The industrial use of plant cell walls (CWs) is impacted by their heterogeneity and dynamics. Indeed, CW composition (e.g. polysaccharides, proteins) displays multiscale spatiotemporal specificities (e.g. evolutionary, developmental, cellular, subcellular). In some instances, this heterogeneity positively impacts CW use (e.g. cotton or flax fibers have been selected for millennia for their mechanical properties, now understood as corresponding to particular cellulose-enriched CWs). In other cases, CW heterogeneity negatively impacts CW use (e.g. the pulp and paper or biofuel industries need abundant homogeneous and easy-to-process CW material). CW heterogeneity is still poorly understood at the subcellular scale. Indeed, CWs may be viewed as the assembly of multiple microdomains that are growingly described through various (immuno)labellings. However, the molecular interactions and functions of these microdomains remain obscure. ‘MicroWall’ will uncover scaffolds of molecular interactions within CW microdomains and will provide functional roles for these polarised CW molecular scaffolds. The molecular components of particular interest will be CW proteins encoded by multigenic families and various patterns of a highly variable CW polysaccharide. The 3 multigenic families particularly studied will be Class III peroxidases (PRXs) for their dual roles of CW loosening or CW stiffening, pectin methylesterases (PMEs) and pectin methylesterase inhibitors (PMEIs) that control the methylesterification degree of homogalacturonan (HG) pectin domains. The specific HG patterns constitute the highly variable CW polysaccharide hypothesized to enable positioning of specific PRXs to specific individual CW microdomains through specific molecular interactions. These specific PRX localisations will contribute to CW dynamics through either polarized CW loosening or stiffening at the position of the individual CW microdomains. MicroWall aims at (i) establishing the proof of concept through the extensive characterization of one particular CW microdomain molecular scaffold involved in Arabidopsis seed development and for which we have convincing preliminary data, and (ii) providing evidence that this example is part of a more universal concept. The first goal of this project (WP1 and 2) will be achieved through the extensive pluridisciplinary characterization of a partially methylesterified HG microdomain created, during Arabidopsis seed development in the outer CW of mucilage secretory cells, by a yet-to-be discovered PME that is regulated by PMEI6. In turn, this HG microdomain is expected to enable PRX36 specific anchoring during seed mucilage secretory cell development. This accurate PRX36 anchoring will sequentially allow (i) loosening this CW microdomain during seed development, (ii) proper rupture of the loosened polarised CW during mature seed imbibition and (iii) correct mucilage release and efficient germination. The second goal of this project will be achieved through the transposition of this proof of concept HG/PMEI6/PRX36 model to flax mucilage secretory cell development (WP1), and through the search of additional similar CW microdomains involving other HG methylesterification patterns and other PRXs, PMEs and PMEIs co-expressed during mucilage secretory cell development (WP3). Indeed, the rationale is that individual members of these multigenic families that are co-expressed in a single cell may have non redundant functions because of their accurate positioning in CW microdomains of this individual cell. Beyond, these examples dedicated to the understanding of CW dynamics during seed mucilage secretory cell development, and since the molecular actors of these scaffolds are universal along plant development and evolution, this fundamental knowledge proposing putative functions for hundreds of protein and polysaccharide CW components, will be crucial for future industrial use of plant CWs in various contexts.

Pectins are a family of highly complex cell wall polysaccharides which play a major role in controlling plant vegetative and reproductive development. They are widely used in the food ingredient sector (i.e. as gelating agent/stabilizers or hydrogels for drug delivery). The simplest and the most abundant pectic block is homogalacturonan (HG), an unbranched polymer of alpha-(1-4) linked D galacturonic acids residues that can be methylesterified at the C6 position. This determines the degree of methylesterification (DM), which is controlled in the cell wall by the large multigenic family of pectin methylesterases (PMEs, 66 isoforms) and determines the susceptibility of HG to degradation by specific enzymes to produce oligogalacturonides (OGs). The properties of HG-derived OGs are related to their i) Degree of Polymerisation, ii) Degree of Methylesterification, and iii) pattern of methylesterification. In planta, oligogalacturonids are notably involved in eliciting plant defense responses against bacterial and fungal pathogens as well as aphids, a property that can be used to engineer dedicated biomass-derived products for plant protection. This approach requires a better knowledge of the structure, the specificity and the mode of action of OGs. This aspect is of importance in the current context of the reduction of chemicals used in phytoprotection (Grenelle de l’Environnement, EcoPhyto-2018). Based on preliminary results showing the relations between the structure of OGs and their biological activity, and using the biochemical specificity of PME enzymes, the project will provide a means by which we will be able to create specific methylester decoration on HG. Using this tool, the project will generate purified OGs of characterized degree of polymerization (DP) and degree/pattern of methylesterification. This will allow the precise structure/properties analysis of these OGs to be determined. In particular, to address the functionality and potential application of the generated OGs, their properties will be tested with regards to: i) responses of plants to bacterial and fungal pathogens and ii) effects on plant growth. The first aspect will notably concerns both model plant (Arabidopsis) and species of agro-economical interest (grape and potato). As an additional outcome, through an external collaboration, the potential prebiotic activity of these compounds will be tested. Altogether, the project will provide an integrated multidisciplinary approach relating the fine structure of pectic oligomers to their potential application. The project brings together three partners (Univ. Amiens, INRA Nantes, Univ./INRA Dijon) with highly complementary expertise, and external collaborator (Univ. Reading). The project fits into the field of three competitivity clusters (Industrie Agro-Ressources, Végépolys and VITAGORA) and has potential application as shown by letters of support (Cargill, Comité Nord, BIVB).

