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Structural and functional study of oil droplet in diatoms



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Published on 4 March 2020
PhD subject
Diatoms, and on a more global scale Chromalveolates, are a major group of microalgae in both freswater and oceans. They are the fruit of a complex evolutive history involving multiple endosymbiosis events, which resulted in a peculiar and complicated cellular structure (Flori et al., 2016, 2017, Maréchal 2019). The large ecological success of diatoms make them one of the most important groups of primer producers in the biosphere, responsible for about 25% of photosynthesis based CO2 capture. Moreover, in response to some environmental cues, they are able to store this carbon as triacylglycerol (TAG), or oil, in specific organelles called Lipid Droplets (LD) (Abida et al., 2015; Dolch et al., 2017a and b; Conte et al., 2018; Hao et al., 2018; Lupette et al., 2018a, 2019; Mulhroth et al., 2017; Winge et al., in press). This ability has raised increasing interest over the recent years as microalgae are a promising source for the production of biofuels as well as for other interesting compounds (e.g. omega-3 fatty acids) (Lupette et al., 2018 b and c; Maréchal 2015, 2017). Yet two major blocks remain: 1) production of TAG is induced by stress conditions, which leads to growth arrest; 2) the cost of oil extraction from microalgae remains prohibiting. Gaining insights into the biogenesis and function of LD in microalgae is thus crucial to bypass those locks.
The hydrophobic core of LD is surrounded by a monolayer of phospholipids containing proteins that can be more or less tightly associated with the LD. In a recent work, we have analyzed the lipid and protein composition of LD in the model diatom Phaeodactylum tricornutum (Lupette et al., 2019), which has allowed us to unravel some interesting candidates to investigate.
The present project aims at characterizing the function of some of LD associated proteins in Phaeodactylum tricornutum. Genes of interest expression will be invalidated using the CRIPR-Cas9 technology, which we have already implemented in the lab. Phenotypes of the mutants will then be analyzed with a focus on lipid content, photosynthesis ability, impact on the cell structure and organization and on LD extractability. All required techniques are fully available in the hosting lab.

Methods
Molecular Biology: cloning techniques (restriction enzymes and Gibson assembly); CRISPR-Cas9 gene editing, DNA and RNA extraction, qRT-PCR
Cell culture: Microalgae culture, growth curves, measurements of photosynthesis parameters, Lipid fluorescence staining
Cell Biology: Light Microscopy (Brightfield, epifluorescence and confocal) and Electronic Microscopy
Biochemistry: Organelle purification (LD, possibly chloroplast), Lipid extraction and analysis, Protein extraction and Western blotting

Hosting team
The hosting team is leader in the field of membrane glycerolipids and storage lipids metabolism in diatoms
Plant & Cell Physiology Lab / Laboratoire de Physiologie Cellulaire et Végétale (LPCV), IRIG, CEA Grenoble, GRENOBLE, France

Reference: SL-DRF-20-0298

Supervisors and contact:
Eric Maréchal and Juliette Salvaing  

Part of a national flagship network focusing on Carbon Cycle Closure 
The PhD project is part of a national flagship program of CEA, focused on carbon cycle closure. The student will have therefore a unique opportunity to join a network of biologists, chemists, engineer and material scientists, addressing one of the most ambitious challenges in the context of energy transition and climate change.

Applications
Interested students should contact Eric Maréchal and Juliette Salvaing and send a CV, motivation letter as well as Licence and Master (or equivalent) mark sheets. Reference of 1 or 2 people to contact should be included as well. Applications should be sent before April 15th. Candidates should have a Master degree in Biology (M2 or equivalent), preferably in Plant biology or Biochemistry, and ideally previous lab experience in at least one of the following fields: cell culture and cell biology, biochemistry, molecular biology.

