The role of CESA protein modification in localisation and function of the cellulose synthase complex
Lead Research Organisation:
University of Manchester
Department Name: Life Sciences
Abstract
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.
Technical Summary
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.
Planned Impact
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.
People |
ORCID iD |
Simon Turner (Principal Investigator) | |
Karl Kadler (Co-Investigator) |
Publications
Campbell L
(2018)
An essential role for abscisic acid in the regulation of xylem fibre differentiation.
in Development (Cambridge, England)
Kumar M
(2015)
Protocol: a medium-throughput method for determination of cellulose content from single stem pieces of Arabidopsis thaliana.
in Plant methods
Kumar M
(2015)
Plant cellulose synthesis: CESA proteins crossing kingdoms
in Phytochemistry
Kumar M
(2018)
Exploiting CELLULOSE SYNTHASE (CESA) Class Specificity to Probe Cellulose Microfibril Biosynthesis.
in Plant physiology
Kumar M
(2016)
Secondary cell walls: biosynthesis and manipulation.
in Journal of experimental botany
Kumar M
(2016)
S-Acylation of the cellulose synthase complex is essential for its plasma membrane localization.
in Science (New York, N.Y.)
Kumar M
(2017)
Functional Analysis of Cellulose Synthase (CESA) Protein Class Specificity.
in Plant physiology
Kumar, M.
(2015)
Encyclopedia of Life Science
Sorek N
(2016)
From the nucleus to the apoplast: building the plant's cell wall.
in Journal of experimental botany
Turner S
(2018)
Cellulose synthase complex organization and cellulose microfibril structure.
in Philosophical transactions. Series A, Mathematical, physical, and engineering sciences
Wightman R
(2013)
SPIRAL2 determines plant microtubule organization by modulating microtubule severing.
in Current biology : CB
Description | Cellulose is the most abundant polymer on the planet, but it has been not been possible to isolate and characterise the large enzyme complex that is responsible for synthesising cellulose in plants. This complex is unique in that it move through the plasma membrane while generating a large number of individual sugar chains that bond together to form a very strong microfibril. We have identified that that many of the subunits of this enzyme complex are modified by the addition of a fatty acid group. We believe that this is essential both for the structure of the enzyme complex and also to help retain the enzyme within the plasma membrane as it make cellulose. However we also consider that these fatty acid modifications are what make the enzyme so hard to purify because they make the enzyme very prone to aggregation. |
Exploitation Route | It will help other understand what is needed to purify a functional cellulose synthesising complex. We have also developed methods to identify sites where fatty acids are added to the protein that is likely to be broadly applicable to many proteins. |
Sectors | Agriculture, Food and Drink,Chemicals,Energy,Pharmaceuticals and Medical Biotechnology |
Description | Institutional Strategic Support Fund |
Amount | £14,155 (GBP) |
Funding ID | 105610/Z/14/Z |
Organisation | Wellcome Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 12/2016 |
End | 03/2017 |
Description | Response mode |
Amount | £456,742 (GBP) |
Funding ID | BB/P01013X/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 04/2017 |
End | 03/2020 |
Description | University Research Grant |
Amount | £246,732 (GBP) |
Funding ID | RPG-2014-309 |
Organisation | The Leverhulme Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 05/2015 |
End | 09/2018 |
Title | measurement of cellulose content |
Description | medium throughput method of cellulose analysis. |
Type Of Material | Technology assay or reagent |
Year Produced | 2015 |
Provided To Others? | Yes |
Impact | Makes public well-developed method to wider community |
Description | Acylation assay |
Organisation | University of Dundee |
Department | Proteomics and Mass Spectrometry Facility |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We carried out much of the genetics and analytical work. |
Collaborator Contribution | They used their expertise in protein S-acylation to analyse the CESA proteins that we provided. |
Impact | Outcome was a joint publication in Science. Multidisciplinary we did the genetics, molecular biology and glycobiology they contributed biochemistry and training in techniques. |
Start Year | 2010 |
Description | FTIR collaboration |
Organisation | University of Manchester |
Department | Faculty of Science and Engineering |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We provide plant material specifically altered in cellulose synthesis. |
Collaborator Contribution | They provided expertise and equipment in the area of FTIR imaging. |
Impact | The collaboration is multidisciplinary our partner works on cancer biology. We used their imaging expertise to look at cellulose deposition in plants. |
Start Year | 2017 |
Description | UBC colaboration |
Organisation | University of British Columbia |
Country | Canada |
Sector | Academic/University |
PI Contribution | We provide specific plant material with altered cellulose biosynthesis. |
Collaborator Contribution | Our partner contributed expertise in the area of microfibril angle and cellulose crystalinity using X-ray diffraction. |
Impact | This collaboration is multidisciplinary. We combined our skills in cellulose synthesis and molecular genetics and UBC contributed expertise and equipment to use X-ray crystallography to measure cellulose crystalinity and microfibril angle. It lead to a publication in plant physiology. |
Start Year | 2017 |
Description | Acylation press release |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Media (as a channel to the public) |
Results and Impact | Press release to follow up on publication in Science magazine |
Year(s) Of Engagement Activity | 2016 |
Description | FLS open day |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | We provided a display to explain a project we have on finding the cause of a disease known as apple rubbery wood. We had samples showing the disease symptoms and display explaining how we were looking to find the causative agent and the importance of different components of wood in causing the diease symptoms |
Year(s) Of Engagement Activity | 2016 |
Description | Royal Society Summer exhibition |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Public/other audiences |
Results and Impact | Exhibition sparked discussion and engagement. large number of requests for seed and for further information about growing Arabidopsis mutants. |
Year(s) Of Engagement Activity | 2013 |
URL | http://sugar-complexity.tumblr.com |
Description | Science spectacular |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | Display to explain project looking at find the causitive agent of apple rubbery wood. The display explained why we would do this and how we could use modern technology to find the agent. There were also displays of diseased material exhibiting symptoms and recent graft of apple onto rootstocks. |
Year(s) Of Engagement Activity | 2016 |