QLK5-1999-01493 COPOL - Integrated control of polysaccharide and lignin biosynthesis to improve cellulose content, availability and fibre quality

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Biocomposites/Boards : Crops for Biocomposites/Boards : Crops for Paper/Pulp : Crops for Solid Biofuels : Paper/Pulp : Quality of Life - 5.1.1 Sustainable Agriculture - Plant Systems : Solid Biofuels

Contract No:	QLK5-1999-31493
Source:	Second Annual Progress Report March 2003

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Progress Report - March 2003

Introduction

A major goal of the Common Agricultural Programme (CAP) is the diversification of agriculture. Manufacturing industry has highlighted the potential benefit of using crop-derived products as renewable raw materials. Biopolymers are a major focus of effort as an alternative to plastics and glass fibre, but progress is at present hampered by a serious deficiency in knowledge of their biosynthesis and assembly. Rapid improvements in the scientific and technological base are necessary in order to exploit this potential and to maintain the competitiveness of the EU in this field. COPOL is designed to capitalise on existing knowledge and advance it in such a way as to improve use of plant fibre and to exploit new wealth creating options.

Objectives

The essential purpose of the programme is the improvement of raw material quality for the non-food use of plant fibre for use in pulp and paper industry, textiles, composites for industry, biomass for energy. This will encourage diversification of agriculture. A second impact will also be indirectly in the food area as there is a need to improve forage digestibility in a number of crops. Basic research is proposed which will lead to an improvement of the genetics resource controlling the nature of the non-cetlulosic components of cell walls and its consequences on fibre properties and processing. The hemicellulose and lignin content of fibres have been recognised for a long time to strongly influence fibre processing and digestibility COPOL would seek to widen the successful demonstration of technology that has modified the biosynthesis and deposition of lignin with beneficial consequences on the fibre properties in the target crop. The proposed programme would seek to understand the biosynthesis of hemicellulosic matrix polysaccharides of the wall to determine their influence on fibre properties. The relative influence of the two non-cellulosic components (hernicellulose and lignin) on fibre properties and quality will be determined in an integrated approach. Co-ordinate biosynthesis of these two types of non-cellulosic components synthesised through distinct pathways will also be studied through analysis of new lines of genetic material.

The programme will consist of gene discovery involved in hemicellulose biosynthesis initially in the model plant tobacco to complement the existing pool of known lignification-specific genes from this plant. Tobacco has been chosen for this purpose since the technology for lignin alteration was developed in this species. Work in this species has demonstrated that there can be a large flexibility in lignin content and even extreme decreases still results in an apparent normal structure of vessels and a normal phenotype for the plant. Preliminary data suggests that compensatory mechanisms for the lack of lignin exist to modify the polysaccharides. These mechanisms are unknown at present and to what extent they may be exploited for industrial purposes. To begin this process of understanding genes will be cloned for various enzymes involved in hemicellulose biosynthesis and modification, particularly xylans. This process of gene discovery will be used in several ways.

• improved knowledge base of the families of enzymes involved in synthesis and modification of cell wall polysaccharides
• acquisition of molecular markers for mapping and breeding
• genetic manipulation to understand the extent of plasticity in the synthesis and assembly of viable cell walls.

Transgenic plants down-regulated in these enzymes will be produced and analysed for effects on fibre quality and extractability. Effects on hemicellulose biosynthesis will also be tested in transgenics having a background of lignin down-regulation. This would allow development of a strategy using combinatorial gene regulation to optimise cellulose extraction from woody and fibre producing species. While it has to be recognised that in future the development of GM crops will be closely scrutinised, even for non-food use, the transgenics still need to be produced to understand the inherent plasticity in fibre components and their association. This know!edge can also be used to direct breeding programmes through the use of molecular markers. In the case of forage, it may still be acceptable as transgenic feedstock since it would be a secondary GM food crop. However in the case of maize which is well mapped, the availability of molecular markers for the targets can be used to direct breeding programmes and identify transposon knockouts without resorting to transgenesis.

A series of technology transfers will be performed. Firstly, poplar for two purposes. One will be modification for improvement in pulping and paper making quality. This will necessitate gene discovery and manipulation, the eventual strategy for which will depend upon the outcomes of the tobacco work. Similarly, manipulation of cell walls as biomass for renewable energy production will be directed in this way but without identical strategies. A second major objective would be transfer of the technology to the monocot, maize to test the effect on forage digestibility. Thirdly, manipulating flax for improved production of textile and industrial fibre used in composites will be an objective aimed at modification of the retting process and the quality of the final product. Commerdally-directed research will therefore increase the knowledge base concerning the ability to produce through molecular breeding and/or genetic modification:

• combinatorially modified poplar for improved pulping and paper making
• high lignin content poplar with improved calorific value
• maize with quantifiable improvements to digestibility
• high cellulose content flax with modified accessibility properties.

