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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...

Pharmacological evidence implicates trans-cinnamic acid as a feedback modulator of the expression and enzymatic activity of the first enzyme in the phenylpropanoid pathway, L-phenylalanine ammonia-lyase (PAL). To test this hypothesis independently of methods that utilize potentially non-specific inhibitors, we generated transgenic tobacco lines with altered activity levels of the second enzyme of the pathway, cinnamic acid 4-hydroxylase (C4H), by sense or antisense expression of an alfalfa C4H cDNA. PAL activity and levels of phenylpropanoid compounds were reduced in leaves and stems of plants in which C4H activity had been genetically down-regulated. However, C4H activity was not reduced in plants in which PAL activity had been down-regulated by gene silencing. In crosses between a tobacco line over-expressing PAL from a bean PAL transgene and a C4H antisense line, progeny populations harboring both the bean PAL sense and C4H antisense transgenes had significantly lower extractable PAL activity than progeny populations harboring the PAL transgene alone. Our data provide genetic evidence for a feedback loop at the entry point into the phenylpropanoid pathway that had previously been inferred from potentially artifactual pharmacological experiments.

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Expression of cysteine proteinase inhibitors (cystatins) in tobacco or other plants has the potential for improving resistance against pathogens and insects that possess cysteine proteinases. A chimeric gene containing a cDNA clone of rice cystatin (oryzacystatin-I; OC-I), the cauliflower mosaic virus 35S promoter, and the nopaline synthase 3' region was introduced into tobacco plants by Agrobacterium tumefaciens. The presence of the chimeric gene in transgenic plants was detected by a polymerase chain reaction-amplified assay, and transcriptional activity was shown by RNA blot analysis. Heated extracts from transgenic tobacco plants, as well as from progeny which were obtained by selfing a primary transformant, contained protein bands that corresponded in molecular mass to OC-I and reacted with antibodies raised against rOC, a recombinant OC-I protein produced by Escherichia coli. Similar bands were absent in extracts from untransformed control plants. OC-I levels reached 0.5% and 0.6% of the total soluble proteins in leaves and roots, respectively, of some progeny. On a fresh weight basis, the OC-I content was higher in leaves (50 micrograms/g) than in roots (30 micrograms/g). OC-I was partially purified from protein extracts of rice seeds and from transgenic tobacco leaves by affinity to anti-rOC antibodies. OC-I from both sources was active against papain.