Light-driven processes: key players of the functional biodiversity in microalgae

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Le système d expression du gène chloroplastique petA chez Chlamydomonas reinhardtii

Depuis l endosymbiose ayant mené au chloroplaste de l algue verte unicellulaire Chlamydomonas reinhardtii, la plupart des gènes de l endosymbiote primitif ont été transférés dans le génome nucléaire de l hôte, qui code donc des sous-unités des complexes de l appareil photosynthétique, mais aussi des facteurs régulant l expression des gènes restés dans le chloroplaste. Le gène chloroplastique petA, codant le cytochrome f, possède deux facteurs spécifiques : MCA1 est responsable de la stabilisation de l ARNm petA, et TCA1 de l activation de sa traduction. D autre part, la synthèse du cytochrome f est réprimée quand celui-ci ne peut s assembler au sein du complexe b6f, par le contrôle par épistasie de synthèse (ou CES). Le mécanisme moléculaire permettant l expression du gène petA et son contrôle par le CES a été largement dévoilé grâce aux travaux présentés dans ce manuscrit : les facteurs MCA1 et TCA1 forment différents complexes de haut poids moléculaires, pouvant contenir l ARNm petA, et le cytochrome f non assemblé signalise la dégradation de MCA1 par les protéases FtsH et ClpP.Par ailleurs, ces travaux ont fortuitement mené à la découverte d une mutation nucléaire dominante, su0, conduisant à la dégradation de l ARNm petA, mais seulement lors de sa traduction. La mutation causant ce phénotype intriguant, indépendante des facteurs MCA1 et TCA1 et du CES, a été localisée dans une région de 300 kb sur le chromosome 15, mais n a pas encore été identifiée.PARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

The Nucleus-Encoded trans-Acting Factor MCA1 Plays a Critical Role in the Regulation of Cytochrome f Synthesis in Chlamydomonas Chloroplasts[W]

This work shows that MCA1, required for the expression of cytochrome f, is degraded by proteolysis upon interaction with unassembled cytochrome f. MCA1 proteolysis appears to be critical for the assembly-dependent regulation of cytochrome f synthesis, known as Control by Epistasy of Synthesis, which tightly couples its expression to that of its assembly partners

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

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 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