Cell proliferation, differentiation and polarity

We have identified several mutants in Arabidopsis thaliana, characterized by ectopic cell proliferation that develop into tumor-like structures in presence of cytokinins. These mutants called pasticcino or pas (italian for small cakes) are also characterized by important developmental and metabolic defects, in particular sugars and lipids accumulation (Faure et al. 1998; Harrar et al. 2003). The three PAS genes were identified and characterized. Surprinsingly, the three PAS proteins were found to belong to families conserved among higher eukaryotes including human.

PASTICCINO1 gene encodes an immunophilin from the FKBP family. This protein is characterized by a peptidyl cis-trans isomerase domain (PPiase) which is known in mamals to be involved in the assembly, the translocation and the activation of signalling complexes (receptors, transcription factors). We showed that the C-terminal domain of PAS1 controls the subcellular distribution of this protein and is required for its interaction with a new member of the plant-specific family of NAC transcription factors (Smyczinski et al. 2006).

PASTICCINO2 was identified by positional cloning and encodes the Arabidopsis member of the protein tyrosine phosphatase-like (Ptpl) family characterized by a mutated PTP active site. PAS2 is able to interact with a phosphorylated cyclin-dependent kinase (CDK) (Da Costa et al. 2006). This family of proteins has a yeast homologue that is essential for cell viability. The absence of yeast PAS2 homologue (PHS1) can be functionally replaced by the Arabidopsis PAS2 protein demonstrating that PAS2 function is conserved between higher and lower eukaryotes (Bellec et al. 2002). PHS1 and PAS2 were recently characterized as the dehydratase involved in very long chain fatty acid elongation complex providing a new and conserved functional link between lipids and cell proliferation (Bach et al. 2008).

PASTICCINO3 was identified as the cytosolic acetyl CoA carboxylase (ACCase, ACC1). The production of malonyl-CoA is involved in anthocyanin production and very long chain fatty acids present in cuticular waxes and sphingolipids. As expected, pas3 mutants showed a complete lack of anthocyanins and also depletion of very long chain fatty acids (VLCFAs) in seeds.

VLCFAs is involved in the synthesis of a specific class of lipids, the sphingolipids that are involved in the regulation of several signalling pathways associated with cell cycle, cell fate, differentiation and apoptosis but are also involved in the formation of membrane microdomains or "rafts" and in the establishment of cell polarity. We are coordinating the ANR blanc grant "SphingopolaR" (2007-2009) that is focusing on the involvement of sphingolipids in the establishment and maintenance of cell polarity and its impact on plant development. This project will not only provide a better knowledge of the physiological role of this essential lipids during plant development but will clarify the role of endomembrane trafficking in initiating and maintaining cell polarity.

Phloem ontogenesis and Cell polarity

The study of the establishment of phloem requires knowing the different sites of initiation of this tissue during development, its position relative to other tissues and the key stages of its formation. To implement this study, we are committed as a first step to develop cytological tools to quickly visualize differentiated tissue in various organs of the plant. The chronology of events leading to the establishment of mature sieve elements (functional transportation phloem) was traced back during the early development of Arabidopsis thaliana. To do this, we have identified in the collection of mutants insertion of Versailles, several markers of the expression of genes involved at various stages of differentiation in the phloem (Bauby et al., 2007). We were able to show that the formation of phloem required several stages during embryogenesis.

A step in determining which assigns them, to future cells from the phloem cells procambial. This position is determined during the acquisition of the symmetry of the embryo from the heart stage. The position of the phloem is determined on the abaxial face of cotyledon and the sagittal axis of the embryo and is opposed to the xylem. The positioning of cells can be modified in some mutants (pin1, pinoïd, rev) or abolished (wol, mp). That determination would be under the control of several parameters 1) initiation of cotyledons 2) the initial number of cells procambium.

-An early stage of differentiation, which is the establishment of different cell types including protophloem at the end of the torpedo stage of embryogenesis. This step led to the formation of two cells from a longitudinal division: protophloem and its adjacent cell.

- A stop of differentiation that occurs during the stages of seed maturation. This step is partially lifted in mutants affected in seed maturation (abi3, lec2, fusca3).

-A replay (resumption) of differentiation after seed imbibition that leads to the differentiation of mature elements with characteristics specific subcellular visible fluorescence. This reboot proceeded along an apico-basal gradient and is concomitant with the elongation of sieve elements.

We have identified a mutant affected in the differentiation of the phloem, named octopus. The mutant has a short primary root and displays early development of adventitious roots at the collar. The cytological analysis showed a lack of continuity of phloem, changes in the division and differentiation of the sieve elements along the root and the hypocotyl. These changes are accompanied by defects of long distance and an accumulation of starch. The gene encodes a higher plants specific protein of unknown function. This gene is expressed early during the determination of vascular cells and its expression is restricted thereafter to areas of phloem differentiation. OCTOPUS protein is localized in the plasma membrane. The functional study of this protein is undertaken to ascertain its role in phloem determination and differentiation.

Cell differentiation and cell polarity during regeneration.

Regeneration is defined as the ability of a group of somatic or germinal cells to differentiate into a new organ (shoot, root, embryo). Physiological conditions that promote plant regeneration have been intensively studied over the last 50 years and have provided the basis for clonal propagation and gene transfer technology. But the cellular and molecular bases of this process remain poorly understood due to the fact that regeneration takes place within an heterogeneous material made up of proliferating and differentiating cells.
We are coordinating the ANR Génoplante project "REGENEOME" that will develop and exploit new methodologies based on the use of Laser Assisted Microdissection (LAM) for transcriptome and chromatin profiling of specific cells isolated at different stages of the regeneration process .

Development of new tools

Protein-protein interactions and subcellular distribution in live cells. In the frame of the EU project AGRON-OMICS (http://www.agron-omics.eu/), we aim in deciphering protein interaction networks involved in leaf growth. We are thus developping medium to high throuput analysis of protein interaction by Bimolecular Fluorescence Complementation (BiFC). We have developped an optimized protocol for transient expression in Arabidopsis seedlings allowing efficient co-expression of fluorescent protein fusions in cotyledon cells (Marion et al. 2008). Subcellular distribution and co-localisation of protein can thus be directly adressed but also systematic BiFC studies
To provide an easy and non invasive way to follow protein interaction in time and space, we are also developping the use of fluorescence anisotropy by confocal microscopy.

Cellular phenotyping of Arabidopsis thaliana and other plants
We modified an existing cytology technique to visualize, using the confocal microscope, the different stages of development in Arabidopsis plant and / or entire organ set. This technique allows us to quickly access the different cell layers without making physical sections with a maximum optical resolution (depending on the lens used). This technique, using customized software to make a 3D visualization of surface or internal descriptions of plant material. It can be combined with the study of gene expression reporter UidA gene within the plant.