References
Maréchal, E (2019) Marine and freshwater plants: challenges and expectations. Front Plant Sci. doi: 10.3389/fpls.2019.01545
Winge P, Sharma A, Mühlroth A, Jouhet J, Marechal E, Alipanah L, Kissen R, Brembu T, Bones A. The Myb like transcription factor Phosphorus Starvation Response (PtPSR) controls conditional P acquisition and remodeling in marine microalgae. New Phytol. In press
Lupette J, Jaussaud A, Seddiki K, Morabito C, Brugière S, Schaller H, Kuntz M, Putaux JL, Jouneau PH, Rébeillé F, Falconet D, Couté Y, Jouhet J, Tardif M, Salvaing J, Maréchal E. (2019) The architecture of lipid droplets in the diatom Phaeodactylum. Algal Res 38:101415
Hao X, Luo L, Jouhet J, Rébeillé F, Maréchal E, Hu H, Pan Y, Tan X, Chen Z, You L, Chen H, Wei F, Gong Y. (2018) Enhanced triacylglycerol production in the diatom Phaeodactylum tricornutum by inactivation of a HotDog-fold thioesterase gene using TALEN-based targeted mutagenesis. Biotech. Biofuels. 11:312
Lupette J, Jaussaud A, Vigor C, Oger C, Galano JM, Reversat G, Vercauteren J, Jouhet J, Durand T, Marechal E. (2018a) Non-enzymatic synthesis of bioactive isoprostanoids in the diatom Phaeodactylum following oxidative stress. Plant Physiol. 178(3):1344-1357
Lupette J., Maréchal E. (2018b) Le potentiel des micro-algues pour la chimie verte et les bioénergies. (L. Kalpa ed) Éditions Matériologiques, Paris, France, 321-338
Lupette J., Maréchal E. (2018c) Phytoplankton glycerolipids, challenging but promising prospects from biomedicine to green chemistry and biofuels. In: Blue Technologies: production and use of marine mole-cules. (S La Barre and S. Bates Eds.) Wiley VCH, Weinheim, Germany, 191-215, ISBN:9783527801718 |DOI:10.1002/9783527801718
Maréchal E. (2017) Les biocarburants. Encyclopédie de l’Environnement. http://www.encyclopedie-environnement.org/ (R. Moreau and J. Sommeria Eds.) Université Grenoble Alpes.
Conte M, Lupette J, Seddiki K, Meï C, Dolch LJ, Gros V, Barette C, Rébeillé F, Jouhet J, Maréchal E (2018) Screening for biologically annotated drugs that trigger triacylglycerol accumulation in the diatom Phaeodactylum. Plant Physiol. 177:532-552
Mühlroth A, Winge P, Assimi A, Jouhet J, Marechal E, Hohmann-Marriott M, Vadstein O, Bones A (2017) Mechanisms of phosphorus acquisition and lipid class remodelling under P limitation in Nanno-chloropsis. Plant Physiol 175(4): 1543-1559
Dolch LJ, Lupette J, Tourcier G, Bedhomme M, Collin S, Magneschi L, Conte M, Seddiki K, Richard C, Corre E, Fourage L, Laeuffer F, Richards R, Reith M, Rébeillé F, Jouhet J, McGinn P, Marechal E. (2017a) Nitric oxide mediates nitrite-sensing and acclimation and triggers a remodeling of lipids. Plant Physiol 175:1407–1423
Flori S, Jouneau PH, Bailleul B, Gallet B, Estrozi L, Moriscot C, Bastien O, Eicke S, Schober A, Río Bártulos C, Maréchal E, Kroth P, Petroutsos D, Zeeman S, Breyton C, Schoehn G, Falconet D. Finazzi G (2017) Plastid thylakoid architecture optimises photosynthesis in diatoms. Nat Commun. 8:15885
Dolch LJ, Rak C, Perin G, Tourcier G, Broughton R, Leterrier M, Morosinotto T, Tellier F, Faure JD, Fal-conet D, Jouhet J, Sayanova O, Beaudoin F, Marechal E. (2017b) A palmitic acid elongase affects eicosapentaenoic acid and plastidial monogalactosyldiacylglycerol levels in Nannochloropsis. Plant Physiol., 173:742-759
Flori S., Jouneau P.H., Finazzi G, Maréchal E*, Falconet D. (2016) Ultrastructure of the periplastidial compartment of the diatom Phaeodactylum tricornutum. Protist. 167(3):254–267 (* corresponding)
Abida H, Dolch LJ, Meï C, Villanova V, Conte M, Block MA, Finazzi G, Bastien O, Tirichine L, Bowler C, Rébeillé F, Petroutsos D, Jouhet J and Maréchal E (2015). Membrane glycerolipid remodeling triggered by nitrogen and phosphorus starvation in Phaeodactylum tricornutum. Plant Physiol, 167(1): 118-136
Maréchal E. (2015) Carburants à base d'algues oléagineuses - Principes, filières, verrous. In Techniques de l'Ingénieur. IN 186, 2015, 1-19

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