Cinnamyl alcohol dehydrogenase (CAD) catalyzes the last step i n the biosynthesis of the lignin precursors, the monolignols. We have down-regulated CAD in transgenic poplar (Popuhs tremula x Populus alba) by both antisense and co-suppression strategies. Severa1 antisense and sense CAD transgenic poplars had an approximately 70% reduced CAD activity that was associated with a red coloration of the xylem tissue. Neither the lignin amount nor the lignin monomeric composition (syringyl/guaiacyl) were significantly modified. However, phloroglucinol-HCI staining was different i n the down-regulated CAD plants, suggesting changes in the number of aldehyde units in the lignin. Furthermore, the reactivity of the cell wall toward alkali treatment was altered: a lower amount of lignin was found i n the insoluble, saponified residue and more lignin could be precipitated from the soluble alkali fraction. Moreover, large amounts of phenolic compounds, vanillin and especially syringaldehyde, were detected in the soluble alkali fraction of the CAD down-regulated poplars. Alkaline pulping experiments on 3-month-old trees showed a reduction of the kappa number without affecting the degree of cellulose degradation. These results indicate that reducing the CAD activity i n trees might be a valuable strategy to optimize certain processes of the wood industry, especially those of the pulp and paper industry.

The composition of lignin in tobacco stems has been altered by genetic engineering. Antisense expression of sequences encoding cinnamyl alcohol dehydrogenase (CAD), the enzyme catalysing the final step in lignin precursor synthesis, leads to the production of a modified lignin in otherwise normal plants. Although Klason and acetyl bromide lignin determinations show little quantitative change in lignin deposition in CAD antisense plants, a number of qualitative changes have been identified. The lignin is altered in both composition and structure and is more susceptible to chemical extraction. Consistent with a block in CAD activity, antisense plants incorporate less cinnamyl alcohol monomers and more cinnamyl aidehyde monomers into lignin than corresponding control plants. Antisense plants with very low levels of CAD activity also show a novel phenotype with the appearance of a red‐brown colour in xylem tissues. A similar phenotype is correlated with altered lignification and improved digestibility in brownmidrib mutants of maize and sorghum. The improved chemical extractability of lignin in CAD antisense plants supports a role for this technology in improving the pulp and paper‐making value of forest trees while the similarity with brown‐midrib mutants suggests a route to more digestible forage crops.

Lignin is a polymeric constituent of the cell wall that needs to be removed during the paper making process. Bispecific caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT) catalyses the O-methylation of caffeic acid and 5-hydroxyferulic acid to ferulic acid and sinapic acid, respectively. These compounds are intermediates in the biosynthesis of the lignin precursors. Therefore, COMTs are potential target enzymes for reducing the amount, or modifying the composition, of lignin in plants. Different antisense and sense constructs have been expressed of a gene encoding a COMT from poplar (Populus trichocarpa X II: deltoides) in a I! tremula X P. alba clone under the control of the cauliflower mosaic virus 35S promoter. From all analysed transformants, four lines transformed with an antisense construct had a reduced COMT activity. Two showed a 50% reduction of COMT activity, which altered only slightly the monomeric composition. In the two other transform ants, the COMT activity was reduced by 95%. In the latter case, the syringyl/guaiacyl ratio (S/G) was reduced by sixfold (due to a decrease of S and an increase of G), as analysed by thioacidolysis. A new component of lignin, the 5-hydroxy-guaiacyl residue, was detected among the thioacidolysis products. Moreover, in contrast to the white/yellow colour of wild-type wood, the xylem of the transgenic lines with a 95% reduction of COMT activity was pale rose. A similar phenotype was observed in brown-midrib mutants of maize and sorghum, known for their altered lignification. Although the lignin composition was consistently modified, the lignin content of the transgenic poplars was similar to that of the controls

SummaryThe monomeric composition of tobacco lignin has been modified by genetic engineering. Sense or antiseose expression of sequences encoding O-methyltransferese (OMT), a lignin biosynthetic enzyme, was shown to modulate enzyme activity. Ten constructs harboring the entire or a partial OMT cDNA were used. Populations of 20 transgenic plants per construct were analyzed for OMT activity and compared with untransformed controls. As expected, expression of only the full-length sense construct led to an increase in OMT activity. An important reduction of activity was found in a variable number of plantlets from all other transgenic populations but the inhibition was sustained through the adult stage only in plants transformed with the complete cDNA. T-DNA genes were shown to be stably integrated into the tobacco genome and to be transmitted to the progeny. By using gene-specific probes, OMT inhibition in stems was correlated to a parallel disappearance of OMT transcripts originating from both the resident gene and the transgene. In contrast, transgene transcripts were detected in leaf tissues where the resident gene is poorly expressed, thus indicating that relative expression of the two OMT genes controls transcript turnover. In stems of inhibited plants, a marked decrease of syringyl units and the appearance of 5-hydroxy guaiacyl units were demonstrated. These two structural features are also characteristic of natural mutants of maize with an improved digestibility compared with wild lines. These data demonstrate the feasibility and the potential benefits of lignin manipulation.