Drought stress impacts negatively on plant growth and product quality and this situation will become worse because of global climatic change. It is therefore important to improve not only our understanding of strategies used by plants to adapt to this stress, but also to learn more about its impact on different plant products. A major transformable plant product is the plant cell wall composed of cellulose, hemicelluloses and, in certain tissues, lignin. This resource (lignocellulosics) is transformed into biofuels and is used in bio-based materials. In this project, we aim to produce comprehensive data via multi-scale –omics analyses on the impact of drought stress – with a major focus on the cell wall - in two plant species (flax and Brachypodium). We have chosen these 2 species because we wish to evaluate the impact of drought stress directly on two major types of valorisable cell wall structure: flax for the use of their long cellulose fibres in composite materials and textiles and Brachypodium as a model bio-fuel species system. The NoStressWall project aims to: i) generate and integrate large amounts of transcriptome, metabolome and proteome data together with comprehensive analyses of cell wall structure and modifications induced by drought stress, ii) use a reverse genetics screen to identify specific mutants in available flax and Brachypodium chemical mutant populations, and iii) initiate preliminary functional characterization of selected mutants. One of the must important final products of this project will be the construction of the up-datable NoStressWall database including information on the multi-omics and cell wall analyses as well as the results of functional analyses of flax and Brachypodium mutants. A parallel monitoring of these data by different systems biology approaches will be performed in order to learn more about biologically relevant events during drought stress in both analysed species. The information will also inform us about the physiology of stress responses and adaptation strategies in higher plants. The identification and functional characterization of drought-stress genes in flax and Brachypodium, together with the wide range of genetic resources/tools that are available from other projects, will accelerate plant breeding programs and allow the development of other strategies such as whole genome and targeted association mapping approaches. The proposed project is essentially fundamental but as drought stress impacts directly on flax fiber quality and Brachypodium biomass production, our results on these species will therefore be of direct interest to breeders, farmers and end-users of fibers (composite materials, textiles) and biomass.

The plant primary cell wall is a complex structure composed of polysaccharides and proteins. It plays a central role in the control of plant growth and development, therefore in the production of biomass. Pectins are major components of the primary cell wall, representing up to one third of the wall dry mass, and are widely used in the food industry, as gelling agents. Among pectins, homogalacturonan (HG) is a homopolymer of ?-1,4-linked-D-galacturonic acid units that can be methylesterified and acetylated. Over the recent years, HG-type pectins have been reported as major actors of the modulation of the mechanical properties of the cell wall. They can mediate changes in growth, through the action of remodeling enzymes that fine-tune the degree of polymerization (DP) - polygalacturonase (PG) or pectin/pectate lyases (PLLs) - or of methyesterification/acetylation (DM/DA) of the polymer - pectin methylesterase, PME or pectin acetylesterase, PAE. Up to now, the combination of developmental biology and biophysics has started to shed light on the cell wall mechanical effectors that mediate changes in cell shape. However, our understanding of the mode of action of pectin remodeling enzymes is impaired by gene redundancy (enzymes are all encoded by multigenic families of 12 to 69 members), as well as by the occurrence of compensation mechanisms among gene families and cell wall polymers. The objectives of the WALLMIME project will be thus to: i) Biochemically characterize a set of HG remodeling enzymes (PME, PAE, PG, PLL), ii) Comprehensively follow their effects upon application on both etiolated hypocotyls and biomimetic model of cell wall, iii) Build a model of the dynamics of HG-pectin remodeling. To tackle these objectives, the WALLMIME project will use two distinct models. On one hand, mimetic membranes/films combining cellulose microfibrils and pectins of various structures (DM, DA, DP), will be used as a simplified well-controlled system to determine the role of pH, ionic strength and calcium ions concentration, which are key effectors of apoplast homeostasis, on the dynamic of the pectic network. On the other hand, Arabidopsis dark-grown hypocotyl, a standard developmental model that is solely characterized by cell elongation, will be used to relate the modifications of the pectin network to phenotypical changes. The WALLMIME project will be organised in 4 scientific Work Packages (WP) and one management WP. The originality of the organization lies in the fact that the three firsts scientific WP (WP1: Consequences of HG-enzymes action on polymer structure and cell integrity, WP2: Consequences of HG-enzymes action on mechanical properties, and WP3: Consequences of HG-enzymes action on elongation/deformation) will be performed on the Arabidopsis hypocotyl and on mimetic cell wall in parallel. This will allow getting the best benefit from the highly complementary expertise of the three partners. WP4 (Integrated model of the dynamics of pectic network) will build a model of the dynamics of pectic network using the experimental data generated in WP1 to 3. Bringing experts from various fields (enzymology, biophysics, polysaccharide chemistry, glycomaterials), WALLMIME will drastically increase our knowledge on how plants can fine-tune their cell wall pectic matrix and how this can mediate changes in plant growth. This is of the utmost importance to understand how plants can respond to the environment and can produce biomass. The project will further challenge the current concepts of plant cell wall and will contribute to setting new standards for dynamic cell wall models. This is currently a highly competitive field for which the project will be an original contribution, as it combines the characterization of the effects of enzymes on plant material and biomimetic models. It will thus contribute to a better understanding of the enzymes’ mode of action on substrates, paving the way for novel use of plant enzymes in glycomaterials.