Since 1998, Fran莽ois Chaumont has developed and directed a research group aiming at answering hypotheses related to the function and regulation of plant aquaporins. In addition, at Marc Boutry retirement in 2016, he joined Catherine Navarre, a senior scientist, in the supervision of research projects aiming to develop an efficient cellular farming platform. Finally, since 2001, he is also the academic referent for projects developed by the senior scientist Marie-Christine Flamand on the genetic diversity of the Walloon fauna.
The team, today
Aquaporin: Two postdocs: Lei Ding and Ana Romina Fox; two PhD students: Estelle Teirlinckx and Maxime Laurent; one part-time technician: Joseph Nader; three master students: 听Jean Fontaine, Yasmina Mesbah, Alexandre Gatot.
Cellular Farming: Senior Scientist: Catherine Navarre; four PhD students: Laurent Bouhon, Nicolas Bailly, Marie Peeters, Mathilde Moens; one technician: Adeline Courtoy; four master students: Romane Vandevelde, Antoine Mercier, Christophe Magidson et Ma毛lle No毛l.
Genetic diversity: Senior scientist: Marie-Christine Flamand; part-time technician, Adrian Neveu
The team also benefits from the important contribution of the FYMO administrative and technician staff: Mich猫le Rochat, Monique Leloup, Ben El Amraoui, Marie-Eve Renard, Marie-Christine Eloy.
About the current projects
Aquaporin projects
Plant growth and development are dependent upon tight regulation of water and small solute transport across cell membranes and tissues. Their diffusion through the membranes can be facilitated by aquaporins (AQPs). Compared with animals, plant AQPs are more diverse, both in terms of number (> 30 isoforms) and functional features. For instance, we recently identified 84 AQP genes in Nicotiana tabacum.
The research projects developed in our laboratory aim at understanding the roles of AQPs in plants and deciphering the molecular mechanisms regulating them, combining complementary approaches, from molecular and cellular biology to genetics, biophysics and physiology. During the last years, we mostly focused on the PIP (plasma membrane intrinsic proteins) subfamily of AQPs. We significantly contributed to a better understanding of their transcriptional regulation, their subcellular localization, their substrate specificity and their physiological roles in root, leaves and, in particular, stomatal complexes.
We are currently analyzing the genetic variability of AQP expression in maize leaves in well-watered and water deficit conditions. 听A genome-wide association studies (GWAS) allowed us to identify which markers were statistically associated with variations in the expression of each AQP gene, and grouped into eQTLs (expression Quantitative Trait Loci). This identified regions where putative regulators of AQP gene expression might be located, and we are currently focusing on transcription factors (TFs). The ability of the TFs to transactivate/repress the AQP promoter activity is assessed using a successfully developed dual-fluorescence reporter system working in maize leaf cells on entire plants or isolated protoplasts. The role of the TFs at the cell and plant levels is also analyzed.
PIP trafficking to and from the plasma membrane includes highly regulated mechanisms involving many protein-protein interactions. Pull down assay using maize suspension cells expressing YFP-PIP2;5, yeast split-ubiquitin and bimolecular fluorescence complementation (BiFC) assays identified the endoplasmic reticulum (ER) resident (VAMP)-associated VAP27-1 as a PIP2;5 interactor. Both proteins localize in close vicinity of ER-plasma membrane contact sites (EPCS) and endocytic structures upon exposure to salt stress conditions. Under normal condition, VAP27s could recruit and stabilize the PIP channels in EPCSs. Upon abiotic stress, this interaction might guide the channels to rapid endocytosis and autophagic degradation. As PIPs also interact with cytoskeleton components, we hypothesized that PIP interactions at EPCS and the cytoskeleton contribute to the structural organization of the cell in response to stimuli.
