GA responses of the <i>brm-1</i> mutant.

<p>(A, B), Elongation of <i>brm-1</i> 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 <i>brm-1</i> 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 <i>brm-1</i> 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).</p

<i>brm</i> mutants show GA-related phenotypic traits and increased sensitivity to paclobutrazol.

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

Concentration of gibberellins in wild type and mutant lines.

<p>The values are ng/g dry weight (s.e.). They are the means of three biological replicates except for the GA₁₂ and GA₉ measurements in <i>ga1-3</i>, for which 2 replicates were used. n.d. – not determined.</p

Gene Ontology (GO) categories statistically over-represented among genes differentially expressed in both <i>ga1-3</i> and <i>brm-1</i> mutants.

<p>Gene Ontology (GO) categories statistically over-represented among genes differentially expressed in both <i>ga1-3</i> and <i>brm-1</i> mutants.</p

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

<p>(A), Germination of the <i>brm-1</i> mutant on 10 µM PAC is rescued by the <i>triple della</i> mutation. The progeny of <i>brm-1/BRM</i> plants were analyzed 10 days after sowing. (B), Phenotypes of 3-week-old plants grown on 2.5 µM PAC. The <i>brm-1/3xdella</i> line shows an intermediate growth phenotype. Bar = 5 mm. (C), RT-qPCR analysis of relative transcript levels of the <i>OFP16, EXP5, CYS2</i> and <i>LTP2</i> genes in 18-d-old wild type, <i>brm-1</i>, <i>ga1-3</i>, <i>ga1-3/brm-1</i>, <i>ga1-3/3xdella</i> and <i>ga1-3/brm-1/3xdella</i> 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 <i>GA3ox1</i> and <i>SCL3</i> 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).</p

<i>ga1-3/brm-1</i> mutant phenotypes.

<p>(A–B), Phenotypes of the <i>ga1-3</i>, <i>brm-1</i> and <i>ga1-3/brm-1</i> 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 <i>brm-1</i>, <i>ga1-3</i> and <i>ga1-3/brm-1</i> 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 <i>ga1-3/brm-1</i> (7 plants). * All <i>ga1-3/brm-1</i> plants except one failed to flower by the end of the experiment (80 days). (F), RT-qPCR analysis of relative transcript levels of <i>GA3ox1</i> and <i>SCL3</i> in 20-d-old wild type, <i>brm-1</i>, <i>ga1-3</i>, and <i>ga1-3/brm-1</i> lines. RT-qPCR data are the means ± s.d. of 3 biological replicates. Transcript levels in the wild type were set to 1.</p

BRM directly regulates the expression of the <i>GA3ox1</i> and <i>SCL3</i> genes.

<p>(A), RT-qPCR analysis of relative transcript levels of GA biosynthesis and signaling genes in 18-d-old wild type, <i>brm-1</i> and <i>brm-3</i> lines. The housekeeping genes <i>PP2A</i> and <i>GAPC</i> 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 <i>GA3ox1</i> and <i>SCL3</i> in wild type and <i>brm-1</i> plants, analyzed by ChIP-qPCR. The signal obtained for the <i>PP2A</i> 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 <i>brm-1</i> sample. The value of ChIP enrichment in <i>brm-1</i> was set to 1. Data are the means ± s.e. from 3 reactions in one ChIP experiment. Similar results were obtained in separate experiments.</p