BioMatNet Item: FAIR-CT95-0424 - Tree Improvement Based on Lignin Engineering (TIMBER)

FAIR-CT95-0424
Tree Improvement Based on Lignin Engineering (TIMBER)

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Economics : FAIR Area 1.3 - Forestry-Wood Chain : Plant Genetics : Pulping : Wood (Lignocellulose)

	Contract No:	FAIR-CT95-0424
	Date Prepared:	September 2000
	Source:	Final Report Executive Summary

Final Report Executive Summary Introduction

Lignin is an important component of wood (1/4 of the total biomass) and a major impediment to the manufacture of paper products. Removal of lignin from pulp chips is a major step in the processing of paper, requiring the use of large amounts of noxious chemicals that must then be disposed of along with the lignin by-products themselves. It may then be critically important to genetically manipulate lignin composition and levels to help the paper-making industry become more efficient and more environmentally friendly. In addition, the improvement of chemical and physical properties of wood may be interesting not only for the pulp industry but also for other industrial utilisation of wood. As a consequence of recent advances in the characterisation of lignification genes, these objectives have become tangible. A previous ECLAIR project ( OPLIGE) had demonstrated for the first time that lignins can be manipulated by genetic engineering.

The present proposal took advantage of the above results in order to explore various avenues for the optimisation of lignin profiles of woody plants. The lignin content of plants may vary to a large extent depending on the species (15% to 36% dry weight for woody plants), and for one and the same species with the environment conditions and/or the specific cultivar. For example a range from 25 to 35% in lignin content was found in Eucalyptus grandis clones and hybrids. These observations suggest that this range of modification can be easily envisaged through genetic engineering even though it is not known if greater reductions can be tolerated by the plant.

Another level of variability of lignins lies in their monomeric composition, that is the relative proportion of p-hydroxyphenyl, guaiacyl and syringyl residues or as demonstrated recently in a previous ECLAIR project, cinnamaldehydes/cinnamyl alcohols ratios. The impact of these changes on lignin properties and extractability is essentially unknown even though preliminary results have shown that the increased aldehyde/alcohol ratio in antisense CAD tobacco and poplar had a positive impact on the extractability of lignins and pulping properties.

Objectives

In short, the general objectives of the TIMBER project aimed:

to provide new woody materials more adapted to the requirements of the pulp industry
to investigate the potential impact of lignin profiles modifications on different properties of plant biomass and consequently on different exploitation of this biomass.

Activities

To reach these general objectives the project envisaged:

the confirmation and the extension to mature trees of the results obtained on young plants through the antisense RNA strategy
the production of a finely tuned lignin content and composition in model and target species through the extensive exploitation of different lignification genes and suitable constructs
the extension of the most interesting results to trees and clones of economic interest for the European industry (including gymnosperms)
the careful investigation of the chemical, mechanical and fuel quality properties of transformed plants
the investigation of the improved pulping properties of the transformed materials through the most up-to-date process of the pulp industry.

The project envisaged the genetic manipulation of four lignification genes: Cafeoyl O-methyltranferase (COMT), Cafeoyl-CoA O-methyltransferase (CCoAOMT), Cinnamoyl-CoA reductase (CCR) and Cinnamyl alcohol dehydrogenase (CAD), (independently or in combination).

This was done as an extension of the previous project that clearly demonstrated that down-regulation of specific genes in the lignification pathway resulted in marked changes in lignin composition and reactivity. Some of these changes appeared particularly interesting for the pulp industry. The target genes COMT, CCR, CAD had been characterised for the first time within this project and their manipulation was performed in model plants tobacco or poplar clones easy to transform but of limited economic interest.

The main goals of the TIMBER project were:

to evaluate the long term effects of some of the previous transformation events through convenient field trials.
to extend the defined technology to woody species of economic importance for the European market (poplar, Eucalyptus, Norway spruce).
to obtain a more finely tuned lignin content and composition through the modulation of expression of new-genes (CCOAOMT) or through the combinatorial exploitation of different genes and promoters.
To evaluate the impact of these transformations on the chemical profiles of lignins, the pulping properties, the biomechanical and energetic characteristics of the plant materials.

The overall strategy was clearly oriented towards the production of improved materials for the pulp industry. The envisaged modifications aimed to reduce the lignin content and/or to alter the lignin composition of wood materials in order to increase pulp yield to reduce energy consumption and the amount of noxious chemicals necessary for lignin extraction in the pulping/bleaching processes. Through the excellent complementary and collaborations between the different specialised groups of the TIMBER network most of the objectives have been attained. The main results and their potential technological exploitation are listed below.

