Contacts :

Group leader Thierry Chardot Senior scientist Pascale Jolivet Research Scientist Sabine d'Andréa Associate Professor AgroParisTech Jacques Snégaroff Research Scientist Celine Boulard Assistant Engineer Bernard Cintrat Technician Carine Deruyffelaere Technician Laure Ayme Master 2 from 15/12/12 to 28/07/12

Pierre Briozzo Professor AgroParisTech Yann Gohon Associate Professor AgroParisTech Marine Froissard Research Scientist Isabelle Bouchez-Mahiout Engineer, ACP Roselyne Tache Technician Michel Canonge Technician Franck Jagic Technician

Germination and seedling establishment require high energy input and the supply of carbon backbones for various biosynthetic pathways such as that of lipids. In effect, the plant organ exhibiting the highest levels of lipid accumulation is seeds. Storage lipids are accumulated in lipid bodies (LBs), also called lipid droplets or oil bodies. LBs structure is conserved among living organisms, with a core composed of neutral lipids surrounded by a monolayer of phospholipids, in which a highly variable number of proteins is embedded. These proteins in microbe or animal LBs are mostly comprised of enzymes, or proteins involved in subcellular transport. Seed LBs contain a limited set of proteins, with few enzymes. In particular, little is known about the dynamics of LBs during seed life cycle. To date, no structure has been determined to high resolution for a protein inserted in a phospholipid monolayer. To identify and characterize proteins contributing to the dynamic and the structure of LBs, we apply genetic, biochemical, molecular, and structural approaches. As models we use oleaginous plants (Arabidopsis thaliana, Brassica napus), and also yeasts (Saccharomyces cerevisae, Yarrowia lipolytica).

Our main goals are:
-to fill in the gaps between the knowledge of LB structure and function in plants and other organisms.
-to determine high resolution structures of selected plant oil body proteins.

In accordance with the principles of “Green Chemistry”, our research aims to modulate amounts and tailor the nature of lipids produced, as well as to facilitate storage lipid extraction.
Our expertise in the field of protein structure determination using crystallographic techniques is available to the scientific community, and we are known for our experience on bacterial kinases and plant oxidases.

Our efforts are focused on two investigative axes: « Structural and functional », and « Proteomic and dynamic ». Plants and microorganisms able to store lipids are used as models for our studies.

Structural and functional investigations
The three-dimensional structure determination of lipid body integral proteins is a challenge. Structural data on these very hydrophobic proteins are still scarce and contradictory. We are using original tools to solubilize and handle these proteins and to determine their structure, in isolation or inserted into lipid bodies. (Gohon 2008).
We have set up a simple and rapid protocol for the specific extraction of oleosins from A. thaliana seeds leading to the extensive identification of lipid body proteins (Andréa 2007b).
Using original amphiphilic polymers (Apols), we are able to maintain soluble S5 oleosin (with a low molecular weight and very hydrophobic) and caleosin (a calcium binding protein that is less hydrophobic). Original protein folds have been observed using synchrotron radiation circular dichroism (Gohon et al. submitted).
We are using similar approaches to study the structure of allergenic wheat proteins (bacterially expressed glutenins and gliadins).

We have expertise in the determination of protein post-translational modifications. We proved that A. thaliana caleosin was phosphorylated (Purkrtova 2007), and that its interfacial properties were modified upon calcium binding (Purkrtova 2008). Using structural proteomic approaches, the topology of proteins at the surface of lipid bodies has also been determined. (Vermachova).
We showed that the original intrinsic properties of AtClo1 protein were sufficient to generate a functional lipid body membrane and to promote over accumulation of storage lipids in yeast oil bodies (Froissard 2009).

Proteomic, dynamic
Using proteomic approaches, we have identified more than 30 different proteins in Y. lipolytica lipid bodies, and proved that their composition was dictated by the carbon sources used for yeast growth (Athenstaed 2006). Yeast lipid bodies contain enzymes from triacylglycerol accumulation and mobilisation pathways, as well as proteins related to intracellular traffic, revealing the dynamics of the organelle. Glycerol-3 phosphate dehydrogenase is a protein found at the surface of lipid bodies. Its inactivation, in strains unable to perform ?-oxidation, redirected carbon fluxes and enabled an accumulation of up to 51 % neutral lipids in yeast (Beopoulos 2008, 2009).

