It is clear that the lignin content and composition of crops and woody species can be manipulated through genetic engineering. In addition to the application-orientated objectives that have triggered most of the research in this area, the impressive amount of data that have been accumulated in the last few years has generated new fundamental concepts about lignin biosynthesis pathways and lignin chemical flexibility. As with other metabolic engineering experiments, the results obtained have also revealed pleiotropic and unexpected effects beyond the targeted pathway. Some of these could be explained through cross-talks between genes because it has been recently shown by G. Pinçon et al. (32) that COMT gene down-regulation has an impact on the expression of other genes of the lignification pathway. Lignin genetic engineering is also a way to explore the subtle regulations occurring during the lignification process. The analysis of our CCR X CAD tobacco double transformants has shown that in addition to a strong reduction in lignin content, the simultaneous down-regulation of these two activities has a significant and specific impact on the nature of lignins synthesized and on the preferential cell localization of the residual lignins. In collaboration with K. Ruel and A. Yoshinaga (33), it was demonstrated that the transgenic lignins were enriched in noncondensed units and that the reduction in lignin content was more important in the fibers than in the vessels.
The new transformed plants may also help better identify the chemical characteristics of lignocelluloses, which are beneficial in wood processing. Indeed, comparisons performed on wild-type and transgenic lines represent a new exploratory approach to reevaluate the criteria important for the efficiency of the pulping/bleaching process (in addition to the classical S/G ratio). Such chemical studies have been initiated by C. Lapierre (personal communication) using a range of sophisticated methods. For example, lignins from CCR and CAD down-regulated plants are enriched in free phenolic groups, which improve their extractability.
At the present stage of progress, and in addition to the evaluation of the stability of the traits in the long term, which has already been discussed, many tests still have to be performed on the existing transgenic material in order to evaluate its industrial usefulness. Simulated pulping experiments have been performed by the CTP (Centre Technique du Papier, Grenoble, France) on tobacco stems and poplar branches and trunks of different transformants with decreased COMT, CCoAOMT, CCR, and CAD activities (6) showing several interesting modifications in terms of kappa number, cellulose yield, and cellulose DP.
The most interesting lignin modifications should now be integrated in the pulping/bleaching process taking into account the constraints and limitations associated with each technological stage. Breeders and chemists involved in the pulp industry are usually looking for genetically engineered trees with the lowest lignin content possible but enough lignin (or the equivalent!) to enable them to withstand environmental hardships (winds, heavy rains, etc.). At the same time, they want to process trees with lignin that would be easier to dissolve, thus requiring less chemicals, less heat, and less retention time in the digester. The resultant pulp should have equal or improved quality (attributes desired in the pulp and in the end-use product, paper) when compared with current bleached kraft market pulp.
From the same biological resource, the quality of the pulp can be improved by modifying intermediate steps in the process and particularly in the cooking and the bleaching stages. However, this improvement is usually associated with a decrease in yield and an increase in the amount of chemicals and energy required. Thus, it is clear that most of the time, a compromise has to be found between opposing objectives. An optimized resource should increase the yield and the quality of the pulp and/or decrease the quantity of energy and chemicals used in the transformation process.
Taking into account these potential limitations, the existing technology should now be rapidly extended to different woody species of economic interest in light of the progress observed in tree genetic transformation [see, for example, D. Clapham et al. (34) and M. Maralejo et al. (35)]. New avenues are also open to optimize and to extend the first results. They include, as partially described, the fine tuning of the lignin content or composition through the use of a combination of genes and the exploitation of transcription factors.
Finally, progress in the characterization of the genes involved in the biosynthesis of the polysaccharides of the cell wall (36) opens the way to targeted genetic manipulation of cellulose and hemicelluloses in plants. A combined modification of polysaccharides and lignin profiles can also be envisaged.
The resulting transformants should increase the natural variability of cell wall components in plants. They should then contribute directly or through integration in classical breeding programs to the improvement of the resource used for pulp and paper making.
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