The role of CESA protein modification in localisation and function of the 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. It is made of long chains of the sugar glucose that bind together. Wood is composed of plant secondary cell walls and a particularly high proportion (up to 70%) of wood is made up of cellulose. Wood is 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. Concerns over 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 huge interest in finding alternative fuel sources. Of particular interest are those fuels that do not contribute to increases in CO2 concentrations and that are sustainable. One potential source is to use biological material known as biomass to make 'biofuels'. The most abundant source of biomass is plant cell walls and it may be possible to use cellulose in the cell wall to make ethanol or other fuels in a similar manner to the sugar cane-derived biofuels produced in Brazil. Although cellulose is very abundant, there are several technological challenges associated with using cellulose including (i) separating it from other parts of the cell wall, (ii) breaking up its strongly bonded structure and (iii) getting plants to make more of it. Surprisingly, the importance of cellulose is not matched by an equal understanding of the processes behind its formation. Cellulose is made at the surface of the cell by a very large enzyme complex that acts like a machine making many chains of sugars which then bond together to form a fibre. The cellulose synthesising machine is unusual as it moves through the cell membrane whilst spinning out cellulose into the cell wall. This presents a paradox since the enzyme complex sits in a membrane that must be fluid enough to allow the complex to move through it, but the complex must be bound tightly enough to prevent the enzyme complex being pushed out of the membrane. We have found that the enzyme complex is extensively modified after is has been made. We want to test the idea that this modification targets the complex to particular regions of the membrane that are specialised for making cellulose and that it is the modification of the protein that is the driving force for moving the complex from inside the cell to the cell membrane. We will test whether this is one of the limiting factors in the cell's ability to synthesise cellulose and we will determine whether increasing the capacity of the cell to modify CESA proteins will increase the cellulose content of the plant, something that would be very useful for applications such as making biofuels. Finally, we want to test our theory which states that the complex moves to the outside of the cell where CESA protein modifications are responsible for insertion of the complex into particular parts of the cell membrane in a process that also causes a large change in the structure of the complex.

Cellulose is the most abundant component of biomass and consequently has huge potential as a source of fermentable sugars for biofuel production. One of the unique aspects of cellulose synthesis is the way in which the cellulose synthase complex (CSC) moves through the plasma membrane. The complex is believed to make ~ 36 chains of (1-4) linked glucose that form the microfibril. The microfibril is believed to be a rigid structure that the CSC is able push against as it moves through the plasma membrane driven by the force of polymerisation of the glucan chains. It has been hard to understand how a plasma membrane can be fluid enough to allow movement of such a large (>4MDa) complex, whilst preventing the complex from being pushed out of the membrane. The only components of the CSC identified to date are the presumed catalytic CESA proteins. We have recently found that the CESA proteins undergo extensively modified by a novel kind of post-translational modification that is essential for cellulose synthesis. Mutants of CESA7, in which these modifications are abolished, are retained within the cell. There are around 36 CESA proteins in a complex and the evidence suggest all CESA proteins undergo post-translation modification and this will have a huge effect on the biophysical properties of the complex. As well as being a major factor in intracellular targeting, CESA protein modification is likely to be essential for embedding the CSC in the plasma membrane and will likely have a major influence on the structure of the complex. We will characterise the role of acylation in cellulose synthesis and specifically the role of acylation in: (i) intracellular trafficking of the CSC, (ii) partitioning of the CSC into specialised regions of the plasma membrane, (iii) determining the structure of the CSC and (iv) limiting rates of cellulose deposition.

Cellulose is a key component of a huge variety of plant-based products. It is fundamental to a number of industries that rely on plant cell walls such as pulp and paper and forage crops. Cellulose is also very important in determining the mechanical properties of the cell wall and particular the cellulose within the woody cell wall. Cellulose within the secondary cell wall is essential for the mechanical support and mechanical strength of many plants including crops and consequently whether crops are vulnerable to lodging or other mechanical failure is dependent upon cellulose. Plant cell walls are at the heart of any sustainable biofuels programme. Biomass is essentially plant cell wall material and the potential yields and gains are very large if we are able to utilise it efficiently. Little investment has been put into biomass optimisation and there are potentially huge benefits that can be gained from improving cell wall material. Better starting material will help all down-stream processing such as cell wall breakdown, fermentation and/or other processing. The biggest component of plant cell wall material is cellulose and as such cellulose is the most abundant biopolymer and represent as huge and currently underutilised resources. One of the barriers to exploitng cellulose is the fact that it is very hard to digest due to the inability of enzyme to access individual glucan chains when they are packed in to an ordered microfibril and surrounded by other polymer such as lignin and xylan. There is considerable natural variation in cellulose crystallinity and it occurs as a much less ordered amorphous form that should be much easier to enzymatically digest. The problem is that we do not know how cellulose microfibrils are formed or how the structure and order of the microfibril in controlled. Ultimately, this programme will be of potential use to anyone with an interest in efficiently utilising cellulose as a source of biomass or for other processes. This will include companies interested in generating the material (biomass crops) as well as those interested in processing it. This proposal aims to generate fundamental information on how the structure of the cellulose synthase complex, how it is assembled and how it trafficked within the cell. It is essentially fundamental research that may have very important applied implication. Cellulose is one of our most under-resourced resources. A real understanding of how it is made and the ability to generate cellulose in vitro could offer the opportunity to develop novel and highly specialised applications. Only a proportion of this project is not directly aimed at generating patentable information for industrial application. This study, however, will generate data that is relevant to many problems that have direct industrial relevance described above. Any opportunities for commercial exploitation will be explored using the Universities commercial arm (UMIP. We already have a precedent for this approach. In a current BBSRC project we patented a discovery related to increasing biomass production. On the basis of this patent we raised further funds for additional proof of concept work to demonstrate that the invention worked in different species. We are currently in the process of finalising a licensing agreement for this patent.