Hugo Meyermans

Modifications in lignin and accumulation of phenolic glucosides in poplar xylem upon down-regulation of caffeoyl-coenzyme A O-methyltransferase, an enzyme involved in lignin biosynthesis.

Caffeoyl-coenzyme AO-methyltransferase (CCoAOMT) methylates, in vitro, caffeoyl-CoA and 5-hydroxyferuloyl-CoA, two possible precursors in monolignol biosynthesis in vivo. To clarify the in vivo role of CCoAOMT in lignin biosynthesis, transgenic poplars with 10% residual CCoAOMT protein levels in the stem xylem were generated. Upon analysis of the xylem, the affected transgenic lines had a 12% reduced Klason lignin content, an 11% increased syringyl/guaiacyl ratio in the noncondensed lignin fraction, and an increase in lignin-attached p-hydroxybenzoate but otherwise a lignin composition similar to that of wild type. Stem xylem of the CCoAOMT-down-regulated lines had a pink-red coloration, which coincided with an enhanced fluorescence of mature vessel cell walls. The reduced production of CCoAOMT caused an accumulation ofO 3-β-d-glucopyranosyl-caffeic acid,O 4-β-d-glucopyranosyl-vanillic acid, andO 4-β-d-glucopyranosyl-sinapic acid (GSA), as authenticated by 1H NMR. Feeding experiments showed thatO 3-β-d-glucopyranosyl-caffeic acid and GSA are storage or detoxification products of caffeic and sinapic acid, respectively. The observation that down-regulation of CCoAOMT decreases lignin amount whereas GSA accumulates to 10% of soluble phenolics indicates that endogenously produced sinapic acid is not a major precursor in syringyl lignin biosynthesis. Our in vivo results support the recently obtained in vitroenzymatic data that suggest that the route from caffeic acid to sinapic acid is not used for lignin biosynthesis.

Wood formation in poplar : identification, characterization, and seasonal variation of xylem proteins

Abstract. 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 α-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.

Applications of molecular genetics for biosynthesis of novel lignins

Abstract For the production of high-quality paper, lignin needs to be removed from cellulose. This process is energy requiring, expensive and toxic. Molecular biology offers tools to generate trees with a modified lignin composition to facilitate the extractability of lignin. We have cloned several genes encoding enzymes in the methylation of the lignin monomers [bi-specific caffeic acid/5-hydroxyferulic acid O -methyltransferase (COMT) and caffeoyl-CoA O -methyltransferase], and encoding enzymes specific for the lignin biosynthesis pathway [cinnamoyl-CoA reductase and cinnamyl alcohol dehydrogenase (CAD)]. The antisense strategy is used to study the effect of a down-regulation of these enzymes on the lignin content and the lignin composition of poplar wood. Our results demonstrate that the monomeric composition of lignin was dramatically affected by down-regulation of COMT. Transgenic poplars with a reduced CAD activity have better pulping properties, due to a higher extractability of the lignin.

Lignin Biosynthesis in Poplar: Genetic Engineering and Effects on Kraft Pulping

ABSTRACT Transgenic poplar, downregulated in cinnamyl alcohol dehydrogenase (CAD) or caffeic acid-O-methyltransferase (COMT) expression have been grown in field trials. Wood of these trees has been evaluated for Kraft pulping. The results show that lignin is more easily extracted from wood of the CAD-downregulated trees, whereas wood from COMT-downregulated trees is less suitable for Kraft pulping. Detailed NMR analyses of lignin from COMT-downregulated poplars reveal the presence of benzodioxane structures, which are derived from coupling of 5-hydroxyconiferyl alcohol with the lignin polymer, showing that monolignols other than p-coumaryl, coniferyl and sinapyl alcohol can be incorporated into lignin. Analysis of poplar downregulated for CCoAOMT shows that CCoAOMT is involved in the synthesis of both syringyl and guaiacyl units, and that sinapic is probably not an important precursor for syringyl lignin synthesis.