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The role of acylation in cellulose synthesis

Reference	BB/P01013X/1
Principal Investigator / Supervisor	Professor Simon Turner
Co-Investigators / Co-Supervisors
Institution	The University of Manchester
Department	School of Biological Sciences
Funding type	Research
Value (£)	456,742
Status	Completed
Type	Research Grant
Start date	01/04/2017
End date	31/03/2020
Duration	36 months

Abstract

S-acylation is the addition of a fatty acid, usually stearate of palmitate to a cysteine residue that dramatically increases the hydrophobicity of the protein. We have recently shown that the cellulose synthase complex is extensively modified by S-acylation of the CESA proteins. A single cellulose synthase complex is likely to contain more than 100 acyl groups which represents S-acylation on an unprecedented scale that will dramatically increases its hydrophobicity and contribute to essentially locking the complex into the plasma membrane during cellulose synthesis. It has also been reported that tubulin is also modified by S-acylation. These discoveries have allowed us to generate several testable hypothesis regarding how acyl modification and membrane partitioning can contribute to co-localisation of all proteins involved in cellulose synthesis and to the alignments of cellulose deposition with the underlying cortical microtubules. We will investigate whether S-acylation of the CESA proteins contributes to the formation of specialized plasma membrane domains that facilitate unimpeded movement of the cellulose synthase complex. Xylem vessels are an excellent place to study plasma membrane microdomains as the patterned cell wall deposition means that domains showing very high cellulose synthase activity are immediately adjacent to regions that are not making any cellulose. We will exploit the inducible VND systems that can be used to drive ectopic vessel formation in Arabidopsis plants and tobacco BY-2 cells. We will perform proteomic analysis using LOPIT, a technique that combines membrane fractionation with multivariate analysis to assign proteins to particular compartments allowing us to separate proteins that localize to sites of cellulose from other domains. We will also address whether the partitioning of proteins is also marked by differences in lipid composition and in particular whether domains synthesizing cellulose are also sterol rich.

Summary

Cellulose is the major component of many plant cells walls and is considered to be the world's most abundant naturally occurring polymer. Cellulose is actually composed on many chains of the sugar (glucose) units bonded together to form something known as the microfibril. These microfibrils have unique physical properties that are exploited by plants. Cellulose microfibrils are ubiquitous among higher plants where they are important in determining how plant cells grow and also determining how strong the plant material is. We already exploit the properties of cellulose to make paper and cotton, however, cellulose has the potential to be used in a wide range of other applications including novel materials and as a renewable source of sugars for the production of biofuels and chemicals. One of the major advantages of using plant based material is that plants obtain their carbon from the atmosphere in form of CO2 and so using plant material such as cellulose is not only renewable, but dramatically reduces net carbon emission into the atmosphere compared to the use of fossil fuels. Cellulose is synthesis by a unique enzyme complex that sits in the plasma membrane that surrounds the contents of every cell. Each cellulose synthase complex makes around 18 chains that bond together to form a microfibril. These microfibrils are rigid structures and so as the complex adds sugars to the growing chains, it is effectively driven along the plasma membrane. Given the large size of the complex, it will cause severe local disruption of the plasma membrane. The plasma membrane is composed of lipids that provide a fluid environment that allows movement of the cellulose synthase complex, but it is essential the cells maintain the integrity of the plasma membrane for its viability. Movement of the cellulose synthase complex is governed by long tubular structures known as microtubules that sit close to the plasma membrane and guide the movement of cellulose synthase complex and henceorientation of the cellulose microfibrils. Orientation of cellulose microfibrils is essential for the growth of plant cells and has a major influence on their physical properties. Although cellulose is very abundant, there are several technological challenges associated with studying cellulose, including separating it from other parts of the cell wall and breaking up its strongly bonded structure. Surprisingly, the vast importance of cellulose is not matched by our understanding of the processes behind its formation. We have become interested in how the individual components of the cellulose synthase complex are modified by the addition of fatty acid groups. These fatty acid groups are very hydrophobic and have a very high affinity for membranes. We believe this has an essential role in locking the cellulose synthase complex into the plasma membrane and preventing it "popping out" as the complex moves through the membrane. The cells are also faced with another logistical problem, as the plasma membrane is crowded with many other components. We now want to look at how the plasma membrane might be partitioned to allow unimpeded movement of the cellulose synthase complex. We will investigate how the addition of hydrophobic fatty acid groups both to the cellulose synthase complex and to the underlying microtubules contributes their co-localisation and the ability of the cell to form membrane partitions at sites of cellulose synthesis. 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 make it easier to breakdown into its constituent sugars that maybe used for biofuels or other industrial applications. It is likely that a better understanding of the local environment in which plants make cellulose may help us to alter other cellulose properties such as microfibril length.

Impact Summary

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 BBSRC-funded networks in biotechnology: Lignocellulosic Biorefinery Network (LBNet) and - Glycoscience Tools for Biotechnology and Bioenergy (IBCarb). 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 5-10 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.

Committee	Research Committee B (Plants, microbes, food & sustainability)
Research Topics	Plant Science
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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