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 (NAD7/49kDa) and Nd9 (NAD9/30kDa) 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.Peer reviewe

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 (NAD7/49kDa) and Nd9 (NAD9/30kDa) 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.Peer reviewe

Starch metabolism in Archaeplastida : the case of the glaucophyte unicellular model algae Cyanophora paradoxa and of the multicellular red algae Chondrus crispus

L'amidon et le glycogène définissent les deux polysaccharides de réserve les plus répandus dans le monde vivant. L'apparition de l'amidon chez les eucaryotes coïncide avec un événement d'endosymbiose unique qui s'est déroulé il y a 1,6 milliards d'années entre une cyanobactérie et un eucaryote primitif. De cet événement sont apparues trois lignées photosynthétiques : les Chloroplastida (plantes terrestres et algues vertes), les Rhodophyceae (algues rouges et organismes qui en sont dérivés par endosymbiose secondaire) et les Glaucophyta. La voie de biosynthèse de l'amidon chloroplastique chez les Chloroplastida est relativement bien caractérisée. Pour celle des Rhodophyceae (amidon cytoplasmique) des études ont déjà été entreprises mais les informations obtenues sont encore incomplètes et non représentatives de la lignée en général. Enfin pour les Glaucophyta, qui ont divergé de façon plus précoce après l'évènement d'endosymbiose aucune information sur le métabolisme de son amidon (cytoplasmique) n'était disponible. Afin d'appréhender la voie de biosynthèse utilisée par ces organismes nous avons entrepris la caractérisation de la voie métabolique de l'amidon de Cyanophora paradoxa (Glaucophyta) ainsi que celle de Chondrus crispus (Rhodophyceae). Ce manuscrit de thèse présente essentiellement des informations sur le métabolisme de l'amidon floridéen chez Cyanophora paradoxa, l'étude entreprise chez Chondrus crispus étant encore à l'état préliminaire.Starch and glycogen both define the most wide-spread storage polysaccharide in the world. The starch appearance in the eukaryotic lineage coincides with a single endosymbiotic event occurring 1,6 billion years ago between a cyanobacteria and a primitive eukaryotic hosto From this event appeared three photosynthetic lines: Chloroplastida (land plants and green algae), Rhodophyceae (red algae and organisms which are diverted from the Rhodophyceae by secondary endosymbiosis) and Glaucophyta. The plastid starch biosynthesis pathway in Chloroplastida is relatively weIl characterized. For the Rhodophyceae (cytosolic starch) studies have already been initiated but the information obtained is still incomplete and not representative of the lineage. Finally for the Glaucophyta, which diverged in a more premature way after the event of endosymbiosis no information about this (cytosolic) starch metabolism was available. To discover the biosynthesis pathway used by these organisms, we decided to perform the complete characterization of the starch biosynthetic pathway for two differents organisms: The Glaucophyta named Cyanophora paradoxa and the Rhodophyceae named Chondrus crispus. This manuscript of PhD mainly presents information on the Floridian starch metabolism of Cyanophora paradoxa. The starch characterisation of Chondrus crispus is currently at the preliminary stage and will require further development

Étude du métabolisme de l'amidon chez les Archaeplastida (le cas de l'algue glaucophyte modèle unicellulaire Cyanophora paradoxa et de l'algue rouge multicellulaire Chondrus crispus)

L'amidon et le glycogène définissent les deux polysaccharides de réserve les plus répandus dans le monde vivant. L'apparition de l'amidon chez les eucaryotes coïncide avec un événement d'endosymbiose unique qui s'est déroulé il y a 1,6 milliards d'années entre une cyanobactérie et un eucaryote primitif. De cet événement sont apparues trois lignées photosynthétiques : les Chloroplastida (plantes terrestres et algues vertes), les Rhodophyceae (algues rouges et organismes qui en sont dérivés par endosymbiose secondaire) et les Glaucophyta. La voie de biosynthèse de l'amidon chloroplastique chez les Chloroplastida est relativement bien caractérisée. Pour celle des Rhodophyceae (amidon cytoplasmique) des études ont déjà été entreprises mais les informations obtenues sont encore incomplètes et non représentatives de la lignée en général. Enfin pour les Glaucophyta, qui ont divergé de façon plus précoce après l'évènement d'endosymbiose aucune information sur le métabolisme de son amidon (cytoplasmique) n'était disponible. Afin d'appréhender la voie de biosynthèse utilisée par ces organismes nous avons entrepris la caractérisation de la voie métabolique de l'amidon de Cyanophora paradoxa (Glaucophyta) ainsi que celle de Chondrus crispus (Rhodophyceae). Ce manuscrit de thèse présente essentiellement des informations sur le métabolisme de l'amidon floridéen chez Cyanophora paradoxa, l'étude entreprise chez Chondrus crispus étant encore à l'état préliminaire.Starch and glycogen both define the most wide-spread storage polysaccharide in the world. The starch appearance in the eukaryotic lineage coincides with a single endosymbiotic event occurring 1,6 billion years ago between a cyanobacteria and a primitive eukaryotic hosto From this event appeared three photosynthetic lines: Chloroplastida (land plants and green algae), Rhodophyceae (red algae and organisms which are diverted from the Rhodophyceae by secondary endosymbiosis) and Glaucophyta. The plastid starch biosynthesis pathway in Chloroplastida is relatively weIl characterized. For the Rhodophyceae (cytosolic starch) studies have already been initiated but the information obtained is still incomplete and not representative of the lineage. Finally for the Glaucophyta, which diverged in a more premature way after the event of endosymbiosis no information about this (cytosolic) starch metabolism was available. To discover the biosynthesis pathway used by these organisms, we decided to perform the complete characterization of the starch biosynthetic pathway for two differents organisms: The Glaucophyta named Cyanophora paradoxa and the Rhodophyceae named Chondrus crispus. This manuscript of PhD mainly presents information on the Floridian starch metabolism of Cyanophora paradoxa. The starch characterisation of Chondrus crispus is currently at the preliminary stage and will require further development.LILLE1-Bib. Electronique (590099901) / SudocSudocFranceF

