Oil accumulation in the model green alga Chlamydomonas reinhardtii: characterization, variability between common laboratory strains and relationship with starch reserves

International audienceBackground: When cultivated under stress conditions, many microalgae species accumulate both starch and oil (triacylglycerols). The model green microalga Chlamydomonas reinhardtii has recently emerged as a model to test genetic engineering or cultivation strategies aiming at increasing lipid yields for biodiesel production. Blocking starch synthesis has been suggested as a way to boost oil accumulation. Here, we characterize the triacylglycerol (TAG) accumulation process in Chlamydomonas and quantify TAGs in various wild-type and starchless strains. Results: In response to nitrogen deficiency, Chlamydomonas reinhardtii produced TAGs enriched in palmitic, oleic and linoleic acids that accumulated in oil-bodies. Oil synthesis was maximal between 2 and 3 days following nitrogen depletion and reached a plateau around day 5. In the first 48 hours of oil deposition, a~80% reduction in the major plastidial membrane lipids occurred. Upon nitrogen re-supply, mobilization of TAGs started after starch degradation but was completed within 24 hours. Comparison of oil content in five common laboratory strains (CC124, CC125, cw15, CC1690 and 11-32A) revealed a high variability, from 2 μg TAG per million cell in CC124 to 11 μg in 11-32A. Quantification of TAGs on a cell basis in three mutants affected in starch synthesis (cw15sta1-2, cw15sta6 and cw15sta7-1) showed that blocking starch synthesis did not result in TAG over-accumulation compared to their direct progenitor, the arginine auxotroph strain 330. Moreover, no significant correlation was found between cellular oil and starch levels among the twenty wild-type, mutants and complemented strains tested. By contrast, cellular oil content was found to increase steeply with salt concentration in the growth medium. At 100 mM NaCl, oil level similar to nitrogen depletion conditions could be reached in CC124 strain. Conclusion: A reference basis for future genetic studies of oil metabolism in Chlamydomonas is provided. Results highlight the importance of using direct progenitors as control strains when assessing the effect of mutations on oil content. They also suggest the existence in Chlamydomonas of complex interplays between oil synthesis, genetic background and stress conditions. Optimization of such interactions is an alternative to targeted metabolic engineering strategies in the search for high oil yields

Caractérisation structurale et fonctionnelle de la nouvelle sous-unité C17 de l'ARN polymérase III de Saccharomyces cerevisiae

Chez tous les eucaryotes, il existe trois formes distinctes d'ARN polymérase nucléaire (polI, II et III), chacune spécialisée dans la transcription d'une classe de gènes et constituée de plus d'une dizaine de sous-unités. Chez S. cerevisiae, la Pol III, qui synthétise des petits ARNs stables et non traduits (ARNt, ARNr 5S...), est composée de 17 sous-unités. L'assemblage séquentiel sur le promoteur des facteurs d'initiation TFIIIC et TFIIIB (complexe de pré-initiation), suivi du recrutement de la Pol III, représente la première e tape de la transcription des gènes de classe III. Dans ce travail, nous caractérisons un polypeptide de 17 kDa associé à la Pol III comme une nouvelle sous-unité de la Pol III essentielle à la viabilité cellulaire et paralogue aux sous-unités A14 (Pol I) et Rpb4 (Pol II). Les sous-unités C17 et C25 forment un hétérodimère stable, semblable aux paires A43-A14 (Pol I) et Rpb7-Rpb4 (Pol II). Nous démontrons que cet hétérodimère est conservé à travers l'évolution avec l'homologue archébactérien RpoF-RpoE. Nous proposons que le couple C17-C25 intervient lors du recrutement de la Pol III au complexe de pré-initiation, via une interaction entre C17 et la sous-unité Brf1 du facteur d'initiation TFIIIB. Par ailleurs, l'identification de nombreux orthologues de C17 révèle une conservation fonctionnelle de cette sous-unité. Nous démontrons ainsi que la protéine orthologue CGRP-RCP, décrite chez les mammifères comme un transducteur de signal, est une sous-unité de la Pol III humaine. L'existence d'un ancêtre commun d'ARN polymérase simplifie notre vision sur l'évolution moléculaire de ces enzymes. Cette étude représente aussi le premier exemple de sous-unité d'ARN polymérase ayant acquis chez les mammifères une fonction additionnelle, indépendante de la transcription.In all eukaryotes, there are three different forms of RNA polymerase (Pol I, II and III), each specialized in the transcription of one class of genes, and composed of more than ten subunits. In S. cerevisiae, Pol III that synthesizes small and stable untranslated RNA (tRNA, rRNA 5S...) contains 17 subunits. The sequential assembly of initiation factors TFIIIB and TFIIIC on the promoter (pre-initiation complex), followed by Pol III recruitment, is the first step of the transcription of class III genes. In this study, we characterize a polypeptide of 17 kDa associated with Pol III as a new Pol III subunit essential for cell viability and paralog of A14 (Pol I) and Rpb4 (Pol II) subunits. The subunits C17 and C25 form a stable heterodimer, counterpart of the A43-A14 (Pol I) and the Rpb7-Rpb4 (Pol II) pairs. We demonstrate that this heterodimer is conserved throughout evolution with RpoF-RpoE archaeal homolog. We propose that the C17-C25 couple is required for Pol III recruitment at pre-initiation complex, via the interaction between C17 and Brfl, a subunit of the initiation factor TFIIIB. Additionally, the identification of several orthologs of C17 reveals a functional conservation of this subunit. We demonstrate that the ortholog protein CGRP-RCP, described in mammals as a signal transductor, is a guenine human Pol III subunit. The existence of a common ancestor of RNA polymerase simplifies our vision of the molecular evolution of these enzymes. This study represents also the first example of a RNA polymerase subunit that acquired a dual function in mammals, unrelated to transcription.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

