Laboratoire de Glycobiologie et Matrice Extracellulaire Végétale





























Topic II. Cell wall glycomolecules and cell growth

Principal investigators: J-C. Mollet, Professor and A. Lehner, Associate Professor.
Participants: P. Lerouge, Professor ; A. Driouich, Professor ; M. Bardor, Professor ; A. Mareck, Associate Professor; M-L Follet-Gueye, Associate Professor; E. Rivet, Associate Professor; M-C. Kiefer-Meyer, Research Engineer; C. Burel, Technician; C. Plasson, Technician. PhD students: J. Dehors (2016-present).

Project: Cell growth is driven by turgor pressure and depends on subtle changes in the extensibility of the primary cell wall. Our project aims to understand the relationships among the enzymes involved in cell wall biosynthesis and remodeling of pectin and xyloglucan, known to modulate the cell wall rigidity, on different aspects of plant cell growth such as elongation, plasticity, signaling... We will mainly focus our work on two models of elongating cells in which are the root and the pollen tube.

Pectins are composed of homogalacturonan (HG), rhamnogalacturonan I and II. HG is composed of a linear chain of α(1,4)-GalA residues synthesised in the Golgi apparatus in a fully methylated form and is then demethylated in the cell wall by Pectin Methyl Esterases (PMEs). Remodeling of HG by pectin-modifying enzymes (PMEs, polygalacturonases and pectate lyases) affects the cell wall plasticity and plant growth (Sénéchal et al., 2014) and is also involved in plant defence. In addition to PMEs, plants produce PME inhibitors (PMEIs) capable in maintaining PMEs in an inactive state during the secretion and/or in the cell wall. Several PMEIs are synthesized in a mature form and others are released from the N-terminal region of PMEs after processing by subtilases (Levesque-Tremblay et al., 2015).

In pollen tubes, our work on PMEs has led to the functional characterization of PME48 and highlighted the involvement of calcium and PMEI as regulators of the cell wall plasticity (Leroux et al., 2015; Paynel et al., 2014). We now would like to focus our investigations on the presence and the role of PME-PMEI pairs and the processing of PME by subtilases and how they control pectin demethylation. Moreover, methanol produced during PME activity and reactive oxygen species (ROS) together with calcium were shown to be involved in the signalling pathway leading to pollen germination, pollen tube growth and pollen tube rupture (Speranza et al., 2012; Potocky et al., 2007; Boisson-Dernier et al., 2013; Hamamura et al., 2014; Duan et al., 2014). ROS are also able to modify the cell wall plasticity. The project will investigate whether a regulatory network exists between the cell wall, cell wall modifying enzymes, calcium and ROS. We will use a combination of functional genetic with mutants impaired in the expression of PME, PMEI, PGase and PLL expressed in pollen (Mollet et al., 2013) and found by proteomic studies (Holmes-Davis et al., 2005; Noir et al., 2005). We will then investigate the impact of the mutation on the cell wall and the signalling of calcium (with plants expressing the calcium sensor yellow cameleon YC3.60, Iwano et al., 2012), ROS with probes such as CM-H2DCFDA, nitroblue tretrazolium (NBT) and singlet oxygen sensor green and NO with 4-Amino-5-Methylamino-2',7'-Difluorofluorescein Diacetate (DAF-FM-DA). ROS and NO will also be quantified using electron paramagnetic resonance (in collaboration with V. Richard, University of Rouen, oxidative stress Platform). Using a pharmacological approach using lanthanum chloride (a calcium-channel blocker), diphenylene iodonium (inhibitor of ROS production by blocking the NADP(H) oxidase), sodium benzoate or sodium salicylate (ROS scavengers), methyl viologen (ROS inducer), H2O2, Carboxy PTIO (NO scavenger), L-NG-Nitroarginine (L-NNA an inhibitor of nitric oxide Synthase, NOS) and sodium nitroprusside (SNP,a  donor of NO), we will investigate their effects on the expression of cell wall remodelling enzyme genes (in collaboration with Pr. J. Pelloux, University Jules Verne Picardie, France), the cell wall composition using cell imaging and biochemical approaches and how it is involved in the regulation of the cell wall mechanical properties (In collaboration with Pr. C. Ringli, University of Zürich, Switzerland).

We recently demonstrated that the glycosyltransferase inhibitor 2F-Fucose (2F-Fuc) was able to stop the root cell elongation by inhibiting RG-II biosynthesis and fucosylation of xyloglucan (Dumont et al., 2015). This fluoro analogue of fucose also had a strong effect on pollen tube growth (unpublished). It is known that rapidly expanding cells respond dramatically to defects in cell wall integrity. Several studies have reported that this response is mediated by a cell wall integrity-sensing mechanism involving cell wall sensors and receptors (Humphrey et al., 2007; Hématy and Höfte, 2008; Boisson-Dernier et al., 2011; Engelsdorf and Hamann, 2014). We would like to use 2F-Fuc for the selection, in an EMS mutant population, of Arabidopsis lines that are resistant to this inhibitor. This would allow the identification and characterization of new candidate proteins involved in the maintenance of the cell wall integrity.

In addition to functional genomics and pharmacological approaches, we will use a new generation of biological tools that are currently under development in the two transverse projects, 1) the click-mediated labelling of cell wall polymers to study the dynamic of glyco-polymer biosynthesis during growth, for instance in growing pollen tubes. 2) The correlative Light Electron Microscopy which permits to image cell processes within the same region of interest. For example, this technique is of particular interest to study whether pectic polysaccharides and their remodelling enzymes are sent to the cell wall within the same Golgi vesicles.

References: Boisson-Dernier et al. (2011) J. Exp. Bot., 62, 1581-1591. Boisson-Dernier et al. (2013) Plos Biol. 11, e1001719. Duan et al. (2014) Nature Com. 5, 3129. Engelsdorf  & Hamann  (2014)Ann. Bot. 114, 1339-1347. Hamamura et al. (2014) Nature Com. 5, 4722. Hématy and Höfte (2008) Curr. Opin. Plant Biol. 11, 321-328. Holmes-Davis et al. (2005) Proteomics 5, 4864–4884. Humphrey et al.  (2007). New Phytol. 176, 7-21. Iwano et al. (2012) Development 139, 4202-4209. Levesque-Tremblay et al. (2015) Planta 242, 791-811. Noir et al. (2005) Biochem. Biophys. Res. Commun. 337, 1257–1266. Potocký et al. (2007) New Phytol. 174, 742-51. Speranza et al. (2012) Plant Biol. 14, 64-76.