Functional dissection of pectic rhamnogalacturonan-I (RG-I) in plant cell walls

Plant cell walls are rigid structures at the surface of all plant cells and are responsible for generating the mechanical properties of growing plants and many products/materials derived from crops such as fruits, vegetables and textile fibres. Cell walls are mechanically strong biomaterials and are highly complex in terms of structure. They are comprised of varied configurations of a range of polysaccharides that include cellulose, hemicelluloses and pectic polysaccharides. The pectic components of plants cell walls are particularly structurally highly complex and are entwined within cell walls with the cellulose and other polysaccharides. The roles of pectin in cell wall assembly and the generation of cell wall properties leading to the properties of plant materials are not understood. A portion of the pectic polysaccharides are a structurally complex and highly variable set of polysaccharides that are known as rhamnogalacturonan-I (RGI) due to the presence of a structural backbone that contains the sugars rhamnose and galacturonic acid. This polysaccharide backbone has side chains rich in arabinose and galactose sugars. The individual structures of RG-I domains appear to vary from cell to cell and several lines of evidence suggest that they have a role in generating cell wall properties influencing factors such as the extensibility and firmness of cells and tissues. The project will develop a new set of tools to identify and track individual RGI molecules using the model plant system Arabidopsis. This work in generating understanding of the profiles of RGI components at a microscale level in a single leaf or root will open up a new area for plant biology with the prospect of controlling the mechanical and textural properties of plant materials. The project will use the analytic methods developed in the first part of the programme to study RGI structure and functions in the context of plant growth. Growing plants have to withstand a range of mechanical stresses - whether this is wind and rain that constantly stress shoots and leaves or roots growing through soil with varying degrees of compactness. Little is known about how cell walls and plant organs respond to these mechanical stresses although preliminary evidence indicates that RGI molecules are involved generating the mechanics of cells and organs and also involved in responses to stress. The tools and approaches for RGI analysis developed in the project will be used to study the variation and modification of RGI molecules subject to mechanical stresses during growth. An understanding of how RGI impacts on cell wall properties such as firmness and elasticity to cell walls will generate important knowledge to understand how cell wall properties are generated. The work will impact on our understanding and capacity for exploitation of a range of plant materials and products from texture of eaten products such as apple and tomato fruits to wheat grain to cotton fibres. It will also provide underpinning understanding of how crop plants respond to mechanical stress imposed by climatic factors and environmental stresses.

Plant cell walls are important components of plant organs that underpin cell adhesion/organ cohesion and biomechanics. Cell walls are composites of complex glycans including the pectic supramacromolecules that are co-extensive with cellulose microfibrils and hemicellulose polysaccharides. The pectic components of cell walls are thought to be important for cell wall assembly, cell expansion and cell wall mechanical properties. A major component of the pectic component of cell walls is the polymer rhamnogalacturonan-I (RGI) that is highly heterogeneous in terms of its structure. This heterogeneity is based on arabinan- and galactan-rich side chains extending from rhamnogalacturonan backbones. The highly variable RGI structures are extensively regulated in relation to meristem and early organ development. Some evidence indicates arabinan-rich forms are associated with elasticity and galactan-rich forms with cell wall stiffness but there is no mechanistic understanding underpinning this current view. The project will develop a new sensitive methodology to generate profiles of individual RGI molecules using high affinity antibody probes in conjunction with chromatographic separations. The project will use Arabidopsis as a model system to dissect RGI profiles and using its genetic resources the project will develop understanding of the synthetic and physiological underpinnings of RGI structures. The imposition of mechanical stresses to Arabidopsis roots and shoots will be used to determine RG-I functions in the generation of biomechanics and response to mechanical stresses and how RG-I polymers are involved in generating specific cell wall properties. In providing a new methodology and approach the project will open up a new area of RGI biology that can be integrated with understanding and approaches to plant development and can also provide important underpinning science for understanding the properties of plant products and plant-derived biomaterials in general.

The routes to impact for this project will have four major strands that will ensure that the outputs and new understanding generated from the project is both widely accessible and channelled towards most appropriate sectors with potential to make a positive impact on their relevant activities.

ACADEMIC ROUTES
Results relating to understanding of individual RGI structures/functions in the context of cell wall architectures, plant growth, development and physiology, biomechanics and crop sciences will be presented by the post-doctoral researcher and the PI at cell wall and plant physiology meetings and published in international journals.

FOOD/FOOD-ADDITIVE INDUSTRIES
The new set of tools and methodologies and understanding of RGI structure-function relations will be of potential interest to a range of food-related industries. The PI has links with pectin companies including CPKelco through a current EU Marie Curie WallTrac ITN and can therefore explore implications of the new extended understanding of RGI and explore routes to inform current industrial understanding and procedures relating to both pectin extractions and also properties of commercially important isolated pectic fractions. Similarly the PI has links through staff (Dr Caroline Orfila) of the Food Science department at Leeds with a range of companies focused in specific plant products for which understanding of texture is of direct relevance (Heinz (beans), Bakkavor (coleslaw)).

FIBRE INDUSTRIES
The PI has current links with Bayer Bioscience in Ghent in the area of cotton fibres through a BBSRC-funded CASE project (2008-2012) and the current EU Marie Curie WallTrac ITN (2011-2015). The PI has strong productive links with Dr Richard Blackburn in the Green Chemistry/Textiles department at Leeds who also has extensive links with fibre/textile companies.

PUBLIC ENGAGEMENT
The research team will be involved in a range of activities in relation to the public understanding of the value of science. This will involve participation in university organised schools events, café scientifiques and exhibitions at large events such as the Great Yorkshire Show where there is a University pavilion with an option for exhibitions and demonstrations. The involvement will range from explaining the basis, rationale and usefulness of the funded work on cell walls to more general presentation of the importance of plant science.
A second strand of the public outreach activity will be to develop specific pages for the Knox Cell Wall Lab website, accessed through plantcellwalls.net, aimed at a more general non-academic audience and to explain the basis and importance of our research projects.