An Rpb4/Rpb7-Like Complex in Yeast RNA Polymerase III Contains the Orthologue of Mammalian CGRP-RCP

The essential C17 subunit of yeast RNA polymerase (Pol) III interacts with Brf1, a component of TFIIIB, suggesting a role for C17 in the initiation step of transcription. The protein sequence of C17 (encoded by RPC17) is conserved from yeasts to humans. However, mammalian homologues of C17 (named CGRP-RCP) are known to be involved in a signal transduction pathway related to G protein-coupled receptors, not in transcription. In the present work, we first establish that human CGRP-RCP is the genuine orthologue of C17. CGRP-RCP was found to functionally replace C17 in Δrpc17 yeast cells; the purified mutant Pol III contained CGRP-RCP and had a decreased specific activity but initiated faithfully. Furthermore, CGRP-RCP was identified by mass spectrometry in a highly purified human Pol III preparation. These results suggest that CGRP-RCP has a dual function in mammals. Next, we demonstrate by genetic and biochemical approaches that C17 forms with C25 (encoded by RPC25) a heterodimer akin to Rpb4/Rpb7 in Pol II. C17 and C25 were found to interact genetically in suppression screens and physically in coimmunopurification and two-hybrid experiments. Sequence analysis and molecular modeling indicated that the C17/C25 heterodimer likely adopts a structure similar to that of the archaeal RpoE/RpoF counterpart of the Rpb4/Rpb7 complex. These RNA polymerase subunits appear to have evolved to meet the distinct requirements of the multiple forms of RNA polymerases

Oil accumulation in the model green alga <it>Chlamydomonas reinhardtii</it>: characterization, variability between common laboratory strains and relationship with starch reserves

