CE20 - Biologie des animaux, des organismes photosynthétiques et des microorganismes

Towards a comprehensive model of plant plasma membrane lipid bilayer – PLAYMOBIL

Purification and complete lipidomic analysis of the Arabidopsis thaliana plasma membrane
Development of digestion and labeling methods for different lipid families coupled with lipidomic analysis to allow the determination of the composition of each of the leaflets
Development of specific molecular models of the identified lipid species and coarse-grained modeling of the leaflets based on the obtained data concerning their composition
Implementation of genetic and pharmacological approaches to analyze the impact of targeted modifications of lipid composition on membrane organization and early plant defense signaling steps

This project used the model plant Arabidopsis thaliana to purify its cell membrane and carry out a complete analysis of its lipid and protein composition. Reference proteomic methods were used, as well as an innovative combination of lipidomic analysis techniques. The analyses carried out identified the complete composition of the plant plasma membrane (over 400 molecular species of lipids and 2100 different proteins) and highlighted the adjustment of lipid and protein characteristics within the membrane. Selective enzymatic digestion methods and the use of specific probes have enabled progress to be made towards identifying the lipids present on each of the leaflets, showing for some of them an asymmetry in their distribution. The biophysical characteristics of each leaflet were analyzed using fluorescent probes whose properties depend on the organization of the molecules between them, and by developing protocols enabling them to be specifically addressed to one or other of the two membrane leaflets. These approaches demonstrated that the outer leaflet of the membrane was more ordered than the inner one. Atomistic modeling approaches have produced the first structure of plant-specific sphingolipids (GlycosylInositolPhosphorylCeramides, GIPC) and analyzed the molecular determinants of lipid chain coupling between leaflets. All-atom simulations of membranes composed of PC, GIPC, PS and cholesterol (with different GIPC and PS fatty acid chain lengths) revealed GIPC mobility from the outer to the inner leaflet, and thus potential interactions with inner leaflet lipids, for all simulations with different GIPC and PS chain lengths. In addition, lipid density analyses confirmed this observation, with the densities of the outer and inner leaflets overlapping beyond the leaflet separation.

The Playmobil project has made available to the community the most complete repertory ever proposed of the lipids and proteins present on plant cell membranes. It led to significant advances in the understanding of the asymmetric composition of membrane sheets, and demonstrated for the first time that they have different physical properties. Finally, it laid the methodological foundations for producing, on the basis of the experimental results obtained, a realistic model of the organization of the lipid bilayer of the cell membrane.
The results concerning the lipidome and proteome of the membrane are the subject of a currently submitted publication. Results concerning both the experimental part and modeling approaches to explore the role of GIPCs and PS in sheet coupling and domain formation will be included in a publication scheduled for submission in the first quarter of 2024. The results concerning the physical properties of each of the membrane sheets, their specific composition and the production of a coarse-grained model directly derived from the experimental data will be brought together in a publication describing the asymmetry of the membrane, to be written and submitted in 2024.

1 Mamode-Cassim et al FEBS Lett. 2020 Nov;594(22):3719-3738. doi: 10.1002/1873-3468.13987
2 Mamode-Cassim et al JBC 2021, 296:100602. doi: 10.1016/j.jbc.2021.100602
3 Genva et al,, Plant J. 2023 Nov 8. doi: 10.1111/tpj.16525. A global LC-MS2 -based methodology to identify and quantify anionic phospholipids in plant samples
4 Bahammou et al MS ID#: BIORXIV/2023/540643 A combined lipidomic and proteomic profiling of Arabidopsis thaliana plasma membrane

Submission summary

In all organisms, the plasma membrane (PM) forms a selective barrier between the cell and the extracellular medium. It is a sensor for modifications of cellular environment, and a platform orchestrating signal transduction allowing translation of external signals in a finely tuned appropriate responses. The cell PM thus plays across kingdoms a critical role in cell physiology. The universal basic PM structure, established from the fluid mosaic membrane model (FMMM), is a lipid bilayer, in which proteins are embedded or associated to via a variety of interactions. Data accumulated since the publication of the FMMM revealed the unexpected and outstanding complexity of PM organization, and the essential role of lipids. Major classes of lipids are shared by all living organisms, but plants exhibit some striking characteristics including the different molecular species of sterols, free or conjugated, or specific sphingolipids. The heterogeneity of PM and the relationships between the dynamics of membrane organization and cell signalling has recently emerged as a key feature of cell biology. In plants, the presence of nano- to micro-scale domains exhibiting different lipid and protein content and biophysical characteristics, was demonstrated, together with the differential ability of plant lipids to generate such biophysical heterogeneity. Moreover dynamic relocalization of lipids and proteins within PM, concomittent with modifications of PM order and fluidity, have been particularly documented in early steps of plant-microorganism interactions. Deciphering the basis of PM organization at the molecular scale thus appears as a key step to understand the ability of plant cells to face the different environmental stress. In this context, a crucial point to describe PM organization is the asymmetry between the PM leaflets, and the remaining unraveled mechanisms of their coupling : we need to understand how the PM is able to ensure its physiological function, coordinating the translation of signals coming from the cell medium in which the outer leaflet is exposed, into appropriate responses through the activation of the cellular machinery facing the inner leaflet. We are currently missing reliable and comprehensive data describing plant PM asymmetry, regarding both its composition and organization. In this project, we will take advantage of the very complementary expertise of the different partners in membrane biochemistry, lipid analysis, cell signaling and modelling to develop an interdisciplinary project addressing this crucial question. The project will use the model plant Arabidopsis thaliana to : 1/ produce the full reference lipidome for the plant PM; 2/ address the question of plant PM asymmetry through the characterization of the lipid composition and in vivo biophysical characteristics of each PM leaflet 3/ combine atomistic and coarse-grained modelling approaches, used in back and forth with data generated by these experiments to provide an unprecedented comprehensive model of the plant PM bilayer. Genetic and pharmacological tools will allow to test in vivo the influence of the different lipids identified (i) on the biophysical characteristics of the inner and outer leaflets, (ii) on the dynamics of proteins differentially anchored to the PM inner and outer leaflets, (iii) on the very early steps of immune signaling. Our project will benefit from state of art methodologies to produce substantial cutting edge results, and also put a strong focus on the production of data which should help the plant community to move a step forward on these questions. Combining results of experimental and modelling approaches the project aims at establishing the foundations for a global understanding of plant PM leaflet asymmetry regarding lipids, which are essential constituents providing the core architecture and instrinsic biophysical features of PM, and shedding light on the wide open question of the molecular coupling between two leaflets.

Project coordination

Francoise Simon-plas (AGROECOLOGIE - UMR 1347)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.


LBM Laboratoire de biogenèse membranaire
GEC Unité de Génie Enzymatique et Cellulaire
UMR Agroécologie AGROECOLOGIE - UMR 1347

Help of the ANR 425,338 euros
Beginning and duration of the scientific project: September 2019 - 42 Months

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