Phenotypic analysis of the <i>zwg</i> mutant.

<p>(A) Comparison of adult wild type Sav-0 plant (left) and <i>zwg</i> mutant (right). (B–D) Comparison of wild type (B) and <i>zwg</i> (C) flowers from different perspectives, note increased organ number and shorter organs in <i>zwg</i>. (E–G) Comparison of wild type and <i>zwg</i> siliques. (H) Comparison of flowering time measured as age or rosette leaf number. (I) Comparison of primary root length in tissue culture at 9 days after germination (dag). (J) Comparison of cell number per cell diameter measured at, above or below the 1^st internode. (K–M) Comparison of transverse stem sections taken below the first internode. (N–O) Gene ontology (GO) classification of genes down-regulated (N) or up-regulated (O) in <i>zwg</i> as compared to wild type, expressed as normalized frequency. Size bars are 1 cm (A), 1 mm (B–G) and 100 µm (K–M).</p

Analysis of random-primed RNA sequencing of wild-type and <i>vip3^zwg</i> mRNAs.

<p>(A) Distribution of the 5′ to 3′ coverage index (i.e. number of reads mapping into the 20% 5′-most region divided by number of reads mapping into the 20% 3′-most region of a transcript) in wild type and <i>vip3^zwg</i>. The box plots indicate maximum, minimum, median and quartile values, as well as the average (wider horizontal bar). Out of range maximum values were added numerically. (B) Relative read abundance in 1% bins cumulated for the 10% of most highly expressed transcripts (n = 2’437). (C) Distribution of the 5′ to 3′ coverage index (box plots, left y axis) and the ratios of median and average between wild type and <i>vip3^zwg</i> (diamonds, right y axis) in nuclear (n = 5’617), chloroplast (n = 34) and prime exosome target (n = 68) genes. Maximum values were added numerically. (D) Distribution of the averages and medians of the 5′ to 3′ coverage index and their wild type to <i>vip3^zwg</i> ratio in random sets drawn from the nuclear or prime exosome target sets. (E) Wild type to <i>vip3^zwg</i> ratio of the 5′ to 3′ end ratios for selected genes as determined by qPCR analyses of three independent RNA samples for each genotype. A value >1 indicates stabilized transcript in <i>vip3^zwg</i>. The respective 5′ to 3′ coverage ratios from the RNA sequencing are given in brackets above. (F) Relative expression levels of <i>FLC</i> in wild type, <i>vip3^zwg</i> and <i>Atski2</i>. n.s.: not significant; *: p<0.05; **: p<0.01; ***: p<0.001.</p

Isolation and characterization of the <i>zwg</i> locus.

<p>(A) Representation of the final mapping interval for <i>zwg</i>, including position of markers and the <i>VIP3</i> polymorphism. (B) Illustration of the 7 bp deletion in the <i>vip3^zwg</i> coding sequence. (C) Conceptual translation of the <i>VIP3</i> wild type and <i>zwg</i> C-terminus. (D) Immunoblot analysis of transgenic GFP-VIP3 or GFP control lines, probed with anti-GFP antibody. Different independent lines are shown, GFP-VIP3 migrates below 75 kDa marker as expected. (E–G) Subcellular localization of GFP-VIP3 fusion protein in nucleus and cytoplasm of differentiated (E) or meristematic (F) root cells, and epidermal leaf cells (G). (H) Gel filtration analysis of GFP-VIP3, fractions pooled for subsequent co-immunoprecipitation of GFP-VIP3 are indicated by bars. (I) VIP3 and AtSKI3 (At1g76630) peptides identified in MALDI-TOF of co-immunoprecipitates obtained from the smallest set of fractions (H). (J) Number of peptides from Paf1c and SKI complex components identified in MALDI-TOF of co-immunoprecipitates obtained from total protein extract of GFP-VIP3 plants. Size bars are 10 µm (E–G).</p

Phenotypes of the <i>Atski2</i> mutant and <i>vip3^zwg</i> rescued by transgenic ScSki8 expression.

