Véronique Larosa

Lack of isocitrate lyase in Chlamydomonas leads to changes in carbon metabolism and in the response to oxidative stress under mixotrophic growth.

Isocitrate lyase is a key enzyme of the glyoxylate cycle. This cycle plays an essential role in cell growth on acetate, and is important for gluconeogenesis as it bypasses the two oxidative steps of the tricarboxylic acid (TCA) cycle in which CO₂ is evolved. In this paper, a null icl mutant of the green microalga Chlamydomonas reinhardtii is described. Our data show that isocitrate lyase is required for growth in darkness on acetate (heterotrophic conditions), as well as for efficient growth in the light when acetate is supplied (mixotrophic conditions). Under these latter conditions, reduced acetate assimilation and concomitant reduced respiration occur, and biomass composition analysis reveals an increase in total fatty acid content, including neutral lipids and free fatty acids. Quantitative proteomic analysis by ¹⁴N/¹⁵N labelling was performed, and more than 1600 proteins were identified. These analyses reveal a strong decrease in the amounts of enzymes of the glyoxylate cycle and gluconeogenesis in parallel with a shift of the TCA cycle towards amino acid synthesis, accompanied by an increase in free amino acids. The decrease of the glyoxylate cycle and gluconeogenesis, as well as the decrease in enzymes involved in β-oxidation of fatty acids in the icl mutant are probably major factors that contribute to remodelling of lipids in the icl mutant. These modifications are probably responsible for the elevation of the response to oxidative stress, with significantly augmented levels and activities of superoxide dismutase and ascorbate peroxidase, and increased resistance to paraquat.

Co-evolution of mitochondrial tRNA import and codon usage determines translational efficiency in the green alga Chlamydomonas.

Mitochondria from diverse phyla, including protozoa, fungi, higher plants, and humans, import tRNAs from the cytosol in order to ensure proper mitochondrial translation. Despite the broad occurrence of this process, our understanding of tRNA import mechanisms is fragmentary, and crucial questions about their regulation remain unanswered. In the unicellular green alga Chlamydomonas, a precise correlation was found between the mitochondrial codon usage and the nature and amount of imported tRNAs. This led to the hypothesis that tRNA import might be a dynamic process able to adapt to the mitochondrial genome content. By manipulating the Chlamydomonas mitochondrial genome, we introduced point mutations in order to modify its codon usage. We find that the codon usage modification results in reduced levels of mitochondrial translation as well as in subsequent decreased levels and activities of respiratory complexes. These effects are linked to the consequential limitations of the pool of tRNAs in mitochondria. This indicates that tRNA mitochondrial import cannot be rapidly regulated in response to a novel genetic context and thus does not appear to be a dynamic process. It rather suggests that the steady-state levels of imported tRNAs in mitochondria result from a co-evolutive adaptation between the tRNA import mechanism and the requirements of the mitochondrial translation machinery.

The ARG9 Gene Encodes the Plastid-Resident N-Acetyl Ornithine Aminotransferase in the Green Alga Chlamydomonas reinhardtii

ABSTRACT Here we report the characterization of the Chlamydomonas reinhardtii gene ARG9, encoding the plastid resident N-acetyl ornithine aminotransferase, which is involved in arginine synthesis. Integration of an engineered ARG9 cassette in the plastid chromosome of the nuclear arg9 mutant restores arginine prototrophy. This suggests that ARG9 could be used as a new selectable marker for plastid transformation.