Results

It has been clearly demonstrated on 2 year old and 4 year old poplar trees produced in field trials either in England or in France that the differences in lignin patterns observed for young poplar plants down-regulated for CAD and COMT activities are conserved. These observations and other related to the maintained expression of the transgenes demonstrate the cumulative and sustained effects of the transformation. In addition, all the data converge to show that the agronomic characteristics of transgenic lines are similar to those of control lines and that in a general way transgenic poplars behave as wild type corroborating the risk assessment of negligible environmental impact. The transfer of the existing technology to poplar OGY cultivar of commercial interest has been successfully performed and preliminary experiments confirm the great interest of CAD down-regulation for improved pulping characteristics.

Techniques for genetic transformation of spruce and eucalyptus have been significantly improved particularly for spruce where routine procedures are available. Antisense constructs housing different lignification genes have been introduced in these woody plants, but due to the length of transformation/regeneration procedure the impact on the lignin profiles is still under study. In addition, lignification genes have been extensively characterised in spruce.

Concerning the exploitation of new genes and combination of genes several important advances have been realised. Caffeoyl-CoA 0-methyltransferase (CCOAOMT) genes have been characterised in the target species and their down-regulation induce a reduction in lignin content. Double transformants resulting from the crossing of individual transformants or from an original strategy involving multiple partial sense constructs have shown complex patterns suggesting subtle interactions between the expression products of different genes and the genes themselves. Interestingly the CCR x CAD double transformants exhibit promising characteristics since they contain a low lignin content and display normal developmental characteristics. In this way they could represent a positive alternative to some transgenic lines (CCR or CCOAOMT down-regulated) in which a decrease in lignin content is associated with an altered size and morphology.

At the moment, the use of tissue specific promoters (xylem specific) has not, at least in our hands, allowed a sufficient expression of the transgenes. The in depth chemical analysis of the transgenic plants was a crucial step in the general strategy of the network for relating the modification of specific genes expression with typical lignin profiles and also for explaining in chemical terms the changes in downstream technological properties of transgenic plants (pulping characteristics, energetic properties). In addition to the decrease in lignin content observed in CCOAOMT and CCR down-regulated plants a similar reduction in lignin content was also detected in transgenic lines with a severely depressed COMT and CAD activity.

The increase in free phenolic groups in lignins is a characteristic of CAD down-regulated plants which may explain the better extractability of lignins, and the incorporation of cinnamaldehydes (particularly sinapaldehyde) in the lignins of CAD deficient lines was demonstrated by GC-MS evaluation of thioacidolysis dimers and / or monomers. The specific composition in monomers of COMT down-regulated plants has been clearly established with a correlative increase in the condensed fraction of lignins making the lignins lees easily extracted during the pulping process.

Different specific chemical signatures for CAD, COMT or CCR deficiency have been defined through a range of sophisticated methods. In addition, HPLC phenolic profiles of transgenic tobacco plants and of transgenic poplar have been obtained showing dramatic differences with the patterns of control plants. These secondary side-effects should be evaluated in more details in the future in the context of the relations between plants and their biotic environment. The pulping properties of the transgenic lines have been analysed on tobacco plants as a model system or on poplar trees through kraft (essentially) or refiner mechanical pulping procedures.

The main results show that CAD down-regulation has immediate utility with very significant improvements at the level of kappa number, pulp yield and cellulose DP. Double transformants with reduced CAD and CCR activity seem also very interesting with improved characteristics in comparison with CAD and CCR individual down-regulated lines. In contrast the OMT down-regulated plants are more difficulty delignified. Other preliminary analyses performed on other single and double transformants merit further developments. In addition to changes in pulping properties the different transgenic lines display significant changes in biomechanical properties and in calorific value. Most transgenic plants have in general lower cutting work and cracking work than the control lines. In addition, fuel quality and energy potential evaluated on poplar of different ages reveal that OMT down-regulated lines seem better for utilisation in charcoal production whereas CAD down-regulated lines are more appropriate for an utilisation in a pyrolysis process to obtain liquid fuels.

Conclusions

In conclusion, the results of the TIMBER project demonstrate that genetic engineering of lignification genes represents a realistic way to improve plant biomass and may have immediate application for increasing the value of paper producing woody species. Further studies may be envisaged both for optimising the induced modifications and for probing, if any, potential in new interactions between the transgenic lines and their environment.

Future work is also necessary to clearly demonstrate the advantages of defined technologies applied to species still difficult to transform genetically.

Finally, one of the main concerns about genetically modified trees is their acceptance by public opinion. The fact that these transformed plants are non food products designed for industrial purposes should facilitate public acceptability. A careful examination of these new transgenic products is then absolutely necessary in order to evaluate the potential impacts, if any, which could outweigh the already demonstrated advantages for competitiveness of the pulp industry. The results of these studies and in parallel the reduced energy consumption and the environmental benefits associated with the use of these transgenic materials should be clearly communicated to the public opinion.