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Biogenesis, dynamics and homeostasis of membrane lipids

Published on 4 July 2017
Head



Éric Maréchal

Phone: 04 38 78 49 85
Fax: 04 38 78 50 91


Juliette Jouhet

Phone: 04 38 7838 55
Fax: 04 38 78 50 91


Address

Laboratoire Physiologie Cellulaire & Végétale
CEA-Grenoble
17 avenue des Martyrs
38 054 Grenoble cedex 9
France


Members

Catherine Albrieux, CNRS Engineer
Alberto Amato, CEA Post-doc
Olivier Bastien, INRA Researcher
Élodie Billey, CEA Post-doc
Maryse Block, CNRS Researcher
Séverine Collin, Total Researcher
Mélissa Conte, CEA Engineer
Younès Dellero, CEA Post-doc
Denis Falconet, CNRS Researcher
Valérie Gros, CNRS Technician
Juliette Jouhet, CNRS Researcher
Morgane Lapeyre, CEA Technician
Mariette Lefranc-Bedhomme, CEA Researcher
Mehdi Lembrouk, CEA Technician
Josselin Lupette, PhD student
Julie Marais, CEA Technician
Éric Maréchal, CNRS Researcher
Coralie Metton, CEA Technician
Fabrice Rébeillé, CEA Researcher
Suzanne Rose, CEA Researcher
Sylvaine Roy, CEA Bioinformatics engineer
Juliette Salvaing, INRA Researcher
Khawla Seddiki, CEA Engineer

June, 2017


Museum of Grenoble, June, 2016


Roscoff, April, 2016


2015



Past members

César Botella, PhD thesis student
Cyrille Botté, Marie-Curie Post-doc
Yoshiki Yamaryo-Botté, Tokyo Institute of Technology Post-doc CR2 CNRS
Céline Cataye, AI ANR
Florian Chevalier, CNRS Post-doc
Olivier Clerc, IE CNRS
Mélissa Conte, AI CEA
Mathilde Cussac, technician
Lina-Juana Dolch, PhD thesis student
Cécile Durand, Fermentalg technician
Aurélie Fangain, Engineer
Marina Leterrier, Fermentalg Post-doc
Coline Mei, PhD thesis student
Morgane Michaud, Post-doc
Christian Morabito, Engineer
Camille Rak, AI CEA
Alexandra Thomas, BTS Anabiotech

Guillaume Tourcier, AI CEA


Research axis: glycerolipid hoeostasis in plastid-containing eukaryotes: algae, protists and plants.

Glycerolipids are a specific group of lipids, consisting of a glycerol backbone to which 1 to 3 fatty acids are linked by an ester bond (Figure 1). Glycerolipids containing three fatty acids are called triacylglycerols, or oils, and accumulate in the form of droplets within the cells. Glycerolipids containing two fatty acids may have a polar head, and are used to build bilayers, forming the matrix of biological membranes. Ten classes of glycerolipids are thus used to build all membranes in eukaryotic cells. Among these lipids:

  • Phospholipids are the major membrane lipids in animal or yeast cells and in non-plastidial membranes of plant cells;
  • Plants are characterized by the presence of a unique class of non-phosphorus membrane lipids, the galactolipids synthesized in the membranes limiting the chloroplast (plastid envelope). Due to their high level in photosynthetic membranes, the galactolipids form the most abundant lipid class in the biosphere.
  • Two intermediates at the crossroads of several glycerolipid synthetic pathways, phosphatidic acid and diacylglycerol, play important metabolic and signaling roles.
  • Triacylglycerols are synthesized by addition of a third fatty acid onto diacylglycerol. Triacylglycerols are used for third generation biofuels.




Figure 1: Glycerolipids, constituents of biological membranes and oils.

In a eukaryotic cell, each membrane compartment (plasma membrane, endoplasmic reticulum, Golgi, vacuole, mitochondria, plastids, etc.) has a specific lipid composition, regulated according to physiological and environmental conditions. For example, under phosphate deprivation, a breakdown of membrane phospholipids is triggered, and galactolipid synthesis increases. Phospholipid breakdown is considered as a phosphate-saving response. Galactolipids are exported from chloroplasts to other cellular membranes usually devoid of galactolipids such as mitochondrial membranes. The coupling processes between phospholipid synthesis (in the endoplasmic reticulum) and galactolipids (in the chloroplast envelope) and the regulatory mechanisms and machineries of lipid transfers are unknown.

The team addresses the following questions:


How are glycerolipids synthesized in photosynthetic cells and how do they participate in the construction of membranes?

