Overexpression lines for rate-limiting enzymes in various phytohormone pathways.

<p>Transgenic lines harboring <i>35S::DWF4</i>, <i>35S::GA5</i> and <i>35S::NCED3</i> constructs. <b><i>A.</i></b> Over-expression was confirmed by RT-PCR with primers specific for <i>DWF4</i>, <i>GA5</i> and <i>NCED3</i>. Primers specific for the elongation factor 1-alpha gene were used as a control. Representative lines are shown. All lines tested showed over-expression of the gene of interest >3 fold. <b><i>B.</i></b> Images of 3-weeks-old plants grown under long days (16 h light/8 h darkness) in the greenhouse. The white bar indicates 1 cm.</p

Student's t-test for flowering-time differences between mutant genotypes.

<p>Listed are pairs of compared genotypes. P values for each pair are provided.</p><p>ø No significant difference P>0.05;</p><p>statistically significant differences:</p><p>***P<0.0001,</p><p>**P<0.001,</p><p>*P<0.05.</p

Simplified hormone biosynthetic pathways.

<p>The hormone biosynthetic pathways of Arabidopsis for gibberellins <b><i>A.</i></b>, ABA <b><i>B.</i></b>, and, brassinolide <b><i>C.</i></b>. The biosynthesis mutants used in this study and sites of their lesions are shown. Also, the biosynthetic genes over-expressed to increase the levels of respective hormones are indicated. <b><i>A.</i></b> The <i>ga1</i> mutant is impaired in the first stage of GA-biosynthesis: the cyclization of geranylgeranyl diphosphate (GGPP) to copalyl diphosphate (CPP). <b><i>B.</i></b> The <i>aba2</i> mutant is blocked at the cis-xanthoxin to ABA-aldehyde conversion. <b><i>C.</i></b> The conversion of 6-Deoxocathasterone/Cathasterone to 6- Deoxoteasterone/teasterone does not occur in the <i>cpd</i> mutant. <b><i>A.</i></b> The <i>GA5</i> gene encodes a GA 20-oxidase that catalyzes the formation of the GA20 and GA9, the final precursors of the bioactive GAs. <b><i>B.</i></b> The <i>NCED3</i> encodes 9-<i>cis</i>-epoxycarotenoid dioxygenase that catalyzes the oxidative cleavage of a 9-<i>cis</i> isomer of epoxycarotenoid (9-<i>cis</i>-violaxanthin or 9’-<i>cis</i>-neoxanthin) to form xanthoxin. <b><i>C.</i></b> The <i>DWF4</i> gene encodes a 22-a hydroxylase (CYP90B1) that catalyzes the conversion of 6- Oxocampestanol/Campestanol to 6-Deoxocathasterone/Cathasterone. IPP, Isopentenyl pyrophosphate. ABA, abscisic acid. Adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014012#pone.0014012-Srivastava1" target="_blank">[49]</a>.</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

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

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

<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

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>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