<p>(A) A phylogenetic tree of SKI2 homologs from various eukaryotic species including AtSKI2. (B) Comparison of adult wild type Col-0 plants (right) and <i>Atski2</i> mutants (left). (C) Close-up of an <i>Atski2</i> mutant. (D–E) Comparison between wild type, <i>Atski2</i> and <i>vip3^zwg</i> mutant flowers at anthesis. (F) Semi-quantitative RT-PCR (increasing number of amplification cycles) of transgenic ScSki8 expression in <i>vip3^zwg</i> plants as compared to the elongation factor 1 gene (<i>EF1</i>). (G–H) Illustration of the rescue of dwarf (G) and flower development (H) phenotypes by transgenic expression of ScSki8 in <i>vip3^zwg</i> mutants. Size bars are 1 cm (B, C, G) and 1 mm (D, E, H).</p

Genomic profiles of Pol II, H3K4me3, and H3K36me3 densities around transcription start sites (TSSs) and polyadenylation sites (PASs) at ZT2.

<p>(A) Average signals over 11,217 genes with nonoverlapping TSSs (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001442#s4" target="_blank">Materials and Methods</a>) for each mark around the TSSs: Pol II (red), H3K4me3 (green), H3K36me3 (blue), and input chromatin (gray). (B) As in (A) but around the PAS. (C) Average Pol II signals over transcripts split by quartile, based on the level of expression as measured by microarrays. Each quartile is represented by a distinct color shading from light (lowest quartile in mRNA expression) to dark (upper quartile in mRNA expression). (D) As in (C) but for the PAS. (E–F) Profiles of input chromatin. Note that the depletion at the PAS only partially explains the dips in panels (B) and (D). Vertical axes have arbitrary units, but the scales on the left and right panels can be compared for the same marks.</p

Amplitude and phase relationships between Pol II signals and mRNA accumulation identify posttranscriptional regulation in mRNA accumulation.

<p>(A) Relative amplitudes (maximum minus minimum, divided by twice the mean after background subtraction; see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001442#s4" target="_blank">Materials and Methods</a>) of oscillations in Pol II promoter signals and mRNA accumulation identify rhythmic mRNAs with relative amplitudes comparable to that of transcription (class 1, gray, 675 genes), long-lived transcripts with damped mRNA rhythms (class 2, orange, 668 genes), and mRNAs where posttranscriptional regulation increases rhythmic amplitude (class 3, red, 217 genes). Light gray genes are all genes that cycle robustly in either Pol II or mRNA accumulation (3,446 genes). <i>Fus</i>, <i>Tfrc</i>, and <i>Spon2</i> are representative of class 3 and <i>Rdbp</i> of class 2 (see panels F–I for qRT-PCR validations). The few values larger than 1 are due to low signals when background subtraction makes trough values negative. (B) Half-lives of the three classes taken from NIH-3T3 fibroblasts <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001442#pbio.1001442-Dolken1" target="_blank">[46]</a> show a significant difference (TukeyHSD <i>p</i> value <10⁻⁶ for class 2 versus class 1, and class 2 versus class 3). (C) Delays in peak mRNA accumulation versus peak Pol II promoter loading for the union of class 1, class 2, and class 3 genes. The dark gray region delimits the range predicted for a model with constant half-lives (0 h delay for very short-lived up to 6 h for very long-lived transcripts). (D–E) For the same genes the ratio of relative amplitudes (<i>B</i> = relative amplitude of Pol II, <i>b</i> = relative amplitude of mRNA) is plotted against the phase delay, together with the prediction for a constant half-life (red line). The trend (in D, median is the blue line and 25% and 75% percentiles are shown as light blue shading) shows that the ratio is centered on one at short delays and decreases for larger delays. The scatter plot (E) highlights genes for which transcript accumulation is explained by a constant half-life (dark gray area represents short and light gray long half-lives), and genes where nonconstant half-lives either suppress (light orange) or enhance (light red) amplitudes in mRNA accumulation. Triangles show core circadian clock genes. (F–I) Transcription and mRNA accumulation for representative genes. Comparison of (i) mRNA levels as measured by gene expression arrays, (ii) promoter Pol II occupancy as measured by ChIP-Seq, and (iii) pre-mRNA and (iv) mRNA levels as measured by qRT-PCR with intronic and exonic probes, respectively. Symbols and lines indicate measurements and cosine fits, respectively. Open symbols and dashed lines show qRT-PCR data (cDNA from <i>n</i> = 4 animals where pooled) with circles for the pre-mRNA and triangles for the mRNA. Continuous lines and filled symbols represent Pol II ChIP-seq (circles) and mRNA Affymetrix data (triangles). Each temporal profile has been scaled to an arbitrary mean for visualization. Pre-mRNA levels closely follow Pol II promoter occupancy, as expected (given the short half-lives of pre-mRNAs). <i>Fus</i> and <i>Spon2</i> (F and H) show higher amplitude in mRNA compared to transcription; <i>Tfrc</i> (G) is transcribed at similar rates around the clock but shows rhythmic mRNA accumulation; <i>Rdbp</i> (I) shows rhythmic transcription but dampened mRNA accumulation.</p

