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Figure 1 The lignin biosynthetic pathway. PAL, phenylalanine ammonia-lyase; C4H, cinnamate-4-hydroxylase; C3H, 4-coumarate-3-hydroxylase; COMT, caffeic acid 3-O-methyltransferase; CCoAOMT, caffeoyl-CoA 3-O-methyltransfer-ase; F5H, ferulate-5-hydroxylase; 4CL, hydroxycinnamate-CoA-ligase; CCR, cinnamoyl-CoA-reductase; CAD, cinnamyl alcohol dehydrogenase. F5H may hydroxylate coniferyl aldehyde and coniferyl alcohol with better efficiency than ferulic wood is used for fuel because the calorific value of condensed lignins should be improved.

Several groups have envisaged down-regulating another methyltrans-ferase (CCoAOMT) considered to be particularly involved in the synthesis of coniferyl alcohol (7) (M. Legrand, personal communication; W. Boeijan, personal communication). CCoAOMT has been shown to be encoded by multigene families in different plants including poplar (8) and tobacco (9), and specific members of these multigene families have been used in antisense or cosuppression experiments.

Reduction of CCoAOMT alone in transgenic tobacco plants resulted in a decreased lignin content but a simultaneous reduction in CCoAOMT and COMT activities induced a further reduction in lignin content, confirming that both enzymes are indeed involved in methylation reactions in lignin biosynthesis (7). Surprisingly, although the transgenic plants showed a 40 to 60% reduction in lignin content, they appeared to grow normally under greenhouse conditions even though they exhibited a deformation of vessel elements. These results have been partly confirmed by M. Legrand's group in Strasbourg (France) (personal communication) on the same material. However, in addition to a reduction in lignin content, CCoAOMT down-regulation induced a decreased growth rate and a dramatic disorganization of vascular tissues (reduction of xylem thickness, reduction of vessel diameter). These discrepancies concerning the growth of the plants have not yet been explained. W. Boerjan et al. (personal communication) obtained poplar lines cosuppressed for CCoAOMT that exhibit a slight reduction in lignin content. It thus appears that CCoAOMT alone or in combination with COMT is an interesting target for lignin genetic engineering, particularly if the developmental effects observed for some transgenic lines can be reduced. From a qualitative point of view, G units were preferentially reduced in the /3-<?-4-linked monomer fraction of these transgenic plants, leading to an interesting increase in the S/G ratio of the lignins.

B. Down-regulation of 4-Coumarate CoA Ligase (4CL)

This enzyme governs a key step of the common phenylpropanoid pathway leading to hydroxycinnamoyl CoAs. A down-regulation of its activity should lead to pleiotropic effects, such as those observed when phenylalanine am-monia-lyase (PAL) and cinnamate-4-hydroxylase (C4H) are down-regulated, if the targeted enzymes (isoforms) are not strictly dedicated to monolignol biosynthesis. However, interesting results have been obtained in Arabidopsis, tobacco, and poplar 4CL down-regulated plants, suggesting that this enzymatic step is an interesting candidate for lignin genetic engineering (10-12). In both Arabidopsis and tobacco, a nearly total block in 4CL ac tivity led to only a modest reduction in total lignin content and the plants were morphologically normal. In contrast, transgenic aspen exhibited a substantial reduction in lignin quantity and a 15% relative increase in cellulose content (13). In addition, tree growth was substantially enhanced and phenolic profiles of the upper leaves and shoot apex were significantly altered in transgenic lines (14). Even though their rationale is not understood at the moment, these secondary effects on plant growth and cellulose content are particularly interesting in the context of the pulp industry and these investigations merit further attention.

