Abstract: Two methylation steps are necessary for the biosynthesis of monolignols, the lignin precursors. Caffeic acid O-methyltransferase (COMT) O-methylates at the C5 position of the phenolic ring. COMT is responsible for the biosynthesis of sinapyl alcohol, the precursor of syringyl lignin units. The O-methylation at the C3 position of the phenolic ring involves the Caffeoyl CoA 3-O-methyltransferase (CCoAOMT). The CCoAOMT 1 gene (At4g34050) is believed to encode the enzyme responsible for the first O-methylation in Arabidopsis thaliana. A CCoAOMT1 promoter-GUS fusion and immunolocalization experiments revealed that this gene is strongly and exclusively expressed in the vascular tissues of stems and roots. An Arabidopsis T-DNA null mutant named ccomt 1 was identified and characterised. The mutant stems are slightly smaller than wild-type stems in short-day growth conditions and has collapsed xylem elements. The lignin content of the stem is low and the S/G ratio is high mainly due to fewer G units. These results suggest that this O-methyltransferase is involved in G-unit biosynthesis but does not act alone to perform this step in monolignol biosynthesis. To determine which O-methyltransferase assists CCoAOMT 1, a comt 1 ccomt1 double mutant was generated and studied. The development of comt 1 ccomt1 is arrested at the plantlet stage in our growth conditions. Lignins of these plantlets are mainly composed of p-hydroxyphenyl units. Moreover, the double mutant does not synthesize sinapoyl malate, a soluble phenolic. These results suggest that CCoAOMT 1 and COMT 1 act together to methylate the C3 position of the phenolic ring of monolignols in Arabidopsis. In addition, they are both involved in the formation of sinapoyl malate and isorhamnetin.

Abstract: The objective of this study was to assess the influence of the peroxidase/coniferyl alcohol (CA) ratio on the dehydrogenation polymer (DHP) synthesis. The soluble and unsoluble fractions of horseradish peroxidase (HRP)-catalyzed CA dehydrogenation mixtures were recovered in various proportions, depending on the polymerization mode (Zutropf ZT/Zulauf ZL) and HRP/CA ratio (1.6-1100purpurogallin U mmol(-1)). The ZL mode yielded 0-57%/initial CA of unsoluble condensed DHPs (thioacidolysis yields <200micromolg(-1)) with a proportion of uncondensed CA end groups increasing with the HRP/CA ratio (7.2-55.5%/total uncondensed CA). Systematically lower polymer yields (0-49%/initial CA) were obtained for the ZT mode. In that mode, a negative correlation was established between the beta-O-4 content (thioacidolysis yields: 222-660micromolg(-1)) and the HRP/CA ratio. In both modes, decreasing the HRP/CA ratio below 18Ummol(-1) favoured an end-wise polymerization process evidenced by the occurrence of tri-, tetra- and pentamers involving at least one beta-O-4 bond. At low ratio, the unsoluble ZT DHP was found to better approximate natural lignins than DHPs previously synthesized with traditional methods. Besides its possible implication in lignin biosynthesis, peroxidase activity is a crucial parameter accounting for the structural variations of in vitro DHPs.

Abstract: An extensive search for maize (Zea mays) genes involved in cell wall biosynthesis and assembly has been performed and 735 sequences have been centralized in a database, MAIZEWALL (http://www.polebio.scsv.ups-tlse.fr/MAIZEWALL). MAIZEWALL contains a bioinformatic analysis for each entry and gene expression data that are accessible via a user-friendly interface. A maize cell wall macroarray composed of a gene-specific tag for each entry was also constructed to monitor global cell wall-related gene expression in different organs and during internode development. By using this macroarray, we identified sets of genes that exhibit organ and internode-stage preferential expression profiles. These data provide a comprehensive fingerprint of cell wall-related gene expression throughout the maize plant. Moreover, an in-depth examination of genes involved in lignin biosynthesis coupled to biochemical and cytological data from different organs and stages of internode development has also been undertaken. These results allow us to trace spatially and developmentally regulated, putative preferential routes of monolignol biosynthesis involving specific gene family members and suggest that, although all of the gene families of the currently accepted monolignol biosynthetic pathway are conserved in maize, there are subtle differences in family size and a high degree of complexity in spatial expression patterns. These differences are in keeping with the diversity of lignified cell types throughout the maize plant.