Physiological Characterization of a Plant Mitochondrial Calcium Uniporter in Vitro and in Vivo

An Arabidopsis homolog of the mammalian mitochondrial calcium uniporter MCU acts as Ca2+ channel, and its absence impacts mitochondrial function and morphology. Over the recent years, several proteins that make up the mitochondrial calcium uniporter complex (MCUC) mediating Ca2+uptake into the mitochondrial matrix have been identified in mammals, including the channel-forming protein MCU. Although six MCU gene homologs are conserved in the model plant Arabidopsis (Arabidopsis thaliana) in which mitochondria can accumulate Ca2+, a functional characterization of plant MCU homologs has been lacking. Using electrophysiology, we show that one isoform, AtMCU1, gives rise to a Ca2+-permeable channel activity that can be observed even in the absence of accessory proteins implicated in the formation of the active mammalian channel. Furthermore, we provide direct evidence that AtMCU1 activity is sensitive to the mitochondrial calcium uniporter inhibitors Ruthenium Red and Gd3+, as well as to the Arabidopsis protein MICU, a regulatory MCUC component. AtMCU1 is prevalently expressed in roots, localizes to mitochondria, and its absence causes mild changes in Ca2+ dynamics as assessed by in vivo measurements in Arabidopsis root tips. Plants either lacking or overexpressing AtMCU1 display root mitochondria with altered ultrastructure and show shorter primary roots under restrictive growth conditions. In summary, our work adds evolutionary depth to the investigation of mitochondrial Ca2+ transport, indicates that AtMCU1, together with MICU as a regulator, represents a functional configuration of the plant mitochondrial Ca2+ uptake complex with differences to the mammalian MCUC, and identifies a new player of the intracellular Ca2+ regulation network in plants.

Reconstruction of a human mitochondrial complex I mutation in the unicellular green alga Chlamydomonas

Defects in complex I (NADH:ubiquinone oxidoreductase (EC 1.6.5.3)) are the most frequent cause of human respiratory disorders. The pathogenicity of a given human mitochondrial mutation can be difficult to demonstrate because the mitochondrial genome harbors large numbers of polymorphic base changes that have no pathogenic significance. In addition, mitochondrial mutations are usually found in the heteroplasmic state, which may hide the biochemical effect of the mutation. We propose that the unicellular green alga Chlamydomonas could be used to study such mutations because (i) respiratory complex-deficient mutants are viable and mitochondrial mutations are found in the homoplasmic state, (ii) transformation of the mitochondrial genome is feasible, and (iii) Chlamydomonas complex I is similar to that of humans. To illustrate this proposal, we introduced a Leu157Pro substitution into the Chlamydomonas ND4 subunit of complex I in two recipient strains by biolistic transformation, demonstrating that site-directed mutagenesis of the Chlamydomonas mitochondrial genome is possible. This substitution did not lead to any respiratory enzyme defects when present in the heteroplasmic state in a patient with chronic progressive external ophthalmoplegia. When present in the homoplasmic state in the alga, the mutation does not prevent assembly of whole complex I (950 kDa) and the NADH dehydrogenase activity of the peripheral arm of the complex is mildly affected. However, the NADH:duroquinone oxidoreductase activity is strongly reduced, suggesting that the substitution could affect binding of ubiquinone to the membrane domain. The in vitro defects correlate with a decrease in dark respiration and growth rate in vivo.

Transformation of the mitochondrial genome.

Although mitochondrial transformation is highly desirable in mammals and plants, it is only possible in two unicellular organisms, the budding yeast Saccharomyces cerevisiae and the unicellular green alga Chlamydomonas reinhardtii. Here, we give an overview of the attempts made to transform mitochondria of mammals and plants and the possible reasons for their failure. This review briefly describes the mitochondrial transformation principles in yeast and describes in more detail the transformation and its applications in Chlamydomonas.

Isolation of Chlamydomonas reinhardtii mutants with altered mitochondrial respiration by chlorophyll fluorescence measurement

The unicellular green alga Chlamydomonas reinhardtii is a model organism for studying energetic metabolism. Most mitochondrial respiratory-deficient mutants characterized to date have been isolated on the basis of their reduced ability to grow in heterotrophic conditions. Mitochondrial deficiencies are usually partly compensated by adjustment of photosynthetic activity and more particularly by transition to state 2. In this work, we explored the opportunity to select mutants impaired in respiration and/or altered in dark metabolism by measuring maximum photosynthetic efficiency by chlorophyll fluorescence analyses (FV/FM). Out of about 2900 hygromycin-resistant insertional mutants generated from wild type or from a mutant strain deficient in state transitions (stt7 strain), 22 were found to grow slowly in heterotrophic conditions and 8 of them also showed a lower FV/FM value. Several disrupted coding sequences were identified, including genes coding for three different subunits of respiratory-chain complex I (NUO9, NUOA9, NUOP4) or for isocitrate lyase (ICL1). Overall, the comparison of respiratory mutants obtained in wild-type or stt7 genetic backgrounds indicated that the FV/FM value can be used to isolate mutants severely impaired in dark metabolism.

