10 research outputs found
Genetic analysis of functional redundancy of BRM ATPase and ATSWI3C subunits of Arabidopsis SWI/SNF chromatin remodelling complexes
A specialized histone H1 variant is required for adaptive responses to complex abiotic stress and related DNA methylation in Arabidopsis
Linker (H1) histones play critical roles in chromatin compaction in higher eukaryotes. They are also the most variable of the histones, with numerous nonallelic variants cooccurring in the same cell. Plants contain a distinct subclass of minor H1 variants that are induced by drought and abscisic acid and have been implicated in mediating adaptive responses to stress. However, how these variants facilitate adaptation remains poorly understood. Here, we show that the single Arabidopsis (Arabidopsis thaliana) stress-inducible variant H1.3 occurs in plants in two separate and most likely autonomous pools: a constitutive guard cell-specific pool and a facultative environmentally controlled pool localized in other tissues. Physiological and transcriptomic analyses of h1.3 null mutants demonstrate that H1.3 is required for both proper stomatal functioning under normal growth conditions and adaptive developmental responses to combined light and water deficiency. Using fluorescence recovery after photobleaching analysis, we show that H1.3 has superfast chromatin dynamics, and in contrast to the main Arabidopsis H1 variants H1.1 and H1.2, it has no stable bound fraction. The results of global occupancy studies demonstrate that, while H1.3 has the same overall binding properties as the main H1 variants, including predominant heterochromatin localization, it differs from them in its preferences for chromatin regions with epigenetic signatures of active and repressed transcription. We also show that H1.3 is required for a substantial part of DNA methylation associated with environmental stress, suggesting that the likely mechanism underlying H1.3 function may be the facilitation of chromatin accessibility by direct competition with the main H1 variants
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
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 GA<sub>12</sub> and GA<sub>9</sub> measurements in <i>ga1-3</i>, for which 2 replicates were used. n.d. – not determined.</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
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
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 GA<sub>4</sub>. Plants were grown on ½ MS medium for 8 days under long-days conditions in the presence or absence of 1 µM GA<sub>4</sub>. 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 GA<sub>3</sub>. 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
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