Expected Achievements

COPOL therefore has potential achievements from the science and technology base and the development of strategies for the production of new genetic material for industrial exploitation. For the increase in the knowledge base, COPOL would lead to:

• gene discovery in a neglected but industrially important area of plant metabolism
• better understanding of the interactions between cell wall products of different pathways
• first studies on the biosynthesis of wall components at an integrated level
• compensation for the lack of and advancing the knowledge of the limits of plasticity or chemical flexibility of the plant cell wall.

For industrial applicability, COPOL would lead to wealth creation and diversification of agriculture through:

• development of woody species for optimised biomass and biofuel production
• continued improvement in the pulping and paper making qualities of woody species
• improvement and consequent expansion of the use of maize as a forage crop
• improvement of the retting process for flax or its replacement with more controlled and efficient processing with the consequent expansion of the industrial use of flax fibre as an indigenous EU crop.

Activities

The work on tobacco and poplar lignification and its modification linked to COPOL continue to be scientific highlights. The analysis of the transgenic plants produced under previous programmes funded nationally and by the EC promise to advance our knowledge of the importance of lignification to the novel use of woody resources in the non-food area. Transgenic plants are available to COPOL that more or less encompass most of the key enzymes in the process of lignification, be it the phenylpropanoid pathway, the lignin pathway proper or of the polymerization process. The novelty of the programme is the further analysis of these plants which is directed to discover pleiotropic effects on the synthesis of the polysaccharide polymers.

COPOL is meant to result through a process of gene discovery and production of transgenic plants in the acquisition of lines manipulated in the synthesis of xylan. This will be important scientifically since some of these genes have not been identified in any species as yet. The targets will affect xylans both quantitatively, since they affect flux, and qualitatively since they affect synthesis of the backbone of the polymer. Transgenic tobacco, maize and poplar are already produced or in the process of being produced for one of the flux enzymes. Once these lines are produced crosses can be considered with lines engineered for lignin to test the hypothesis of the limits to manipulation of cell walls which underpins the socioeconomic rationale of COPOL. It is hoped that production of such material will provide the impetus for the generic research in gene discovery necessary for the completion of COPOL. Of the three remaining targets in xylan biosynthesis, one appears promising for early completion while more work is required on the remaining two targets. However the critical mass of partners involved and the resource available would guarantee rapid progress once the critical breakthrough is made and extension from the model crop, tobacco, to the commerdat targets, maize, poplar and flax.

Conclusion

COPOL continues to work towards testing the hypothesis that hemicellulose and lignin biosynthesis are co-regulated and any alterations in one affects the other. These processes have consequences on the availability of cellulose as a resource both quantitatively and qualitatively. This, in turn, has important commercial consequences for wood pulping and paper making, industrial fibre, renewable energy resources and forage digestibility.

COPOL has a biological resource in lines of tobacco and poplar altered for structural genes in lignin biosynthesis. Progress has been so good, except in a single case, so that other lines, not previously envisaged, have been added to the programme. During the first year it also became rapidly apparent that maize, with the availability of transposon_tagged lines is also a powerfu resource for rapid progress in identifying genes knocked out for lignin and hemicellujose biosynthesis. In the case of flax, the technology is being implemented to do the same. A number of partners are well placed to advance the gene discovery and genetic manipulation necessary to drive the programme, while other partners have the skills to examine the changes occurring as a consequence, compliant with the need to define any unintended effects of transgenesis. Ethical and social aspects of the research are therefore fully incorporated into the overall programme. A number of subcontractors are in place to examine possible exploitation of developments within COPOL although it is still envisaged as a proof of concept study in its lifetime. Exploitation is covered by a consortium agreement signed by all partners.

Scientifically the third period has seen a number of highlights, and further publications are planned. Tobacco lines manipulated for monolignol biosynthesis continue to be analysed in depth, by the introduction of transcriptome analysis, giving added insight to regulatory processes. A proteomic study of secondary walls has also been carried out, showing similar promise for gene discovery and identification of pleiotropic effects. These lines are being resolved with respect to the cell wall composition and hence the fibre phenotype. These studies should show whether the concept of co-regulation holds in the model species, tobacco.

The analysis of lignin down-regulation in tobacco modified in the polymerisation process, continues to bring added value to the success of the programme.

The rapidity of the maize screening-programme augurs well for rapid progress in producing a panel of lines for digestibility studies. The identification of four mutant lines for different hemicellujose biosynthesis genes is a significant advance. These are being distributed amongst the participants and their analysis will also add to the testing of the proof of concept of co-regulation of lignin and hemicellulose. Metabolomic work on poplar is highlighting changes in phenolic metabolism as a result of transgenesis, an important aspect of defining the consequences of genetic manipulation. Indeed, the work being undertaken on transgenic lines of poplar will constitute one of the most complete descriptions of transgenesis in any one species. Much of this work is funded by EPV and COPOL is a significant contributor.

Overall, new genes have been discovered which through screening of databases have proved to be unique to the consortium and shared by them, covered by Intellectual Property.