Molecular characterization of Chlamydomonas reinhardtii telomeres and telomerase mutants

International audienceTelomeres are repeated sequences found at the end of the linear chromosomes of most eukaryotes and are required for chromosome integrity. Expression of the reverse-transcriptase telo-merase allows for extension of telomeric repeats to counteract natural telomere shortening. Although Chlamydomonas rein-hardtii, a photosynthetic unicellular green alga, is widely used as a model organism in photosynthesis and flagella research, and for biotechnological applications, the biology of its telomeres has not been investigated in depth. Here, we show that the C. rein-hardtii (TTTTAGGG) n telomeric repeats are mostly nondegenerate and that the telomeres form a protective structure, with a subset ending with a 39 overhang and another subset presenting a blunt end. Although telomere size and length distributions are stable under various standard growth conditions, they vary substantially between 12 genetically close reference strains. Finally, we identify CrTERT, the gene encoding the catalytic subunit of telomerase and show that telomeres shorten progressively in mutants of this gene. Telomerase mutants eventually enter replicative senescence, demonstrating that telomerase is required for long-term maintenance of telomeres in C. reinhardtii

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

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Les antennes photosynthétiques chez Chlamydomonas reinhardtii : biogénèse, fonction et régulation

Photosynthesis is the biological process that converts light into chemical energy. It occurs within the chloroplasts of plant cells. Inside these organelles, light energy is absorbed by chlorophyll antennas (light-harvesting complexes or LHC), composed of proteins and pigments, chlorophylls and carotenoids. While chloroplasts possess their own genome, most of the genes have been transferred to the nucleus, thus implying the presence of an efficient protein import and assembly machinery within this organelle. The functioning of the photosynthetic apparatus is also finely regulated, particularly to prevent damage that could be caused by excess light energy. The aim of this thesis was to investigate how the synthesis and assembly processes of photosynthetic antennas and the photoprotective and acclimation responses to changes in light conditions, to further characterize their interactions. In this study, we have collected some observations concerning the synchronization of the two biosynthesis pathways, chlorophyll and LHC proteins, within the chloroplast. Understanding this synchronization will be crucial in order to understand how organisms adapt to natural variations in light intensity. We used the eukaryotic microalga Chlamydomonas reinhardtii as a biological model for its exceptional genetic features. For instance, the analysis of mutants has demonstrated the significance of proteins like cpSRP43 and ALB3.1 in the biogenesis of photosynthetic antennae while also suggesting an alternative pathway. A genetic analysis has precisely identified unexpected genomic rearrangements in a mutant strain of Chlamydomonas commonly used for studies on Chlamydomonas’ acclimation to environmental changes. Finally, we have uncovered the significance of a new genetic context –which is yet to be dissected- that specifies the variable properties of the main photoprotection mechanism in this microalgae.La photosynthèse est le processus biologique qui convertit la lumière en énergie chimique. Elle se déroule dans les chloroplastes des cellules végétales. À l'intérieur de ces organites, l'énergie lumineuse est captée par des antennes chlorophylliennes, connues sous le nom de complexe de récolte de lumière (Light harvesting complex ou LHC), constituées de protéines et de pigments, chlorophylles et caroténoïdes. Bien que les chloroplastes possèdent leur propre génome, la plupart des gènes ont été transférés dans le noyau, ce qui implique la présence d’une machinerie efficace d’import et d’assemblage des protéines dans cet organite. Le fonctionnement de l’appareil photosynthétique est également finement régulé, notamment pour éviter les dommages qui pourraient être causés par un excès d’énergie lumineuse. L’objectif de cette thèse a été d’étudier les processus de synthèse et d’assemblage des antennes photosynthétiques et les réponses de photoprotection et d’acclimatation devant des changements de régime lumineux, dans la perspective plus lointaine d’en comprendre les interactions. Dans cette étude nous avons par exemple recueilli quelques observations relatives à la synchronisation des deux voies de biosynthèse, de la chlorophylle et des protéines LHC, au sein du chloroplaste. La compréhension de cette synchronisation sera essentielle pour comprendre comment les organismes s'adaptent aux variations naturelles de l'intensité lumineuse. Nous avons utilisé la micro-algue eucaryote Chlamydomonas reinhardtii comme modèle biologique pour ses caractéristiques génétiques exceptionnelles. L’analyse de mutants a, par exemple, démontré l’importance des protéines telles que cpSRP43 et ALB3.1 dans la biogénèse des antennes photosynthétiques, tout en suggérant une voie alternative. Une analyse génétique a précisément identifié des réarrangements génomiques inattendus dans une souche mutante de Chlamydomonas couramment utilisée pour les études d’acclimatation aux changements environnementaux de Chlamydomonas. Enfin nous avons mis à jour l’importance d’un nouveau contexte génétique -qui reste à disséquer- qui spécifie les propriétés variables du principal mécanisme de photoprotection chez cette micro-algue

