Unravelling the organisation, composition and dynamics of the plant cellulose synthase complex

Cellulose is the major component of many plant cells walls and is considered to be the world's most abundant naturally occurring polymer and is a major component of the natural world all around us. Wood is composed of plant cell walls and a particularly high proportion (up to 70%) of wood is made up of cellulose. Cellulose is also important in determining the mechanical properties of crop plants and consequently important in preventing cereals and other crops from falling over (lodging). For industry, the properties of the secondary walls directly determine the properties of the manufactured products, for example paper quality and the fibres used in textiles. Additionally, the pressing need to increase the proportion of our raw materials that are biodegradable can be filled by using natural plant fibres instead of synthetic fibres in materials such as fibreglass.
Global warming and its links to rising carbon emissions, due to the use of fossil fuels, such as petrol, coupled with diminishing worldwide fossil fuel reserves has generated a huge interest in finding alternative fuel sources that do not contribute to increases in CO2 concentrations and are sustainable. One potential source is to use biological material, "biofuels". One possibility is to use cellulose to make ethanol or other fuels in the same way that sugar from cane has been used in Brazil. Although cellulose is very abundant there are several technological challenges associated with using cellulose, including separating it from other parts of the cell wall, breaking up its strongly bonded structure and how to get plants to make more cellulose. Surprisingly, the vast importance of cellulose is not matched by an equal understanding of the processes behind its formation. We know some of the components that make up the plant's cellulose synthesising machinery, termed the cellulose synthase complex, which resides in lipid membranes at the cells surface, but we don't understand where in the machinery these components reside nor how each component contributes to making cellulose.
Membrane proteins are particularly hard to study as to make them soluble it is sometimes necessary to add harsh detergents that also break up interaction between proteins. One alternative is to chemically crosslink the proteins together before using detergents, but this can become complicated to unravel if large numbers of proteins in complex all become bound together. To simplify this problem we have modified one of the proteins such that we will now be able to look at the potential of different parts of the protein to form crosslinks. By looking at each region separately, it should simplify the analysis and also tells us where different proteins bind to one another. Using this information we will get an idea of how the entire protein complex that makes cellulose is organised. This is important because it is the organisation of this complex that determines the structure of the cellulose microfibril. We will also use a recently-developed technique that allows us to label proteins that bind to, or are close to components of the cellulose synthase complex in their native environment. By comparing these results with those obtained from studying cellulose synthesis in different cell types will enable us to help distinguish what are essential core components required to make cellulose from those that may only bind transiently and may be involved in localisation of the complex of in moving it around the cell.
Ultimately this work should provide a framework that we can use to make changes that may alter the properties of the cellulose that it produces. It is already known that some mutations reduce the crystalinity of the cellulose and so makes it easier to breakdown into its constituent sugars that maybe used for biofuel or other industrial applications. This work should provide important information about how to make much more dramatic improvements.

The crystalline structure of plant cellulose microfibrils endow its amazing structural properties that make is so useful to plants. However, this also means it is very hard to breakdown and use for biofuel or other industrial applications. CESA proteins are the catalytic components of the cellulose synthase complex (CSC). A rational approach to modify cellulose synthesis in order to improve its properties for industrial use is precluded by our poor knowledge of the composition and organization of the CSC. This project aims to address this knowledge gap by exploiting recent breakthroughs we have made in this area coupled with the application of novel methods for looking at proteins interactions. We have developed purification methods, based upon biotinylation that allow us to purify CESA proteins even in the presence of strong detergents. This allows us to overcome problems associated with purification of the CSC and can be coupled with chemical crosslinking to identify interacting proteins. Furthermore, analysis of the CESA proteins suggests that there are many cysteines that are not essential and can be removed. Consequently, we will also take a targeted cross-linking approach based upon removing non-essential cysteines from CESA proteins that will simplify both interpretation of the crosslinking data and help identify specific sites at which the proteins interact. In a complementary approach we will also use recently developed methods for looking at proteins in their cellular context known as proximity- dependent biotin identification. By combining these approaches and looking at both primary and secondary cell wall cellulose biosynthesis we aim to understand how different CESA proteins are organized to make up the rosette, what the constituents of a core complex required to make cellulose are, and identify components that are involved in trafficking and/or localization of the CSC.

This project is essentially fundamental research aimed at answering central questions about how plants make cellulose. It is probable, however, that the outcomes of this work will offer opportunities to alter cellulose biosynthesis and so represent a means of altering the structure and physical properties of the cellulose microfibril. This could be done, for example, by reducing crystalinity that would allow the cellulose to be digested more easily and so improve the ease by which it may be converted into sugars that could be used for biofuel production or as a source of renewable material for chemical production. In other instances, however, such as in biomaterial production it may be preferable to generate cellulose with longer chain lengths and increased crystalinity. We will maximize the impact of this work, by taking advantage of the fact that the PI is part of 2 recently funded networks in biotechnology: Plant Biomass Biorefinery Network (PBBNet) and IBCarb - Glycoscience Tools for Biotechnology and Bioenergy. We will use the Network meeting to engage with the Industrial members as well as other academic to understand the best means of maximising the industrial application of this work, by improving our understanding of what currently limits its utilisation and work together with them to consider how the outcomes of this project can help to achieve this end. As part of an EU project we were able to identify a mutant in xylan biosynthesis (irx15) that caused a very large increase in sugar release, comparable to the best lignin mutants. We would use the same collaborators who are also part of the biotechnology networks to ensure we were able to explore the benefits of any material that we develop.
Altered cellulose is not the immediate aim of the project, but it is envisioned that information gained as part of this project would subsequently be used to generate altered cellulose with a 5 year time frame. Once the benefits have been established fully commercialising any discoveries would then follow.
Targets for cellulosic biofuel production are huge and so information on how it is synthesised and how its structure may be modified is potentially of enormous interest to the very many industrial concerns with interests in this area.