H3K4me3 and H3K36me3 marks vary during the diurnal cycle with reduced amplitude as compared to Pol II occupancy.

<p>(A) H3K4me3 promoter levels versus Pol II promoter occupancy at ZT2. Two populations can be identified from the densities: silent (or weakly active) promoters (lower left cloud) and active promoters (fainter cloud shifted above the diagonal and to the right). Bimodality in both signals is clearly seen in the projections (histograms). The cross sign, also shown in panels D and E, indicates background levels estimated from the lower maxima of the histograms. (B) H3K36me3 levels (quantified over the most 3′-proximal 40% of gene bodies) versus Pol II body occupancy at ZT2. Two populations can be identified from the densities: silent (or weakly transcribed) genes (lower left cloud) and transcribed genes. (C) H3K36me3 levels as in (B) versus H3K4me3 promoter levels at ZT2. This comparison shows the two classes most clearly, indicating that the large majority of genes harboring H3K4me3 marks are transcribed. In (A–C), data are shown for ZT2, but all time points looked identical. (D–E) Temporal profiles of H3K4me3 and H3K36me3 marks, and promoter Pol II occupancy for some core clock genes (D) and selected output genes (E). Left, temporal profile for promoter Pol II occupancy (red), H3K4me3 marks (green), and H3K36me3 marks (blue) together with cosine fits. Right, the cosine fits for Pol II promoter occupancy and H3K4me3 plotted against each other in the coordinates of panel A. ZT times are color-coded (see color bar). Crosses indicate background levels. Note that levels of H3K4me3 remain relatively high at the troughs of transcription (as measured by Pol II density). (F) Genome-wide temporal analysis showing that H3K4me3 modifications at promoters show compressed amplitudes compared to Pol II promoter occupancy (compare with <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001442#pbio-1001442-g003" target="_blank">Figure 3E</a>). Each line shows the average orientation and amplitude of changes during a diurnal cycle for genes in regions of a grid. The nonbinned plot is shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001442#pbio.1001442.s005" target="_blank">Figure S5</a>.</p

Temporal relationships of Pol II, H3K4me3, H3K36me3 profiles, and mRNA accumulation in mouse liver.