Inactivation of genes coding for mitochondrial Nd7 and Nd9 complex I subunits in Chlamydomonas reinhardtii. Impact of complex I loss on respiration and energetic metabolism

In Chlamydomonas, unlike in flowering plants, genes coding for Nd7 (NAD7/49 kDa) and Nd9 (NAD9/30 kDa) core subunits of mitochondrial respiratory-chain complex I are nucleus-encoded. Both genes possess all the features that facilitate their expression and proper import of the polypeptides in mitochondria. By inactivating their expression by RNA interference or insertional mutagenesis, we show that both subunits are required for complex I assembly and activity. Inactivation of complex I impairs the cell growth rate, reduces the respiratory rate, leads to lower intracellular ROS production and lower expression of ROS scavenging enzymes, and is associated to a diminished capacity to concentrate CO2 without compromising photosynthetic capacity.

Complexes I in the Green Lineage

In land plants and green algae, mitochondria and chloroplasts were acquired sequentially through primary endosymbiotic events with a α-proteobacterium and a cyanobacterium, respectively. The inner membrane of the mitochondria harbors the enzyme complexes of the respiratory chain, the largest of them being the rotenone-sensitive NADH:ubiquinone oxidoreductase or complex I. In the thylakoid membrane of the chloroplast, besides the photosynthetic machinery, a light-independent respiratory-chain inherited from cyanobacteria drives electrons from NAD(P)H to oxygen. In most plants and algae, it comprises a homolog of bacterial complex I (NAD(P)H:plastoquinone (PQ) oxidoreductase) and a PQ oxidase (PTOX). This chapter will be thus dedicated to similarities and peculiarities of plant mitochondrial complex I compared to the well studied enzyme in mammals and fungi, as well as to the structure and role of a complex I homolog in chloroplast.

Transformation and Nucleic Acid Delivery to Mitochondria

Genomic, transcriptomic and proteomic approaches have yielded considerable information, which impacted our understanding of the interactions between the nucleus and the mitochondria. Plant mitochondrial (mt) genomes are very large (220–2,000 kb) and often occur as complex pools of recombined molecules whose stoichiometry is tightly controlled by the nucleus. Unlike their mammalian and fungal counterparts, plant mt transcripts undergo complex post-transcriptional modifications such as editing and trans-splicing. Due to the impossibility to stably transform plant mitochondria and hence to manipulate mt gene expression, the genetic regulation of plant mt genomes has remained poorly understood. In this chapter, we will review the experimental data concerning the unicellular green alga Chlamydomonas reinhardtii, the only photosynthetic organism for which mt transformation has been achieved. Although Chlamydomonas harbors an extremely compact linear mt genome (15.8 kb) that differs from the one typically found in vascular plants, this system could bring novel insights on the role of the few subunits of the respiratory chain that are encoded in the mt genome. This is particularly relevant for the nd genes, which encode subunits of complex I since the yeast Saccharomyces cerevisiae, the other unicellular organism where mt transformation is performed nearly at will, is deprived of complex I. Moreover, because the Chlamydomonas mt genome only encodes three tRNAs, genetic manipulation of the organellar genome is a promising avenue to dissect the import of cytosolic tRNAs, a process that is now known to take place in plant and also human mitochondria. We also present alternative approaches such as the in vitro import of DNA or RNA and electroporation of isolated mitochondria followed by in organello synthesis that have been developed. These approaches have generated fruitful information about transcription and post-transcriptional processing of plant mt RNAs.