The phenylpropanoid pathway provides precursors for the biosynthesis of soluble secondary metabolites and lignin in plants. Ferulate-5-hydroxylase (F5H) catalyzes an irreversible hydroxylation step in this pathway that diverts ferulic acid away from guaiacyl lignin biosynthesis and toward sinapic acid and syringyl lignin. This fact led us to postulate that F5H was a potential regulatory step in the determination of lignin monomer composition. To test this hypothesis, we have used Arabidopsis to examine the impact of F5H overexpression. Arabidopsis is a useful model system in which to study lignification because in wild-type plants, guaiacyl and syringyl lignins are deposited in a tissue-specific fashion, while the F5H-deficient fah1 mutant accumulates only guaiacyl lignin. Here we show that ectopic overexpression of F5H in Arabidopsis abolishes tissue-specific lignin monomer accumulation. Surprisingly, overexpression of F5H under the control of the lignification-associated cinnamate-4-hydroxylase promoter, but not the commonly employed caulif lower mosaic virus 35S promoter, generates a lignin that is almost entirely comprised of syringylpropane units. These experiments demonstrate that modification of F5H expression may enable engineering of lignin monomer composition in agronomically important plant species.

Cinnamate-4-hydroxylase (C4H) is the first Cyt P450-dependent monooxygenase of the phenylpropanoid pathway. To study the expression of this gene in Arabidopsis thaliana, a C 4 H cDNA clone from the Arabidopsis expressed sequence tag database was identified and used to isolate its corresponding genomic clone. The entire C4H coding sequence plus 2.9 kb of its promoter were isolated on a 5.4-kb Hindlll fragment of this cosmid. lnspection of the promoter sequence revealed the presence of a number of putative regulatory motifs previously identified in the promoters of other phenylpropanoid pathway genes. The expression of C4H was analyzed by RNA blot hybridization analysis and in transgenic Arabidopsis carrying a C4H-P-glucuronidase transcriptional fusion. C4H message accumulation was light-dependent, but was detectable even in dark-grown seedlings. Consistent with these data, C4H mRNA was accumulated to light-grown levels in etiolated detl-7 mutant seedlings. C4H is widely expressed in various Arabidopsis tissues, particularly in roots and cells undergoing lignification. The C4H-driven /3-glucuronidase expression accurately reflected the tissue-specificity and woundinducibility of the C4H promoter indicated by RNA blot hybridization analysis. A modest increase in C4H expression was observed in the tt8 mutant of Arabidopsis.The phenylpropanoid pathway gives rise to a wide array of metabolites. These compounds participate in many plantdefense responses (Nicholson and Hammerschmidt, 1992) and absorb potentially damaging UV-B radiation (Caldwell et al., 1983;Li et al., 1993;Landry et al., 1995). The pathway also generates the monomers required for lignin biosynthesis: ferulic acid and sinapic acid (Lewis and Yamamoto, 1990). The purification of the soluble enzymes of the phenylpropanoid pathway over the last 10 years has permitted the cloning of their respective genes. These include the genes encoding Phe ammonia-lyase, p-coumarate COA ligase, caffeic acidl5-hydroxyferulic acid, O-methyltransferase, chalcone synthase, and chalcone isomerase. In comparison, the genes encoding two of the Cyt P450-dependent monooxygenases (P450s) in this pathway, C4H and ferulate-5-