 days under long-days conditions in the presence or absence of 1 µM GA₄. GA application caused considerable elongation of the hypocotyls, but had little effect on brm-1 root growth. Bar = 5 mm. (B), Hypocotyl length of plants grown as in A. Presented data are the means of 12 measurements ± s.d. (C), Flowering of brm-1 plants in response to exogenous gibberellins. Plants were grown in soil under short-day conditions and treated with 10 µM GA₃. At least 15 plants of each line/condition were scored. Data are the means ± s.d. Asterisks indicate significant differences from the wild type plants (p<0.01).

Figure 3. BRM directly regulates the expression of the GA3ox1 and SCL3 genes.

(A), RT-qPCR analysis of relative transcript levels of GA biosynthesis and signaling genes in 18-d-old wild type, brm-1 and brm-3 lines. The housekeeping genes PP2A and GAPC were used as normalization controls. RT-qPCR data are the means ± s.d. of 3 biological replicates. Transcript levels in the wild type were set to 1. Asterisks indicate significant differences from the wild type plants with p<0.05 (*) or p<0.01 (**). (B), Simplified model of the GA signaling pathway. (C), BRM recruitment to the promoters of GA3ox1 and SCL3 in wild type and brm-1 plants, analyzed by ChIP-qPCR. The signal obtained for the PP2A promoter region was used to normalize the qPCR results in each sample. Distal (d) and proximal (p) promoter sequences relative to the start codon of each gene were analyzed. Fold enrichment of each region in the wild type was calculated relative to the brm-1 sample. The value of ChIP enrichment in brm-1 was set to 1. Data are the means ± s.e. from 3 reactions in one ChIP experiment. Similar results were obtained in separate experiments.

Figure 4. ga1-3/brm-1 mutant phenotypes.

(A–B), Phenotypes of the ga1-3, brm-1 and ga1-3/brm-1 mutants grown on MS medium (18-d-old seedlings, A) or in soil (22-d-old plants, B). Bars = 10 mm. (C–F), Quantitative characterization of brm-1, ga1-3 and ga1-3/brm-1 mutants: root length of 18-d-old seedlings (C), rosette diameter at maturity (D) and flowering time under LD conditions (E). Data are the means ± s.d., 10 plants of each line were scored, except for ga1-3/brm-1 (7 plants). * All ga1-3/brm-1 plants except one failed to flower by the end of the experiment (80 days). (F), RT-qPCR analysis of relative transcript levels of GA3ox1 and SCL3 in 20-d-old wild type, brm-1, ga1-3, and ga1-3/brm-1 lines. RT-qPCR data are the means ± s.d. of 3 biological replicates. Transcript levels in the wild type were set to 1.

Figure 5. Transcriptional profile of brm-1 overlaps with that of ga1-3.

(A), Overlap between differentially regulated genes in mutants brm-1, ga1-3 and ga1-3/brm-1 identified in microarray data, shown by a Venn diagram. (B), Genes up- and down-regulated in all three mutants, shown by a heat map. The color scale represents normalized expression levels. (C), 94% of the genes commonly mis-expressed in all three mutants show expression changes in a similar direction. Green – genes mis-regulated only in brm-1; blue – genes mis-regulated in brm-1, ga1-3 and ga1-3/brm-1; orange – genes mis-regulated in a similar direction in all three mutants; gray – genes mis-regulated in an opposite direction in one of the mutants.

Figure 6. BRM acts through distinct mechanisms to regulate GA-mediated responses.

(A), Germination of the brm-1 mutant on 10 µM PAC is rescued by the triple della mutation. The progeny of brm-1/BRM plants were analyzed 10 days after sowing. (B), Phenotypes of 3-week-old plants grown on 2.5 µM PAC. The brm-1/3xdella line shows an intermediate growth phenotype. Bar = 5 mm. (C), RT-qPCR analysis of relative transcript levels of the OFP16, EXP5, CYS2 and LTP2 genes in 18-d-old wild type, brm-1, ga1-3, ga1-3/brm-1, ga1-3/3xdella and ga1-3/brm-1/3xdella lines. Transcript levels in the wild type were set to 1. Data are the means ± s.d. of 3 biological replicates. (D), Model of the role of BRM in regulating the expression of GA-responsive genes. BRM positively regulates the GA3ox1 and SCL3 genes involved in GA biosynthesis and signaling, and probably through this influences the expression of many GA-responsive genes in the opposite manner to DELLA repressors. In addition, BRM seems to act on a subset of GA-responsive genes independently of DELLA repressors. Also in this case, the effect exerted by BRM is typically in the opposite direction to that of DELLAs and is observed both for genes up- and down-regulated by the SWI/SNF complex (blue and red lines, respectively).