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The impact of lignins of various origins on filter paper hydrolysis by fungal cellulase was evaluated. Powdered lignins were added to enzyme incubations, either as isolated or after thorough hydroxypropylation of phenolic sites. Extent of cellulose hydrolysis was reduced by 14−60% by the addition of up to 15% lignin to the substrate. Unmodified lignins were more detrimental to cellulose hydrolysis than hydroxypropylated lignins. The inhibitory effect of lignin addition was only partially overcome by a 10-fold increase in cellulase activity, suggesting inhibitory lignin interactions with both substrate and enzyme. Preincubation of cellulase with underivatized lignins resulted in reduced enzyme activity and soluble protein concentration in the supernatant, suggesting protein precipitation with lignin rather than reduced activity of a lignin−enzyme complex as the inhibitory mechanism. Two further experiments showed that the negative impact of lignin on cellulose hydrolysis can be counteracted by addition of various N compounds and by ammoniation. Keywords: Lignin; cellulose degradation; cellulase inhibition; free phenolic group; ammoniation; PEG; PVP; N compounds

The phenylpropanoid pathway is responsible for the synthesis of a large range of natural products in plants, including flavonoids (pigments and UV protectants), the structural polymer lignin, and antimicrobial furanocoumarin and isoflavonoid phytoalexins (Hahlbrock and Scheel, 1989; Dixon and Paiva, 1995). Salicylic acid, which is involved in the establishment of both local and systemic plant defense responses, is also a product of this pathway (Klessig and Malamy, 1994). Although the importance of phenylpropanoid natural products makes the pathway an obvious target for plant improvement by metabolic engineering, little is known about the control of flux into the various branches of the pathway. Many phenylpropanoid

We analyzed lignin content and composition in transgenic tobacco (Nicotiana tabacum) lines altered in the expression of the early phenylpropanoid biosynthetic enzymes L-phenylalanine ammonia-lyase and cinnamate 4-hydroxylase (C4H). The reduction of C4H activity by antisense expression or sense suppression resulted in reduced levels of Klason lignin, accompanied by a decreased syringyl/guaiacyl monomer ratio as determined by pyrolysis gas chromatography/mass spectrometry. Similar reduction of lignin levels by down-regulation of i-phenylalanine ammonia-lyase, the enzyme preceding C4H in the central phenylpropanoid pathway, did not result in a decreased syringyl/guaiacyl ratio. Rather, analysis of lignin methoxyl content and pyrolysis suggested an increased syringyl/guaiacyl ratio. One possible explanation of these results is that monolignol biosynthesis from L-phenylalanine might occur by more than one route, even at the early stages of the core phenylpropanoid pathway, prior to the formation of specific monolignol precursors.There is currently intense interest in modifying the content and / or composition of the cell wall structural polymer lignin as a means of improving the efficiency of the paper pulping process for forest trees or of increasing digestibility of forages for ruminant animals (Whetten and Sederoff, 1991; Boudet and Grima-Pettenati, 1996; Campbell and Sederoff, 1996).Recent studies have concentrated on attempts to downregulate the levels of enzymes involved in the reactions specific for lignin monomer synthesis by expression of homologous or heterologous antisense genes in transgenic plants (Dwivedi et al., 1994;Halpin et al., 1994;Ni et al., 1994; Atanassova et al., 1995;Van Doorsselaere et al., 1995;Sewalt et al., 1997). Although the biosynthetic pathway to lignin monomers is relatively well understood, involving consecutive hydroxylation and O-methylation reactions leading from p-coumaric acid via ferulic acid (the monomethoxylated precursor of the G residues of lignin) to sinapic acid (the dimethoxylated precursor of the S residues of lignin), it has recently been suggested that parallel pathways of monomer hydroxylation and methylation could occur at the level of the COA thioesters (Ye et al., 1994) or even at the level of the aldehydes formed after the first reduction of the COA thioesters (Matsui et al., 1994; Fig. 1).The existence of a metabolic grid for the O-methylation of monolignols would complicate the interpretation of experiments in which a single enzyme of the pathway was down-regulated. Indeed, severa1 reports of the effects of antisense inhibition of enzymes involved in the late reactions of monolignol biosynthesis have presented unpredicted and sometimes contradictory results. Ni et al. (1994) reported that modest down-regulation of COMT activity in transgenic tobacco (Nicofiana fabacum) leads to a small reduction in lignin content with no significant change in lignin composition. However, other groups have shown that strong down-regulation of COMT in tobacco or poplar (Populu...

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Cell wall digestibility, lignin content, and lignin composition were analyzed in transgenic tobacco altered in the expression of the phenylpropanoid biosynthetic enzymes caffeic acid 3-O-methyltransferase (COMT) and L-phenylalanine ammonia-lyase (PAL). Reduction of COMT activity by antisense technology resulted in reduced lignin content accompanied by an increased syringyl (S)/ guaiacyl (G) monomer ratio, as determined by pyrolysis/GC/MS and measurement of lignin methoxyl content by wet chemistry. These results resemble those obtained by reduction of flux of lignin precursors into the phenylpropanoid pathway by PAL suppression, which results in drastically reduced lignin with sharply increased methoxyl content. Enzymatic digestibility of cell walls from stem internodes was improved in the transgenic lines and was highly negatively correlated with lignin concentration (r ) -0.97). Although lignin composition was also affected, lignin concentration was the overriding factor influencing cell wall digestibility. The results provide a basis for new strategies for lignin modification to improve digestibility of forages.