Respiratory enzyme activities of T11-10, T11-10/2, and T22-11 transformants.

<p>Respiratory activities were measured on membrane fractions of T11-10, T11-10/2 and T22-11 mutants. NADH:Duroquinone corresponds to the rotenone-sensitive NADH:duroquinone oxidoreductase activity (nmol of NADH oxidized min⁻¹ mg of proteins⁻¹); NADH:Ferricyanide corresponds to the NADH:Fe(CN)₆³⁻ oxidoreductase activity (nmol of K3Fe(CN)₆³⁻ reduced min⁻¹ mg of proteins⁻¹); CII+III corresponds to the succinate:cytochrome <i>c</i> oxidoreductase activity (nmol cytochrome <i>c</i> reduced min⁻¹ mg of proteins⁻¹); CIV corresponds to the cytochrome <i>c</i> oxidase activity (nmol of cytochrome <i>c</i> oxidized min⁻¹ mg of proteins⁻¹). Asterisks indicate statistically significantly differences using Student <i>t</i> test with a significance threshold of 0.05. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002946#s2" target="_blank">Results</a> are means of 3 to 6 independent experiments.</p

Total respiration and doubling time in T11-10, T11-10/2, and T22-11 transformants.

<p>Dark whole-cell respiratory rates are expressed in nmol of O₂ min⁻¹ 10⁻⁷ cells ± SD (mean of 3 independent experiments). Doubling times were measured in heterotrophic conditions (D) and mixotrophic conditions (L) and are expressed in hours ± SD (mean of 3 independent experiments). Asterisks indicate statistically significantly differences using Student <i>t</i> test with a significance threshold of 0.05.</p

Molecular characterization of the transformants.

<p>(A) Schematic map of the <i>Chlamydomonas reinhardtii</i> mitochondrial genome. Boxes represent protein-coding genes: (<i>cob</i>) apocytochrome <i>b</i> of complex III; (<i>nd1</i>, <i>2</i>, <i>4</i>, <i>5</i> and <i>6</i>) subunits of complex I; (<i>cox1</i>) subunit 1 of complex IV; (<i>rtl</i>) reverse transcriptase-like protein. W, Q, and M represent tRNAs for Trp, Glu, and Met, respectively. The bidirectional origin of transcription between <i>nd5</i> and <i>cox1</i> genes is represented by a dashed vertical line and two horizontal arrows. Terminal inverted repeats are shown by short arrows and <i>Sac</i>I digestion site at position 5.5 kb (GenBank u03843 numbering) is indicated. Region where modifications on the <i>nd4</i> gene were found on T11-10, T11-10/2 and T22-11 transformants is indicated in grey. Position and name of primers are indicated above the map. Primers with a star are specific for the modified gene version (for primer sequence see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002946#pgen.1002946.s004" target="_blank">Table S1</a>). Positions of the <i>dum11</i> and <i>dum22</i> deletions are shown. Mitochondrial DNA fragments contained in pND4-LP, pCucob and pCumut are schematized. Grey boxes represent the modified genes where GGC/GGT codons were changed in GGG codons. (B) Detection of the <i>cob</i> gene in transformants obtained after biolistic transformation with pND4-LP (T-ND4-LP) and pCucob (T-cucob) constructs. PCR analyses were performed with cobF/cobR (1) and telF/cobR (2) pair primers. (C) Detection of the mutated and the wild-type <i>nd4</i> genes on T11-10, T11-10/2 and T22-11 transformants. PCR analyses were performed with 4F2*/4R2 and 4F2/4R2 pair primers for the modified <i>nd4</i> gene (<i>nd4*</i>) and for wild-type <i>nd4</i> gene (<i>nd4</i>) respectively. (D–E) Reconstitution of complete mitochondrial genome in T11-10, T11-10/2 and T22-11 transformants. Southern blot analyses were performed (D) on total DNA with the <i>nd6</i> PCR probe and (E) on <i>Sac</i>I digested DNA with <i>nd4</i> and <i>nd6</i> PCR probes. (F) Transcript levels of <i>nd4</i> and <i>nd6</i> genes in T11-10, T11-10/2 and T22-11 transformants. Northern blot analyses were performed on total RNA with <i>nd4</i> and <i>nd6</i> PCR probes. Loadings of rRNAs are shown.</p

Analysis of the import status of mitochondrial tRNAs in T22-11 transformant.

<p>(A) Northern blot analysis of mitochondrial tRNAs extracted from the T22-WT strain and T22-11 transformant. Hybridizations were performed with radiolabeled oligonucleotides specific for cytosolic tRNA^Gly(GCC) (G1), tRNA^Gly(UCC) (G2), tRNA^Gly(CCC) (G3), tRNA^Val(AAC) (V) and tRNA^Leu(AAG) (L); for mitochondrial tRNA^Met (M mt), tRNA^Gln (Q mt) and for the mitochondrial L3a rRNA (L3a mt). (B) Signals were quantified and normalized with the L3a mt signal. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002946#s2" target="_blank">Results</a> are the means of 3 to 5 independent experiments and correspond to the percentage of variation of tRNA steady-state levels in the T22-11 transformant as compared to the T22-WT strain. Asterisks indicate statistically significant differences using Student <i>t</i> test with a significance threshold of 0.05.</p

Confocal microscopy of mitochondria from T22-WT, T22-11, and <i>dum22</i> stained with MitoTracker dyes.

<p>Visualization in T22-WT, T22-11 and <i>dum22</i> strains (A) of mitochondria by MitoTracker dye, (B) of chloroplast by chlorophyll autofluorescence and (C) of the overlapping images in <i>Chlamydomonas</i> cells. The white line corresponds to 5 µm.</p

Mitochondrial <i>in organello</i> protein synthesis of T22-11 transformant.

<p>(A) Mitochondrial proteins of T22-WT and T22-11 (25 µg) were loaded on SDS-PAGE after mitochondrial <i>in organello</i> protein synthesis and stained with Coomassie Blue. (B) Mitochondrial protein samples (10 µg) coming from the same experiment as described in (A) were separated by SDS-PAGE, blotted and probed with antisera against the VdacI <i>Chlamydomonas</i> protein. (C) Mitochondrial translated proteins from experiment (A) were visualized by autoradiography. Expected migration of the eight mitochondrial proteins are indicated. Major bands obtained in the <i>in organello</i> protein synthesis are indicated from b1 to b9. (D) Major bands (b1 to b9) were quantified. The histogram corresponds to the percentage of variation in the T22-11 transformant as compared to the T22-WT strain. The experiment was repeated two times and showed the same decrease of the annotated bands.</p