Diurnal transcriptomics on bioreactor cultures of <i>Chlamydomonas</i>.

<p>The effect of 12 h light/12 h dark cycles on organelle (and selected nuclear) transcripts was analyzed by qRT-PCR. <i>C. reinhardtii</i> cells were grown in six independent bioreactor runs (run numbers 1, 2, 3, 15, 16, 20) and harvested at the time points indicated. Data obtained for plastome transcripts is shown in the upper portion of the heatmap, chondriome transcripts in the central region, and nuclear transcripts in the lower region. Within each genome, transcripts are listed alphabetically. Data are normalized to housekeeping transcripts and then to the average across all samples for that gene. Red boxes indicate up-regulation, green boxes: down-regulation, grey boxes: no data. Visualization by Multiexperiment Viewer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108760#pone.0108760-Saeed1" target="_blank">[38]</a>.</p

Confirmation of the diurnal expression patterns observed in the pRT-PCR experiments by northern blot analyses.

<p>To avoid variation from RNA loading, each membrane was hybridized and stripped multiple times. The order of probing corresponds to the vertical order of the images (and the increasing signal intensity known to be obtained with the different probes). The ethidium bromide fluorescence (EtBr) remaining on the membrane after blotting is shown as a loading control.</p

Comparison of microarray and qRT-PCR-based quantification of transcripts in a diurnal rhythm.

<p>Changes in organelle (chloroplast and mitochondrial) transcript levels between time points L2 and D2 in a wild-type strain of <i>C. reinhardtii</i> (CC-124) were determined by microarray and by qRT-PCR. The resulting fold-changes correlate with an R² value of 0.66. It is lower than 1.0, mainly because the microarray produces less accurate results for lowly expressed genes. All data points represent the average of three independent experiments.</p

k-means clustering of diurnal qRT-PCR data for chloroplast transcripts (left) and a subset of nuclear transcripts (right).

<p>The averaged data for six biological replicates were grouped into three clusters with the Pearson correlation as the distance metric. Gene profiles are colored according to the gene product’s function. Green: photosynthesis-related genes, blue: ribosome-related genes, red: plastid-encoded RNA polymerase genes, grey: miscellaneous genes. Clustering and visualization by Multiexperiment Viewer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108760#pone.0108760-Saeed1" target="_blank">[38]</a>.</p

Behavior of functionally related chloroplast transcripts in diurnal conditions.

<p>The average peak time of all transcripts belonging to the three functional classes (red: plastid-encoded RNA polymerase genes, PEP; blue: ribosome-related genes; green: photosynthesis-related genes) is shown for each bioreactor experiment. The intensity of the circle represents the number of transcripts peaking at the time, and the black x represents the average of all transcripts. The yellow bar represents the 12 h light period, flanked by dark periods (grey bars).</p

Key bioreactor parameters from a representative bioreactor run (R2) in diurnal conditions.

<p>Turbidity (navy blue) slightly decreased during the dark period and increased during the light. The rapid peak after dusk and rapid drop after dawn are technical artefacts of the turbidimeter. The peaks in the weight graph correspond to the sampling times, when the fermenter was disturbed. Dissolved oxygen concentration increases from ca. 80% to ca. 150% very rapidly in the light. The pH of the culture is controlled by titration of acid or base, temperature is controlled by dynamic cooling to maintain reasonably stable values for these parameters. Samples were taken every 4 h, these points are indicated and also visible as peaks in the weight data (purple). Yellow bars represents the 12 h light period (L), flanked by dark periods (D; grey bars).</p

Comparison of enrichment of regulatory elements at the <i>Cah1</i> locus.

<p>Quantitative PCR analysis of FAIRE fragments was performed for the <i>Cah1</i> locus, with primers designed for covering coding and non-coding regions, represented by the amplicon identifier (Table 1). Enrichment of FAIRE fragments within the <i>Cah1</i> locus was analyzed by comparing the chromatin from cells cultured under HC and LC condition. Enrichment observed for the 5´ upstream and 3´ downstream regions of the gene locus is more pronounced in cells cultivated at LC, indicating that these regions may have regulatory functions. </p

Quantitative PCR of FAIRE fragments derived from the <i>Cah1</i> locus.

<p>(A) Quantitative PCR analysis was performed for ten DNA segments of the <i>Cah1</i> locus and enrichment of each fragment was measured. Red areas show the location of the DNA segments amplified and quantified. Exons are indicated by blue boxes and introns by dashed lines. (B) DNA segments located in the promoter region (amplicons B and C) and in a region located in the last exon of the <i>Cah1</i> gene, representing the 3´ UTR (amplicons I and J), were found to be enriched. De-crosslinked chromatin was used as control for calculating the enrichment of genomic fragments in the FAIRE samples. The enrichment was determined as 2^-ΔΔCt (cf. Material and Methods) and error bars indicate the standard deviation (SD) from three biological replicates. The reference region H is indicated by a star (*) in (A) and refers to the region where the C_t value ratio between FAIRE and control samples was the lowest. The C_t values of the reference region were used for the calculation of the FAIRE enrichment as a normalization parameter. The position of regulatory elements of the LC response previously identified by Kucho et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079909#B24" target="_blank">24</a>] is indicated. </p

Cumulative histogram of the approximate distances of FAIRE summits from their next closest annotated gene.

<p>Genomic coordinates were used to calculate the distances between FAIRE summits and the start and stop codons of their nearest gene transcripts. The number of transcripts with at least one FAIRE peak assigned was counted and the distances to the start or stop codons computed. The gray line indicates the cumulative percentage of the transcripts to which FAIRE peaks were assigned. As seen, approximately 80% of the FAIRE summits were located within 1.5 kb up- or downstream of the transcript´s start or stop codon. FAIRE peaks were detected using MACS tool v. 1.4.0beta, with a <i>p</i>-value cutoff equal to 10^-05.</p

Web FAIRE-seq GenomeBrowser.

<p>FAIRE-seq reads mapped to the <i>Chlamydomonas</i> genome annotation v.4 were made accessible through a web GenomeBrowser (<a href="http://bce.uniandes.edu.co/gb2/gbrowse/chlamydomonas_v4/" target="_blank">http://bce.uniandes.edu.co/gb2/gbrowse/Chlamydomonas_v4/</a>). (A) <i>Cah1</i> locus. The annotated <i>Cah1</i> transcript is included; the yellow horizontal boxes indicate annotated exons and the connecting black lines indicate introns. Positions of FAIRE peaks are indicated as green horizontal bars, FAIRE summits as vertical red bars. Sequence coverage along the <i>Cah1</i> locus is indicated by the gray vertical bars; the darkness of the gray bars is proportional to the abundance of the sequencing reads mapped to this particular location. (B) Broader view of the genomic location where <i>Cah1</i> is annotated. Other transcripts in the proximity of the <i>Cah1</i> locus are shown, together with their corresponding features.</p