Most enzymes involved in lipid syntheses are known in plants (Arabidopsis). Knowledge is more fragmented in algae (Chlamydomonas) and in protists possessing plastids limited by four membranes (Chromera, Pheodactylum, etc.). We seek to identify missing enzymes, inter-membrane lipid transport systems and regulators. We integrate information and biological data to design models of metabolic networks (from experimental biology to systems biology).
Our efforts have focused on the synthesis of galactolipids in the plastid envelope. We seek to understand how the galactolipids contribute to the construction of thylakoids, the largest membrane system of the biosphere, organized in the form of flattened cisternae inside chloroplasts, and containing photosystems that capture light energy. We consider these issues by using biophysical methods, particularly reflection / neutron diffraction.


How is the metabolism of glycerolipids coordinated at whole cell level?

We study the coordination of lipid synthetic pathways within the plastid envelope and at the whole cell level. We focus on coupling factors, including signaling lipids that are produced in a membrane and act in another membrane compartment. In particular, we analyze the role of phosphatidic acid in regulating the synthesis of galactolipids.



Figure 2A. The synthesis of phospholipid and oils (triacylglycerols) takes place in the reticulum.



Figure 2B. The synthesis of galactolipids is localized in the envelope that limits plastids.
A dialogue between the chloroplast and endomembrane system allows the coordination of lipid synthesis pathways in the different membranes. This dialogue requires machineries to transfer lipids (dashed arrows). The diacylglycerol (DAG) and phosphatidic acid (PA) intermediates are at the intersection of all pathways.


How does this "system" adapt in response to environmental variations?

We consider the metabolic pathways and trafficking of lipids as a "system", and we whish to identify and characterize important regulatory processes by studying the physiological and metabolic responses in the following contexts:
• in mutants (genetics);
• under conditions that promote the synthesis of galactolipids, such as phosphate deprivation (physio-ecology);
• using small molecules we have developed and which block the synthesis of galactolipids (chemical genetics).

We use in particular a novel class of inhibitor obtained by high-throughput screening and chemical optimization (in collaboration with Bernard Rousseau, iBiTec-S, SMMCB, CEA Saclay). These molecules, called galvestines, are used as tools for research, allowing a study of membrane lipid homeostasis.



Figure 3: Some effects of Galvestine-1 at the levels of the target enzyme (MGDG synthase, MGD), sub-cellular development (biogenesis of chloroplasts), leaf development or pollen tube growth.

How to model this metabolism in silico?

Combining knowledge of the lipid system and experimental data, we have initiated:

• The development of a new in silico representation of metabolic pathways, dedicated to the synthesis of fatty acids. The biosynthetic pathways currently available in the KEGG and MetaCyc databases do not cover the very large molecular diversity of fatty acids (8 to > 30 carbons, even of odd numbers of carbons, linear or branched chains, with double bonds, oxidations, rings, etc..) and do not account for redundant synthesis pathways. We developed a pilot database called AcylUniverse, automatically populated by virtual enzymes mimicking the enzymatic activities found in nature. AcylUniverse outperforms KEGG and MetaCyc, allows the design of scenarios for interpreting lipidomic data and will be useful in the field of agrochemistry.
• The development of numerical approaches to simulate and understand metabolic fluxes and pathways of lipid synthesis. We seek to provide new conceptual tools and methods to understand how lipid synthesis in membranes are related to the construction of membranes.


How to control this system in microalgae and photosynthetic protists and produce oil for third generation biofuels and biomedical applications.

Triacylglycerols are a natural source of fatty acids that can be used:

- As a substitute for petroleum fuels (particularly unsaturated, linear, short chain fatty acids)
- As a source of biomolecules for health (especially omega-3 desaturated or oxidized fatty acids).
The current paradigm is that microalgae will be a source of oils for these future applications, based on the accumulation of triacylglycerol in algal cells when deprived in nutrients, especially nitrogen. This accumulation of oil combines neo-syntheses and remodeling of membrane lipids. We use eco-physiology, genetics and chemical genetic approaches to optimize simultaneously the biomass yield and the production and quality of oil. We conduct our research on diatom (Pheodactylum) and chromerids (Chromera). This work is achieved in close collaboration with Giovanni Finazzi (team 01, LPCV), and is supported by grants from the BioEnergy program of the CEA Life Science Division and from the National Agency for Reseach (ANR Bio-Materials and Energy - DiaDomOil).


The biodiversity of photosynthetic organisms, capturing atmospheric CO2 and converting it into organic molecules, comes from a very complex evolutionary history.