Compared to microbial or animal lipid bodies, (Athenstaedt 2006), LBs from A. thaliana or B. napus mature seed seem to be in a quiescent state (Jolivet 2004, 2009, d’Andréa 2007b, Popluechai submitted). Rapeseed LB proteins follow a sequential expression pattern, so it is likely that LBs undergo an ordered maturation scheme (Jolivet, unpublished).

Significant collaborations, contracts

Main collaborations: UMR INRA-INSA LISBP Toulouse, ESPCI ParisTech, ENSCP Paris, Institut Pasteur, Synchrotron Soleil, UMR AgroParisTech-INRA Micalis Grignon, UMR AgroCampus INRA APBV Rennes, UR INRA BIA Nantes, University Paris XI, Graz, Prague, Olomouc, Pittsburg; proteomic facility PAPPSO Le Moulon, CETIOM Pessac.

ANR Programs
-Genoplante GNP0036 Genobodies (2006-2008, 6 partners) “Structural and functional study of oil and protein storage bodies in A. thaliana and in B. napus: towards environmentally friendly oil and protein extraction process ?” . Coordination: T. chardot
-CP2D 08-CP2D-19 SOPOL (2009-2011, 5 partners) «Solubilization of seed oil bodies integral proteins by amphipols: structural studies for valorization » Coordination: T. Chardot
Participations
-GPLA-07-006C Genergy, (2008-2011). “Improvement of the oil yield of the rapeseed crop in the context of bio fuel production”. Coordination : N Nesi, INRA APBV Rennes.
-ANR 07-BIOE-008 Lipicaero (2008-2010) «Production microbienne de LIPIdes spécifiques à usage bioCarburant pour l’AEROnautique Approche intégrée de Physiologie au Procédé » Coordination C. Jouve, INSA LISBP, Toulouse
-ANR-08-ALIA-014-02 : Programme ALimentation et Industrie Alimentaires (ALIA), projet Predexpitope : « Prédiction in silico d’épitopes d’allergènes alimentaires et validation expérimentale sur les allergènes du blé. » Coordination S. Denery, INRA BIA Nantes.
-Prevalorisation programm « ArcoPress » (INRA Transfert). Coordination M. Miquel, IJPB Versailles
-Program CAER « Carburant pour l’Aéronautique », (2010-2013) Coordination C. Jouve, INSA LISBP, Toulouse.

Popluechai, S., Froissard, M., Jolivet, P., Breviario, D., Gatehouse, A.M.R., O’Donnell, A. G., Chardot, T. and Kohli, A. (2011) Understanding Jatropha curcas seed oil storage components: characterization of seed oilbody oleosins. Plant Phys Biochem ,49 352-356.

Jolivet, P., Boulard, C., Bellamy, A., Valot, B., d’Andréa, S., Zivy, M., Nesi, N., and Chardot, T. (2011). Oil body proteins sequentially accumulate throughout seed development in Brassica napus. Journal of Plant Physiology. Accepted

Gohon, Y., Vindigni, JD., Pallier, A., Celia, H., Giuliani, A., Wien, F., Tribet, C., Chardot T., Pierre Briozzo, (2011) High water solubility and folding in amphipols of integral proteins with large hydrophobic regions: oleosin and caleosin from seed lipid bodies. BBA, 1088: 707-716

Savoire R, Quinsac A, Chardot T, Miquel M., Nesi N, Lanoisellé J-L, Vorobiev E. (2010) Micro-pressing of rapeseed (Brassica napus L.) and Arabidopsis thaliana seeds for evaluation of the oil extractability. OCL, 17(2): 115-119

Agrawal GK, Bourguignon Rolland N, Ephritikhine G, Ferro M, Jaquinod M, Alexiou KG, Chardot T, Chakraborty N, Jolivet P, Doonan J, Rakwal R (2010). Plant organelle proteomics: collaborating for optimal function. Mass Spect Review (Sous presse)

Tylichová M, Kopecný D, Moréra S, Briozzo P, Lenobel R, Snégaroff J, Šebela M (2010). Structural and functional characterization of plant aminoaldehyde dehydrogenases with a broad specificity for natural and synthetic aminoaldehydes. J Mol Biol, 396 : 870–882.

Le Bon C, Boulard C, Jolivet P (2009). Association d’un microscope à un système de capture d’image. Analyse d’images par le logiciel ImageJ. Le cahier technique de l’INRA Numéro spécial « Des développements méthodologiques en imagerie à l’INRA » : 63-68

Beopoulos, A.; Chardot, T.; Nicaud, J. M. (2009) Yarrowia lipolytica: A model and a tool to understand the mechanisms implicated in lipid accumulation. Biochimie 91, 692-96

Froissard, M., S. D'Andréa, Boulard T., Chardot, T. (2009) Heterologous expression of AtClo1, a plant oil body protein, induces lipid accumulation in yeast. FEMS Yeast Res, 9 :: 428-38.

