Jan Van Doorsselaere

Molecular Phenotyping of the pal1 and pal2 Mutants of Arabidopsis thaliana Reveals Far-Reaching Consequences on Phenylpropanoid, Amino Acid, and Carbohydrate Metabolism

The first enzyme of the phenylpropanoid pathway, Phe ammonia-lyase (PAL), is encoded by four genes in Arabidopsis thaliana. Whereas PAL function is well established in various plants, an insight into the functional significance of individual gene family members is lacking. We show that in the absence of clear phenotypic alterations in the Arabidopsis pal1 and pal2 single mutants and with limited phenotypic alterations in the pal1 pal2 double mutant, significant modifications occur in the transcriptome and metabolome of the pal mutants. The disruption of PAL led to transcriptomic adaptation of components of the phenylpropanoid biosynthesis, carbohydrate metabolism, and amino acid metabolism, revealing complex interactions at the level of gene expression between these pathways. Corresponding biochemical changes included a decrease in the three major flavonol glycosides, glycosylated vanillic acid, scopolin, and two novel feruloyl malates coupled to coniferyl alcohol. Moreover, Phe overaccumulated in the double mutant, and the levels of many other amino acids were significantly imbalanced. The lignin content was significantly reduced, and the syringyl/guaiacyl ratio of lignin monomers had increased. Together, from the molecular phenotype, common and specific functions of PAL1 and PAL2 are delineated, and PAL1 is qualified as being more important for the generation of phenylpropanoids.

Elucidation of new structures in lignins of CAD- and COMT-deficient plants by NMR.

Studying lignin-biosynthetic-pathway mutants and transgenics provides insights into plant responses to perturbations of the lignification system, and enhances our understanding of normal lignification. When enzymes late in the pathway are downregulated, significant changes in the composition and structure of lignin may result. NMR spectroscopy provides powerful diagnostic tools for elucidating structures in the difficult lignin polymer, hinting at the chemical and biochemical changes that have occurred. COMT (caffeic acid O-methyl transferase) downregulation in poplar results in the incorporation of 5-hydroxyconiferyl alcohol into lignins via typical radical coupling reactions, but post-coupling quinone methide internal trapping reactions produce novel benzodioxane units in the lignin. CAD (cinnamyl alcohol dehydrogenase) downregulation results in the incorporation of the hydroxycinnamyl aldehyde monolignol precursors intimately into the polymer. Sinapyl aldehyde cross-couples 8-O-4 with both guaiacyl and syringyl units in the growing polymer, whereas coniferyl aldehyde cross-couples 8-O-4 only with syringyl units, reflecting simple chemical cross-coupling propensities. The incorporation of hydroxycinnamyl aldehyde and 5-hydroxyconiferyl alcohol monomers indicates that these monolignol intermediates are secreted to the cell wall for lignification. The recognition that novel units can incorporate into lignins portends significantly expanded opportunities for engineering the composition and consequent properties of lignin for improved utilization of valuable plant resources.

NMR Evidence for benzodioxane structures resulting from incorporation of 5-hydroxyconiferyl alcohol into lignins of O-methyltransferase-deficient poplars

Benzodioxane structures are produced in lignins of transgenic poplar plants deficient in COMT, anO-methyltransferase required to produce lignin syringyl units. They result from incorporation of 5-hydroxyconiferyl alcohol into the monomer supply and confirm that phenols other than the three traditional monolignols can be integrated into plant lignins.

Expression of a poplar cDNA encoding a ferulate-5-hydroxylase/coniferaldehyde 5-hydroxylase increases S lignin deposition in Arabidopsis thaliana

Ferulate-5-hydroxylase or coniferaldehyde 5-hydroxylase (F5H or Cald5H; CYP84A1) is the enzyme responsible for the last hydroxylation of the syringyl-type lignin precursors. A cDNA clone highly homologous to the Arabidopsis thaliana F5H/Cald5H cDNA was identified during the random sequencing of a poplar (Populus trichocarpa) differentiating xylem cDNA library. Present as a multigenic family in poplar, this gene (PopF5H) was found to be highly expressed in lignified tissues supporting its role in lignin biosynthesis. When placed under the control of the CaMV 35S promoter, the poplar F5H was able to complement the A. thaliana fah1-2 mutation. Overexpression in wild-type A. thaliana substantially increased the level of syringyl units in lignins. These transgenic lines were also typified by substantial levels of S-units in root lignins, whereas these units are present in low amounts in wild-type A. thaliana. These results support that PopF5H is a functional homolog of the A. thaliana F5H gene.

One-step purification and characterization of a lignin-specific O-methyltransferase from poplar

O-Methyltransferases (OMT; EC 2.1.1.6) play an important role in the synthesis of lignin precursors by catalyzing the O-methylation of o-diphenolic substrates such as caffeic acid (CA) and 5-hydroxyferulic acid (5OH). Here, we report on the purification of a lignin-specific OMT (38 kDa) from poplar (Populus trichocarpa x P. deltoides). The OMT was purified from xylem by a single affinity chromatography step on adenosine agarose. The enzyme uses both CA and 5OH as substrates. We previously have reported the cloning of a corresponding OMT cDNA [Dumas et al., Plant Physiol. 98 (1992) 796-797]. Expression of this OMT cDNA in Escherichia coli further confirmed the identity of the clone. Genomic hybridization demonstrates the presence of one or two OMT genes per haploid poplar genome. RNA gel blot hybridization shows high levels of steady-state OMT mRNA in the xylem of young poplar trees, as compared to the levels in leaves.

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.