Photosynthetic organisms, from algae to vascular plants, have a unique cellular organization characterized by the presence of a chloroplast. This organelle contains stacked membranes in large numbers, also called thylakoids, in which are inserted in the photosystems. It is estimated that 1 m2 of leaves contains 2.5 hectares of photosynthetic membranes, more than twice the size of a football stadium. The photosynthetic surface capturing solar energy by phytoplankton has not been evaluated, but it is certain that this biological membrane structure has a colossal dimension at the scale of the biosphere. In fact, photosynthetic organisms have that faculty to capture CO2 after conversion of light energy into chemical energy and reducing power, and carbon thus removed from the atmosphere feeds the metabolism and all pathways synthesizing organic biomolecules. Biomass represented by the phytoplankton of the oceans accounts for only 1-2% of plant carbon in the biosphere, but these organisms fix about 40% of the CO2. Biomass built by these primary producers is at the basis of the food chain, with finally a return of carbon to the atmosphere as CO2 by the respiration process. Large volumes of biomass have formed deposits over geological times and are currently used as oil called "fossil fuels", leading to a CO2 emission with a time lag and unbalanced cycle. The optimization of biomass production by humans, with appropriate quality to replace fossil fuels would not only compensate for the depletion of this resource in the coming decades, but also would synchronize the capture and emission of atmospheric CO2. A major effort has been conducted in the search for solutions to produce and use biomass, starting with trials using agro-resources. Proof of concept for conversion of biomass grown on land (sugar cane, rapeseed, etc.) for biofuels of first and second generation was made, but the competition between cultivated plants for food and feed and bioenergy is not sustainable, and the use of polluting inputs causes environmental issues. Moreover, the corresponding economic models are not sustainable in the long term. A promising solution is to use algae, especially microalgae, because this resource is theoretically easy to grow in open or closed systems, and can accumulate organic compounds readily convertible into fossil fuel substitutes, i.e. triacylglycerol or oil. Nevertheless, although the proof of concept has been provided that it was possible to amplify the accumulation of oil in these organisms, up to 80% of dry weight in our laboratory conditions, the biomass produced is still too low and the economic viability of an industry based on these algo-resources is much debated.

Chromalveolates (or Chromalveolata).
Chromalveolates are a phylum comprising protists whose emergence follows a secondary endosymbiosis with a red alga and whose cells often contain membrane sacs apposed under the plasma membrane (these membrane cisternae are also called alveoli). This super-group was defined in 1981 by T. Cavalier Smith, and is considered in the classification revised in 2005 as one of the 6 major eukaryotic taxa. Chromalveolata include among other phyla: Apicomplexa, Chromerids, Diatoms and Haptophytes among which Eustigmatophytes. The refined phylogeny of this group is still controversial and requires a major effort of research to which we contribute.

Biological models studied by the team.
One of the strengths of the Plant Cell & Physiology Laboratory in CEA Grenoble lies in the study of photosynthetic organisms using a variety of well defined models, ranging from unicellular algae (with an important scientific production on Chlamydomonas) to vascular plants (Arabidopsis model). The team also aims to define the general principles that are common to Chromalveolata, regarding the organization of glycerolipid metabolism and the production of oil, based on comparative studies using other models, Phaeodactylum and Nannochloropsis, and in collaboration with laboratories that were pioneers in the corresponding genome sequencing, respectively Chris Bowler at ENS Paris and Shawn Starkenberg at Los Alamos National Laboratory, USA. Together with Chromera, and Arabidopsis which is our reference for higher plants, these organisms are thus study models for our team.



Figure 4: Models of photosynthetic organisms and Chromalveolata studied in the laboratory of Plant Cell Physiology, and more specifically in the team (framed with dashed lines)

The team is affiliated to:

- Institut Carnot LISA (Lipides pour la Santé et l'Industrie / Lipids for Health and Industry)
- Institut Multidisciplinaire de Biochimie des Lipides (GIS IMBL)
- Groupe d'Etude et de Recherche en Lipidomique (GDR GERLI)
- LABEX GRAL
- OCEANOMICS consortium (Biotechnologie et bioressources pour la valorisation des écosystèmes marins planctoniques)


Collaborative projects are supported by:

- The National Agency for Research / ANR :
ANR PlasmoExpress, coord L. Bréhélin, Montpellier
ANR RéGal, coll. A. Girard-Egrot, Lyon and C. Breton, Grenoble
ANR DiaDomOil, coll. C. Bowler and A. Falciatore, Paris and Fermentalg, Libourne, label "pôle de compétitivité" AXELERA

- Institut Carnot LISA (coll. C. Joffre, Bordeaux)

- CEA Life Science Division, BioEnergy program (coll. JC Cintrat, iBiTec-Saclay and MO Fauvarque, LBGE-iRTSV)

- EU FP7 Marie-Curie (coll. G. McFadden, Melbourne, Australia).




Our team hosts collaborative projects with

• the Fermentalg company. Read the March 3, 2014 Press release entitled: Fermentalg and the CEA optimize the production of microalgae in a mixotrophic environment.

Seminar

Podcast 'Workshops of the Information' about the seminar regarding the use of light to decipher the life architectures (in french).
  

Keywords

Glycerolipids, membrane lipids, galactolipids, oil, biofuel, plants, algae, protists, Chromalveolates, Diatoms, Apicomplexa, Lipidomices, Physiology, Chemical genetics, Systems Biology, Bioinformatics.