Effect of salt stress on ABA and carotenoid accumulation.

<p>(A) ABA quantification by LC-MS of root and shoot tissues from plants either treated (+) or not (−) with 200 mMNaCl for 5 h. The indicated samples were incubated with 20 µM norflurazon (NFZ) 48 h before and during the salt treatment. (B) Levels of β,βxanthophylls (β,β-x: neoxanthin and violaxanthin), β,β carotenes (β,β-c: β-carotene), and β,ε xanthophylls (β,ε-x: lutein) in root and shoot tissues separated from plants either treated (+) or not (−) with NaCl for 5 h. Data correspond to the mean and standard deviation of n = 3 independent samples. Asterisks mark statistically significant differences (p<0.01) relative to mock-treated controls.</p

Analysis of <i>Arabidopsis PSY</i> promoter activity in roots.

<p>Representative images from transgenic <i>PSY:GUS-GFP</i> (A, C, D, G–J) and control <i>35S:GUS-GFP</i> (B, E, F) seedlings are shown. (A) GUS staining of <i>PSY:GUS-GFP</i> seedlings. (B) GUS staining of <i>35S:GUS-GFP</i> seedlings. (C) Bright field image of the region boxed in blue in A. (D) GFP fluorescence of the root shown in C. (E) Bright field image of the region boxed in blue in B. (F) GFP fluorescence of the root shown in E. (G) GUS staining of the upper region of the root shown in C. (H) Cross-section of the root region shown in G. (I) Magnification of the region boxed in G. (J) Close up of the stele area in a GUS-stained and resin-embedded section. ep, epidermis; c, cortex; s, stele; en, endodermis; pe, pericycle; p, phloem; pc, phloem companion cells; x, xylem.</p

J20 and ClpC1 are required for normal DXS degradation.

<p>(A) WT plants and mutants defective in ClpR1 or ClpC1 were grown for one week on medium lacking the protein synthesis inhibitor cycloheximide and then treated with the inhibitor for the indicated times. DXS protein levels detected by immunoblot analysis are represented relative to those before treatment. (B) Protein extracts from <i>Nicotiana benthamiana</i> plants transiently producing DXS-GFP alone or together with a MYC-tagged ClpC1 protein were used for immunoprecipitation (IP) with anti-MYC antibodies (αMYC) and further immunoblot (IB) analysis with anti-GFP or anti-MYC sera. Immunoblot analyses of the extracts before immunoprecipitation (Input samples) are also shown. (C) Protein degradation rates of DXS in WT plants and mutants defective in J20 or ClpB3. The experiment was performed as described in (A). Mean and SEM of n≥3 experiments are shown in (A) and (C). Asterisks mark statistically significant differences (<i>t</i> test: p<0.05) relative to WT samples.</p

DXS is primarily degraded by the Clp protease.

<p>Columns represent DXS protein and transcript levels in 10-day-old WT plants and single mutants defective in the indicated subunits of plastidial proteases (A) or regulatory components of the Clp protease complex (B). Data correspond to the mean and SEM values of n≥3 independent experiments and are represented relative to the levels in WT plants. Asterisks mark statistically significant differences (<i>t</i> test: p<0.05) relative to WT samples. Representative images of immunoblot analyses with the indicated antibodies and a Coomassie blue staining of the blots (loading control, LC) are also shown in (B).</p

Photopigment gene expression response to an End of Day (EOD)-Far Red (FR) light treatment in Col-0.

<p>Expression levels for <i>LHCA4</i>, <i>PORC</i> and <i>VDE</i> in Col-0 plants treated without (−FR) or with (+FR) a saturating FR pulse (3000 µmol) at the end of the day (EOD) and collected at T15. Samples were grown as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004416#pgen-1004416-g006" target="_blank">Figure 6</a>, and used for gene expression measurements by qPCR. Levels are expressed relative to Col-0 (-FR) T15 sample and normalized against <i>ACT7</i> expression. Error bars represent ± SE of biological triplicates.</p

Chromatin immunoprecitation assays for 35S::HA-HY5, 35S::TAP-PIF1 and 35S::PIF4-HA grown in red-diurnals at 17°C and 27°C.

<p>Two week old seedlings grown for one week in white diurnal cycles (12 h light/12 h dark, 80 µmol m⁻² s⁻¹) at 22°C, followed by one week under Red diurnal cycles (12 h light/12 h dark, 40 µmol m⁻² s⁻¹) were used for the experiment. On the last day, samples were taken 2 or 3 h after the lights came on (T2/T3) and 8 h after the lights were off (T20). ChIP was carried out using antibodies against the tag (anti-HA or anti-MYC). 35S::GUS-HA (labelled HA) or a 35S::GFP-TAP (TAP) lines were used as controls. A non-antibody control sample was processed in parallel in each case (white bars). Immunoprecipitated DNA was analysed by qPCR using specific primers covering the G-box containing region in the promoters for the indicated genes (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004416#pgen.1004416.s010" target="_blank">Table S1</a> for primer information). The assay was carried out in triplicates. Error Bars represent ±SE. (<b>A</b>) ChIP results for <i>PSY</i>. (<b>B</b>) ChIP results for <i>LHCA4</i>.</p