C. Cinnamoyl CoA Reductase (CCR) Down-regulation

As the first committed enzyme in monolignol biosynthesis, CCR channels phenylpropanoid metabolites into the biosynthesis of lignins. Significant down-regulation of CCR activity in tobacco was obtained by ectopic expression of the homologous tobacco antisense gene (15). The CCR down-regulated tobacco plants with a moderate decrease of CCR activity exhibited only a very slight reduction in lignin content and had a normal phenotype, whereas the most severely inhibited transformant showed a 50% reduction in lignin content and an abnormal, heritable phenotype (reduced growth, abnormal leaf morphology). In these plants, the hypolignified xylem vessels were unable to withstand the compressive forces generated during transpiration and tended to collapse inward. In these transformants, the yield of thioacidolysis products was reduced, reflecting a lower proportion of ¡3-0-4 bonds in lignin. However, this uncondensed lignin fraction was enriched in S units. A substantial increase in G units containing free phenolic groups was also observed (C. Lapierre, personal communication). In addition, an increase of cell wall-linked phenolics released by mild alkaline hydrolysis occurred, the main enrichment concerning ferulic and sinapic acids and ace-tosyringone. It was suggested that the incorporation of ferulic acid into the cell wall was responsible for the brown-orange coloration observed as a consequence of CCR silencing (15). Additional studies using 13C nuclear magnetic resonance (NMR) revealed the presence of ferulate tyramine in cell walls of the CCR antisense line (16). These different phenolic compounds could be an integral part of the lignin polymer because, at least in grasses, Jacquet et al. (17) and Ralph et al. (18) have shown that ferulate esters can be converted to phenoxy radicals that copolymerize with lignin polymers. It is clear that CCR silencing induces a marked reorientation of phenolic metabolism, resulting in a new partitioning of phenolic precursors into lignins and other related phenolic sinks.

Simulated pulping experiments performed on CCR down-regulated tobacco plants obtained in our laboratory or in collaboration with Zeneca (19)

revealed a significant decrease in kappa number with little modification of other parameters such as cellulose yield or cellulose degree of polymerization (DP).

Taken together, these data suggest that CCR controls the entry of carbon flux into the lignin pathway. However, the resulting effects—reduction in lignin, increase in cell wall-associated phenolics and in soluble phenolics, potential decrease in other monolignol-derived compounds such as lignans and/or dehydroconiferyl alcohol glucosides—may have an impact on plant development. Whatever the molecular mechanisms involved, it is clear that these results illustrate the unexpected effects that can be associated with a dramatic decrease in lignin content (and associated responses). A compromise should thus be attained between a moderate reduction in lignin content and normal development.

D. Cinnamyl Alcohol Dehydrogenase (CAD) Down-regulation

CAD down-regulation was one of the most impressive successes of lignin genetic engineering research a few years ago (20). Since that time, the results initially obtained on tobacco have been confirmed by independent reports of CAD suppression in tobacco and have been extended to poplar (6,2123). The plants with a strong reduction of CAD activity have normal development and a novel red color of the xylem at certain developmental stages. Their lignin content is not (tobacco) or slightly (poplar) altered but their lignins are enriched in coniferyl and sinapaldehydes, the aldehyde substrates of CAD. The newly formed lignins, which have been characterized in detail by NMR (16), are more easily extracted from antisense plant samples by sodium hydroxide or thioglycolic acid, suggesting that the structural changes in the antisense lignin make it more extractable.

Kraft pulping experiments have shown more extensive delignification of low-CAD tobacco and poplar plants when compared with control plants, demonstrating the benefit of this genetic manipulation for pulp and paper making (6), and field trials of these CAD down-regulated poplars are currently being performed in France and in the United Kingdom. A confirmation of the effects of CAD down-regulation has been indirectly obtained from the study of a CAD-deficient Pin us taeda mutant (24) that accumulates aldehyde components and substantial levels of dehydroconiferyl alcohol in its lignins. Here again, the mutant plant stands normally and has a normal phenotype (except for the red coloration of xylem) and the resulting lignins are more easily extracted. These is no doubt that these CAD-deficient plants are particularly promising for the pulp industry.

E. Double Transformants

In order to combine the advantages of the down-regulation of individual genes and to yield new and useful lignins, several groups have envisaged producing double and triple transformants by crossing existing plants with single antisense genes or using transgenes containing multiple genes under the control of a single promoter.

Our recent results on CCR/CAD down-regulated plants illustrate the potential interest of this approach. By crossing homozygous tobacco lines down-regulated for CCR and for CAD, we have obtained hybrid lines down-regulated for both enzymes. If the hybrid exhibits intermediate values between the two parents for some characteristics, its lignin content is surprisingly lower than for the CCR down-regulated homozygous line. However, in contrast to this parent line, its size and morphology are not affected. This hybrid line with only 40% of residual lignin was processed in simulated kraft pulping experiments by the Centre Technique du Papier, Grenoble (France) (M. Petit-Conil, personal communication), and a 35% decrease in kappa number, a 24% increase in yield, and a 13% increase in cellulose DP were observed. These interesting results, which have been confirmed several times on tobacco plants obtained in culture-room conditions, should now be extended to other species grown in natural conditions. They suggest that the CCR and CAD transgenes work in synergy in a still undefined way to give rise to new lignin profiles without altering plant morphology.

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