Abstract Background When cultivated under stress conditions, many microalgae species accumulate both starch and oil (triacylglycerols). The model green microalga Chlamydomonas reinhardtii has recently emerged as a model to test genetic engineering or cultivation strategies aiming at increasing lipid yields for biodiesel production. Blocking starch synthesis has been suggested as a way to boost oil accumulation. Here, we characterize the triacylglycerol (TAG) accumulation process in Chlamydomonas and quantify TAGs in various wild-type and starchless strains. Results In response to nitrogen deficiency, Chlamydomonas reinhardtii produced TAGs enriched in palmitic, oleic and linoleic acids that accumulated in oil-bodies. Oil synthesis was maximal between 2 and 3 days following nitrogen depletion and reached a plateau around day 5. In the first 48 hours of oil deposition, a ~80% reduction in the major plastidial membrane lipids occurred. Upon nitrogen re-supply, mobilization of TAGs started after starch degradation but was completed within 24 hours. Comparison of oil content in five common laboratory strains (CC124, CC125, cw15, CC1690 and 11-32A) revealed a high variability, from 2 μg TAG per million cell in CC124 to 11 μg in 11-32A. Quantification of TAGs on a cell basis in three mutants affected in starch synthesis (cw15sta1-2, cw15sta6 and cw15sta7-1) showed that blocking starch synthesis did not result in TAG over-accumulation compared to their direct progenitor, the arginine auxotroph strain 330. Moreover, no significant correlation was found between cellular oil and starch levels among the twenty wild-type, mutants and complemented strains tested. By contrast, cellular oil content was found to increase steeply with salt concentration in the growth medium. At 100 mM NaCl, oil level similar to nitrogen depletion conditions could be reached in CC124 strain. Conclusion A reference basis for future genetic studies of oil metabolism in Chlamydomonas is provided. Results highlight the importance of using direct progenitors as control strains when assessing the effect of mutations on oil content. They also suggest the existence in Chlamydomonas of complex interplays between oil synthesis, genetic background and stress conditions. Optimization of such interactions is an alternative to targeted metabolic engineering strategies in the search for high oil yields.</p

Identification and comparative genomic analysis of signaling and regulatory components in the diatom Thalassiosira pseudonana

Diatoms are unicellular brown algae that likely arose from the endocytobiosis of a red alga into a single-celled heterotroph and that constitute an algal class of major importance in phytoplankton communities around the globe. The first whole-genome sequence from a diatom species, Thalassiosira pseudonana Hasle et Heimdal, was recently reported, and features that are central to diatom physiology and ecology, such as silicon and nitrogen metabolism, iron uptake, and carbon concentration mechanisms, were described. Following this initial study, the basic cellular systems controlling cell signaling, gene expression, cytoskeletal structures, and response to stress have been cataloged in an attempt to obtain a global view of the molecular foundations that sustain such an ecologically successful group of organisms. Comparative analysis with several microbial, plant, and metazoan complete genome sequences allowed the identification of putative membrane receptors, signaling proteins, and other components of central interest to diatom ecophysiology and evolution. Thalassiosira pseudonana likely perceives light through a novel phytochrome and several cryptochrome photoreceptors; it may lack the conserved RHO small-GTPase subfamily of cell-polarity regulators, despite undergoing polarized cell-wall synthesis; and it possesses an unusually large number of heat-shock transcription factors, which may indicate the central importance of transcriptional responses to environmental stress. The availability of the complete gene repertoire will permit a detailed biochemical and genetic analysis of how diatoms prosper in aquatic environments and will contribute to the understanding of eukaryotic evolution

The Phaeodactylum genome reveals the dynamic nature and multi-lineage evolutionary history of diatom genomes

Diatoms are photosynthetic secondary endosymbionts found throughout marine and freshwater environments, and are believed to be responsible for around one-fifth of the primary productivity on Earth. The genome sequence of the marine centric diatom Thalassiosira pseudonana was recently reported, revealing a wealth of information about diatom biology. Here we report the complete genome sequence of the pennate diatom Phaeodactylum tricornutum and compare it with that of T. pseudonana to clarify evolutionary origins, functional significance and ubiquity of these features throughout diatoms. In spite of the fact that the pennate and centric lineages have only been diverging for 90 million years, their genome structures are dramatically different and a substantial fraction of genes (40percent) are not shared by these representatives of the two lineages. Analysis of molecular divergence compared with yeasts and metazoans reveals rapid rates of gene diversification in diatoms. Contributing factors include selective gene family expansions, differential losses and gains of genes and introns, and differential mobilization of transposable elements. Most significantly, we document the presence of hundreds of genes from bacteria. More than 300 of these gene transfers are found in both diatoms, attesting to their ancient origins, and many are likely to provide novel possibilities for metabolite management and for perception of environmental signals. These findings go a long way towards explaining the incredible diversity and success of the diatoms in contemporary oceans