Stomata are pores formed allowing CO2 uptake for photosynthesis at the expense of water loss by transpiration. While most stomata are formed by two kidney-shaped guard cells, the graminoid stomata of grasses consist of two dumbbell-shaped guard cells (GCs) flanked by two subsidiary cells (SCs). Grass stomata can open and/or close more rapidly than non-grass stomata providing these plants with more efficient photosynthesis and water use.听 We hypothesize that PIPs play several key roles in the differential regulation of GCs and SCs during stomatal opening/closure. We recently showed that PIP2;5 is a key actor increasing the response of stomatal closure to water deficit, due to its water and hydrogen peroxide channel activities. We recently demonstrated that cell specific CRISPR-TSKO technology to knock-down PIP gene expression specifically in GCs or SCs was possible. We are currently targeting specific PIP genes for knock down in these cells and characterizing their roles in stomatal dynamics.
Cellular Farming
Plant cell suspensions, in particular Nicotiana tabacum Bright Yellow-2 (BY-2), are established as a platform for producing recombinant glycoproteins such as antibodies, enzymes, and viral glycoproteins. The main reasons are the absence of contamination risk by human pathogens, the easily implementable GMP compatible culture conditions, and the presence of typical eukaryote post-translational modifications like N-glycosylation.
Plants have a glycosylation machinery close to that of mammalian cells, with, however, two non-human glycans, core a(1,3)-fucose and b(1,2)-xylose. By CRISPR-Cas9 editing, we succeeded in inactivating the genes involved in this non-mammalian glycosylation, thus allowing for the production of humanized glycoproteins. To illustrate the flexibility of the BY-2 expression system, we recently obtained transgenic lines that allow for the production of glycoproteins with simplified N-glycosylation profile. This constitutes very efficient tools to decipher the role of glycans on the immune response or on the attachment of the SARS-Cov-2 spike 听ectodomain to the mammalian cell receptors.
However, plant cells lack the capacity to perform mucin-type O-glycosylation, the most abundant type of mammalian O-glycosylation, while having their own specific pathways such as hydroxyproline O-glycosylation. We are currently glycoengineering BY-2 cells in order to humanize their O-glycoproteome by introducing selected enzymes of the mucin-type O-glycosylation, and knocking out the proline-4-hydroxylase genes, catalyzing the first step of hydroxyproline O-glycosylation.
In spite of the flexibility and ease of the BY-2 host system, we face two issues. First, the production yields are usually lower than those obtained in other expression systems. This is often related to RNA silencing, an autoregulation process repressing the expression of foreign genetic elements. RNA silencing specificity is ensured by the production of small interfering RNAs from aberrant transcripts, notably catalyzed by the RNA-dependent-RNA-polymerases RDR1 and RDR6. We generated BY-2 cell lines inactivated for RDR1 and RDR6 activities via a multiplex CRISPR/Cas9 strategy and are evaluating them for transient production of reporter proteins.
The second issue is the partial proteolytic degradation of the recombinant glycoproteins. In particular, the production of the viral ectodomains gB from human cytomegalovirus and the spike ectodomain from SARS-CoV-2 secreted in different glycoengineered BY-2 cells revealed strong proteolytic degradation during secretion in the culture medium. We are currently identifying and investigating the role of proteases in the secretory pathway of N. tabacum BY-2 cells, combining proteomics, gene editing and expression of glycoproteins.
Genetic Diversity
These projects aim at characterizing the population biodiversity of wild animal species in defined geographic locations of the Wallonia region in close collaboration with the laboratory of the wild fauna and cynegetic and the unit of hydrobiology (DEMNA) from the Walloon administration.听 We apply the microsatellite molecular marker method together with the sequencing of mitochondrial DNA fragments for estimating the genetic diversity of populations (ungulates, wild boars, fish and crayfish) in the Walloon region. We determined the genetic phylogeny, biodiversity, and impact of stocking on trout (Salmo trutta), European grayling (Thymallus thymallus) and red-legged crayfish (Astacus astacus) in the Walloon rivers. We identified several still native populations of these three species that should be protected from stocking and we are currently analyzing the genetic impact of Fario trout restocking in a tributary of the Lesse (ri de Chicheron). The origin of the salmon returning from the North Sea to the Meuse for reproduction is also determined each year.
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