Genetic dissection of floridean starch synthesis in the cytosol of the model dinoflagellate Crypthecodinium cohnii

Starch defines an insoluble semicrystalline form of storage polysaccharides restricted to Archaeplastida (red and green algae, land plants, and glaucophytes) and some secondary endosymbiosis derivatives of the latter. While green algae and land-plants store starch in plastids by using an ADP-glucose-based pathway related to that of cyanobacteria, red algae, glaucophytes, cryptophytes, dinoflagellates, and apicomplexa parasites store a similar type of polysaccharide named floridean starch in their cytosol or periplast. These organisms are suspected to store their floridean starch from UDP-glucose in a fashion similar to heterotrophic eukaryotes. However, experimental proof of this suspicion has never been produced. Dinoflagellates define an important group of both photoautotrophic and heterotrophic protists. We now report the selection and characterization of a low starch mutant of the heterotrophic dinoflagellate Crypthecodinium cohnii. We show that the sta1-1 mutation of C. cohnii leads to a modification of the UDP-glucose-specific soluble starch synthase activity that correlates with a decrease in starch content and an alteration of amylopectin structure. These experimental results validate the UDP-glucose-based pathway proposed for floridean starch synthesis

Nature of the periplastidial pathway of starch synthesis in the cryptophyte Guillardia theta.

The nature of the periplastidial pathway of starch biosynthesis was investigated with the model cryptophyte Guillardia theta. The storage polysaccharide granules were shown to be composed of both amylose and amylopectin fractions with a chain length distribution and crystalline organization very similar to those of starch from green algae and land plants. Most starch granules displayed a shape consistent with biosynthesis occurring around the pyrenoid through the rhodoplast membranes. A protein with significant similarity to the amylose-synthesizing granule-bound starch synthase 1 from green plants was found as the major polypeptide bound to the polysaccharide matrix. N-terminal sequencing of the mature protein proved that the precursor protein carries a nonfunctional transit peptide in its bipartite topogenic signal sequence which is cleaved without yielding transport of the enzyme across the two inner plastid membranes. The enzyme was shown to display similar affinities for ADP and UDP-glucose, while the V(max) measured with UDP-glucose was twofold higher. The granule-bound starch synthase from Guillardia theta was demonstrated to be responsible for the synthesis of long glucan chains and therefore to be the functional equivalent of the amylose-synthesizing enzyme of green plants. Preliminary characterization of the starch pathway suggests that Guillardia theta utilizes a UDP-glucose-based pathway to synthesize starch

Pathway of Cytosolic Starch Synthesis in the Model Glaucophyte Cyanophora paradoxa▿

The nature of the cytoplasmic pathway of starch biosynthesis was investigated in the model glaucophyte Cyanophora paradoxa. The storage polysaccharide granules are shown to be composed of both amylose and amylopectin fractions, with a chain length distribution and crystalline organization similar to those of green algae and land plant starch. A preliminary characterization of the starch pathway demonstrates that Cyanophora paradoxa contains several UDP-glucose-utilizing soluble starch synthase activities related to those of the Rhodophyceae. In addition, Cyanophora paradoxa synthesizes amylose with a granule-bound starch synthase displaying a preference for UDP-glucose. A debranching enzyme of isoamylase specificity and multiple starch phosphorylases also are evidenced in the model glaucophyte. The picture emerging from our biochemical and molecular characterizations consists of the presence of a UDP-glucose-based pathway similar to that recently proposed for the red algae, the cryptophytes, and the alveolates. The correlative presence of isoamylase and starch among photosynthetic eukaryotes is discussed

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 14 N/15 N 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 beta-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

The Heterotrophic Dinoflagellate Crypthecodinium cohnii Defines a Model Genetic System To Investigate Cytoplasmic Starch Synthesis▿ †

The nature of the cytoplasmic pathway of starch biosynthesis was investigated in the model heterotrophic dinoflagellate Crypthecodinium cohnii. The storage polysaccharide granules were shown to be composed of both amylose and amylopectin fractions with a chain length distribution and crystalline organization very similar to those of green algae and land plant starch. Preliminary characterization of the starch pathway demonstrated that C. cohnii contains multiple forms of soluble starch synthases and one major 110-kDa granule-bound starch synthase. All purified enzymes displayed a marked substrate preference for UDP-glucose. At variance with most other microorganisms, the accumulation of starch in the dinoflagellate occurs during early and mid-log phase, with little or no synthesis witnessed when approaching stationary phase. In order to establish a genetic system allowing the study of cytoplasmic starch metabolism in eukaryotes, we describe the isolation of marker mutations and the successful selection of random recombinant populations after homothallic crosses