Baud, S., Dichow, N. R., Kelemen, Z., d'Andréa, S., To, A., Berger, N., Canonge, M., Kronenberger, J., Viterbo, D., Dubreucq, B., Lepiniec, L., Chardot, T., Miquel, M. (2009) Regulation of HSD1 in seeds of Arabidopsis thaliana. Plant Cell Physiol, 50 : 1463-78.

Jolivet, P., Boulard, C., Bellamy, A., Larre, C., Barre, M., Rogniaux, H., d'Andréa, S., Chardot, T., Nesi, N. (2009) Protein composition of oil bodies from mature Brassica napus seeds. Proteomics, 9 : 3268-84.

Purkrtova, Z., Jolivet, P., Miquel, M., and Chardot, T. (2008). Structure and function of seed lipid-body-associated proteins. C R Biol 331, 746-754.

Purkrtova, Z., Le Bon, C., Kralova, B., Ropers, M. H., Anton, M., Chardot, T. (2008) Caleosin of Arabidopsis thaliana: Effect of Calcium on Functional and Structural Properties. J Agric Food Chem 56 : 11217-11224.

Kopeckný D., Šebela M., Briozzo, P., Spíchal L., Houba-Hérin N., Mašek V., Joly N., Madzak C., Laloue M. (2008) Mechanism-based inhibitors of cytokinin oxidase/dehydrogenase attack FAD cofactor. J. Mol. Biol, 380 : 886-899

Jolivet, P., Negroni, L., d´Andréa, S., Chardot, T., (2008). Oil bodies. Plant Proteomics: Technologies, Strategies, and Applications. G. K. A. R. Rakwal, John Wiley & Sons, Inc.

Gohon, Y., Dahmane, T., Ruigrok, R. W. H., Schuck, P., Charvolin, D., Rappaport, F., Timmins, P., Engelman, D. M., Tribet, C., Popot, J-L., Ebel, C. (2008) Bacteriorhodopsin/amphipol complexes: structural and functional properties. Biophys J, 94 : 3523-3537.

Meyer P., Evrin C., Briozzo, P., Joly, N., Bârzu, O., Gilles, A.M. (2008) Structural and functional characterization of Escherichia coli UMP kinase in complex with its allosteric regulator GTP. J Biol Chem, 283 : 36011-36018

Beopoulos, A., Mrozova, Z., Theveniau, F., Le Dall, M.-T., Hapala, I., Papanicolaou, S., Chardot, T., Nicaud, J.-M. (2008) Mastering lipid accumulation in the yeast Yarrowia lipolytica. Appl Env Microbiol, 74: 7779-89.

d'Andréa, S., Jolivet, P., Boulard, C., Larre, C., Froissard, M., Chardot, T. (2007a) Selective One-Step Extraction of Arabidopsis thaliana Seed Oleosins Using Organic Solvents. J Agric Food Chem, 55: 10008-15.

d'Andréa, S., Canonge, M., Beopoulos, A., Jolivet, P., Hartmann, M. A., Miquel, M., Lepiniec, L., Chardot, T. (2007b) At5g50600 encodes a member of the short-chain dehydrogenase reductase superfamily with 11beta- and 17beta-hydroxysteroid dehydrogenase activities associated with Arabidopsis thaliana seed oil bodies. Biochimie, 89 : 222-229.

Purkrtova, Z., S. d'Andréa, Jolivet, P., Lipovova, P., Kralova, B., Kodicek, M., Chardot, T. (2007) Structural properties of caleosin: a MS and CD study. Arch Biochem Biophys, 464 : 335-43.

Athenstaedt, K., Jolivet, P.; Boulard, C., Negroni, L., Zivy, M., Nicaud, J.-M., Chardot, T. (2006) Lipid particle composition of the yeast Yarrowia lipolytica depends on the carbon source. Proteomics, 6: 1450-1459.

Jolivet, P., Roux, E., d'Andréa, S., Davanture, M., Negroni, L., Zivy, M., Chardot, T. (2004) Protein composition of oil bodies in Arabidopsis thaliana ecotype WS. Plant Phys Biochem, 42: 501-509.