<p>(A) Phase histograms for cyclic genes. A selection of 284 genes (<i>p</i><0.004, FDR = 0.3) showing cyclic patterns in all marks (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001442#s4" target="_blank">Materials and Methods</a>) were fitted with a cosine function and the phase (peak time of the fit) was computed. These phases show a bimodal distribution for Pol II occupancy in promoters and gene bodies with maxima around ZT9 and ZT21, as well as in mRNA accumulation with a phase delay of approximately 3 h. (B) Phases for the same genes are represented in pairs, with color shade indicating <i>p</i> value (lower <i>p</i> values are darker) for the 24-h rhythm of the Pol II promoter signal. Relative to the phase of Pol II in promoters, we find high concordance for Pol II occupancy phases in gene bodies, an average delay of 1.3 h for H3K4me3 phases, and more spread H3K36me3 and mRNA phases with an average delay of about 3 h. Colored lines are mean-square regressions with intercepts corresponding to the average delays, as indicated in color. The thin dashed lines indicate ±2 h delays. (C) Temporal cross-correlation analysis. Using the same gene selection, we applied Fourier interpolation to obtain a continuous time trace (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001442#pbio-1001442-g003" target="_blank">Figure 3C,D</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001442#pbio-1001442-g004" target="_blank">Figure 4D,E</a>) and computed average cross-correlations between each mark and the corresponding Pol II promoter trace. Pol II occupancies in promoters and gene bodies are well-correlated and simultaneous, and H3K4me3 lags by about 1 h on average, whereas mRNA and H3K36me3 are phase-delayed by about 3 h. The same figure is shown for a more permissive selection (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001442#pbio.1001442.s008" target="_blank">Figure S8</a>, <i>n</i> = 752, <i>p</i><0.018, FDR = 0.5).</p

Overview of denitrification capacity of <i>P</i>. <i>veronii</i> 1YdBTEX2.

<p>(A) Overnight growth of <i>P</i>. <i>veronii</i> 1YdBTEX2 wild type (WT) and the Δ<i>nar</i> mutant in presence (+O₂, left) or absence of air but with 15 mM nitrate supplemented medium (+NO₃,–O₂, right panel) conditions. Note the gas formation in the right panel of the WT incubation. (B) Gene regions predicted for denitrification in the <i>P</i>. <i>veronii</i> 1YdBTEX2 chromosome 1 with trivial gene names indicated. Black bar represents the deleted region in <i>P</i>. <i>veronii</i> Δ<i>nar</i>.</p

Circular maps of the replicons encompassing the <i>P</i>. <i>veronii</i> 1YdBTEX2 genome.

<p>(A) Chromosome 1 (chr1) with indication of possible genomic islands (GEI) and prophages (pf). The outermost circles show the location and orientation of predicted coding regions (blue and cyan), followed by tRNA (olive green) and rRNA genes (black), predicted regions of genome plasticity (blue-green-brown) islands and prophages (grey). The inner circles represent BLASTN comparisons with the close relatives <i>P</i>. <i>fluorescens</i> SBW25 (red, Acc. No. AM181176.4), <i>P</i>. <i>trivialis</i> strain IHBB745 (deep pink, CP011507.1), <i>P</i>. <i>syringae</i> pv. syringae B728a (dark purple, CP000075.1), <i>P</i>. <i>putida</i> KT2440 (light purple, AE015451.1) and <i>P</i>. <i>knackmussii</i> B13 (persian green, HG322950). GC skew (dark magenta and yellow green) is shown in the most central circle. (B) As A, but for the chromosome 2 replicon (chr2). Inner circles, from outwards to inwards, predicted transposons (dark purple) and <i>tra</i> genes (green), regions of genome plasticity (blue-green-brown) and prophages (grey), followed by BLASTN comparisons to <i>P</i>. <i>fluorescens</i> SBW25 plasmid pQB103 (red, AM235768.1, NC_009444.1), <i>Pseudomonas stutzeri</i> strain 19SMN4 plasmid pLIB119 (deep pink, CP007510.1), <i>Pseudomonas mandelii</i> JR-1 plasmid (dark purple, CP005961.1) and <i>Pseudomonas resinovorans</i> NBRC 106553 plasmid pCAR1.3 (Persian green, AP013069.1). (C) As B, but for the plasmid replicon. The inner circles represent the BLASTN comparisons with <i>P</i>. <i>putida</i> S12 plasmid pTTS12 (red, CP009975.1), and <i>Pseudomonas</i> sp. VLB120 plasmid pSTY (purple, CP003962.1). Plots generated with DNAPlotter [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165850#pone.0165850.ref046" target="_blank">46</a>].</p