Lignin is, second to cellulose, the most abundant organic compound in the terrestrial biosphere. In different tree species, lignin content varies between 15 and 36% of the dry weight of wood (1). Lignin is a major constituent of cell walls of fibers and tracheary elements and provides these cells rigidity for structural support and impermeability for water transport. For the production of high-quality paper, lignin is considered as a negative factor because it must be extracted from the cellulose fraction by energy-requiring and polluting methods. For this reason, there is considerable interest in modifying lignin by genetic engineering to improve its extractability from wood (2-5).Lignin monomer biosynthesis starts with the deamination of phenylalanine to produce cinnamic acid (Fig. 1). Further enzymatic reactions include the hydroxylation of the aromatic ring, the methylation of selected phenolic hydroxyl groups, the activation of the cinnamic acids to cinnamoyl-CoA esters, and the reduction of these esters to cinnamaldehydes and cinnamyl alcohols. The precise order in which these reactions occur is not yet fully resolved. In dicotyledonous plants, lignin is composed mainly of guaiacyl (G) 1 and syringyl (S) units that are monomethoxylated (C-3) and dimethoxylated (C-3, C-5) and derived from coniferyl alcohol and sinapyl alcohol, respectively. The lignin monomers are transported to the cell wall and are subsequently polymerized, resulting in the deposition of a crosslinked polymer. Although most of the lignin biosynthesis enzymes have been characterized at the molecular level, their precise role in determining lignin amount and composition still needs to be clarified.Based on in vitro data, it has been generally accepted that the methylation reactions in lignin biosynthesis occur exclusively at the cinnamic acid level and that they are catalyzed by a bispecific caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT) (1). However, the analysis of transgenic tobacco and poplar with suppressed COMT activity has shown that

Caffeoyl coenzyme A-3-O-methyltransferase (CCoAOMT) plays an important role in lignin biosynthesis and is encoded by two genes in poplar (Populus trichocarpa). Here, we describe the expression pattern conferred by the two CCoAOMTpromoters when fused to the gus-coding sequence in transgenic poplar (Populus tremula × Populus alba). Both genes were expressed similarly in xylem and differentially in phloem. In xylem, expression was preferentially observed in vessels and contact rays, whereas expression was barely detectable in storage rays and fibers, suggesting different routes to monolignol biosynthesis in the different xylem types. Furthermore, after wounding, fungal infection, and bending, the expression of both genes was induced concomitantly with de novo lignin deposition. Importantly, upon bending and leaning of the stem, the cell-specific expression pattern was lost, and both genes were expressed in all cell types of the xylem. CCoAOMT promoter activity correlated well with the presence of the CCoAOMT protein, as shown by immunolocalization. These expression data may explain, at least in part, the heterogeneity in lignin composition that is observed between cell types and upon different environmental conditions.

Proteins that are preferentially produced in developing xylem may play a substantial role in xylogenesis. To reveal the identity of these proteins, comparative two-dimensional polyacrylamide gel electrophoresis was performed on young differentiating xylem, mature xylem, and bark of poplar (Populus trichocarpa Hook. cv. 'Trichobel') harvested at different times of the year. The most-abundant xylem proteins were identified by microsequence analysis. For 17 of these proteins a putative function could be assigned based on similarity with previously characterized proteins, and for 15 out of these corresponding expressed sequence tags (ESTs) were found in the poplar EST database. The identified xylem-preferential proteins, defined by comparing the protein patterns from xylem and bark, were all involved in the phenylpropanoid pathway: two caffeoyl-coenzyme A O-methyltransferases (CCoAOMT), one phenylcoumaran benzylic ether reductase (PCBER), one bispecific caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT), five S-adenosyl-L-methionine synthetases, and one homologue of glycine hydroxymethyltransferase (GHMT). Remarkably, the biological function of the two most-abundant xylem-preferential proteins (PCBER and a GHMT homologue) remains unclear. In addition, several housekeeping enzymes were identified: two enolases, two glutamine synthetases, one 70-kDa heat-shock cognate, one calreticulin, and one alpha-tubulin. In comparison to the xylem-preferential proteins, the housekeeping proteins were expressed at significant levels in the bark as well. Also, several additional protein spots were detected for CCoAOMT, PCBER, and COMT by immunoblot. Our data show that for the study of xylogenesis, two-dimensional protein gel comparisons combined with systematic protein sequencing may yield information complementary to that from EST sequencing strategies.