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

Dual functions of the nucleus-encoded factor TDA1 in trapping and translation activation of atpA transcripts in Chlamydomonas reinhardtii chloroplasts

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MRL1, a Conserved Pentatricopeptide Repeat Protein, Is Required for Stabilization of rbcL mRNA in Chlamydomonas and Arabidopsis[C][W]

The expression of the chloroplast genome requires specialized proteins that are coded in the nucleus and imported into the organelle. We have identified such a protein that binds the leading end of the mRNA for the most abundant chloroplast enzyme. The function of this novel stabilization factor is conserved from green algae to land plants

Antenna size reduction as a strategy to increase biomass productivity: a great potential not yet realized

de Mooij T, Janssen M, Cerezo-Chinarro O, et al. Antenna size reduction as a strategy to increase biomass productivity: a great potential not yet realized. Journal of Applied Phycology. 2014;27(3):1063-1077.A major limitation in achieving high photosynthetic efficiency in microalgae mass cultures is the fact that the intensity of direct sunlight greatly exceeds the photosynthetic capacity of the cells. Due to the high pigment content of algal cells, the light absorption rate surpasses the much slower conversion rate to biochemical energy. The excess of light energy is predominantly dissipated as heat, decreasing the light use efficiency of the culture. Algae with a truncated antenna system could substantially increase biomass productivity of mass cultures because oversaturation of the photosystems and concomitant dissipation of light energy, are minimized. In this study, we measured the areal biomass productivity of wild-type strain cultures and four promising antenna size mutant cultures of Chlamydomonas reinhardtii. This was performed under simulated mass culture conditions. The strains were cultivated in turbidostat controlled lab-scale panel photobioreactors at an incident light intensity of 1500 μmol photons m-2s-1. The mutant cultures did not exhibit the expected higher productivity. The greatest mutant culture productivity values were approximate to those of the wild-type productivity of 1.9 g m-2h-1. The high sensitivity to abrupt light shifts indicated that the mutant cultures experienced reduced fitness and higher susceptibility to photodamage. This can possibly be explained by impaired photo protection mechanisms induced by the antenna complex alterations, or by unintended side effects of the genetic modifications. Still, if these effects could be eliminated, the principle of antenna size reduction is a promising strategy to increase productivity. Selection criteria for the future creation of antenna size mutants should, therefore, include tolerance to high light conditions

Chloroplast dysfunction causes multiple defects in cell cycle progression in the Arabidopsis crumpled leaf mutant

The majority of research on cell cycle regulation is focused on the nuclear events that govern the replication and segregation of the genome between the two daughter cells. However, eukaryotic cells contain several compartmentalized organelles with specialized functions, and coordination among these organelles is required for proper cell cycle progression, as evidenced by the isolation of several mutants in which both organelle function and overall plant development were affected. To investigate how chloroplast dysfunction affects the cell cycle, we analyzed the crumpled leaf (crl) mutant of Arabidopsis (Arabidopsis thaliana), which is deficient for a chloroplastic protein and displays particularly severe developmental defects. In the crl mutant, we reveal that cell cycle regulation is altered drastically and that meristematic cells prematurely enter differentiation, leading to reduced plant stature and early endoreduplication in the leaves. This response is due to the repression of several key cell cycle regulators as well as constitutive activation of stress-response genes, among them the cell cycle inhibitor SIAMESE-RELATED5. One unique feature of the crl mutant is that it produces aplastidic cells in several organs, including the root tip. By investigating the consequence of the absence of plastids on cell cycle progression, we showed that nuclear DNA replication occurs in aplastidic cells in the root tip, which opens future research prospects regarding the dialogue between plastids and the nucleus during cell cycle regulation in higher plants