Abstract: In Arabidopsis thaliana, silencing of hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT), a lignin biosynthetic gene, results in a strong reduction of plant growth. We show that, in HCT-silenced plants, lignin synthesis repression leads to the redirection of the metabolic flux into flavonoids through chalcone synthase activity. Several flavonol glycosides and acylated anthocyanin were shown to accumulate in higher amounts in silenced plants. By contrast, sinapoylmalate levels were barely affected, suggesting that the synthesis of that phenylpropanoid compound might be HCT-independent. The growth phenotype of HCT-silenced plants was shown to be controlled by light and to depend on chalcone synthase expression. Histochemical analysis of silenced stem tissues demonstrated altered tracheary elements. The level of plant growth reduction of HCT-deficient plants was correlated with the inhibition of auxin transport. Suppression of flavonoid accumulation by chalcone synthase repression in HCT-deficient plants restored normal auxin transport and wild-type plant growth. By contrast, the lignin structure of the plants simultaneously repressed for HCT and chalcone synthase remained as severely altered as in HCT-silenced plants, with a large predominance of nonmethoxylated H units. These data demonstrate that the reduced size phenotype of HCT-silenced plants is not due to the alteration of lignin synthesis but to flavonoid accumulation.

Abstract: Cytochromes P450 monooxygenases from the CYP98 family catalyze the meta-hydroxylation step in the phenylpropanoid biosynthetic pathway. The ref8 Arabidopsis (Arabidopsis thaliana) mutant, with a point mutation in the CYP98A3 gene, was previously described to show developmental defects, changes in lignin composition, and lack of soluble sinapoyl esters. We isolated a T-DNA insertion mutant in CYP98A3 and show that this mutation leads to a more drastic inhibition of plant development and inhibition of cell growth. Similar to the ref8 mutant, the insertion mutant has reduced lignin content, with stem lignin essentially made of p-hydroxyphenyl units and trace amounts of guaiacyl and syringyl units. However, its roots display an ectopic lignification and a substantial proportion of guaiacyl and syringyl units, suggesting the occurrence of an alternative CYP98A3-independent meta-hydroxylation mechanism active mainly in the roots. Relative to the control, mutant plantlets produce very low amounts of sinapoyl esters, but accumulate flavonol glycosides. Reduced cell growth seems correlated with alterations in the abundance of cell wall polysaccharides, in particular decrease in crystalline cellulose, and profound modifications in gene expression and homeostasis reminiscent of a stress response. CYP98A3 thus constitutes a critical bottleneck in the phenylpropanoid pathway and in the synthesis of compounds controlling plant development. CYP98A3 cosuppressed lines show a gradation of developmental defects and changes in lignin content (40% reduction) and structure (prominent frequency of p-hydroxyphenyl units), but content in foliar sinapoyl esters is similar to the control. The purple coloration of their leaves is correlated to the accumulation of sinapoylated anthocyanins.

Abstract: The cinnamyl alcohol dehydrogenase (AtCAD) multigene family in Arabidopsis is composed of nine genes. Our previous studies focused on the two isoforms AtCAD C and AtCAD D which show a high homology to those related to lignification in other plants. This study focuses on the seven other Arabidopsis CAD for which functions are not yet elucidated. Their expression patterns were determined in different parts of Arabidopsis. Only CAD 1 protein can be detected in elongating stems, flowers, and siliques using Western-blot analysis. Tissue specific expression of CAD 1, B1, and G genes was determined using their promoters fused to the GUS reporter gene. CAD 1 expression was observed in primary xylem in accordance with a potential role in lignification. Arabidopsis T-DNA mutants knockout for the different genes CAD genes were characterized. Their stems displayed no substantial reduction of CAD activities for coniferyl and sinapyl alcohols as well as no modifications of lignin quantity and structure in mature inflorescence stems. Only a small reduction of lignin content could be observed in elongating stems of Atcad 1 mutant. These CAD genes in combination with the CAD D promoter were used to complement a CAD double mutant severely altered in lignification (cad c cad d). The expression of AtCAD A, B1, B2, F, and G had no effect on restoring a normal lignin profile of this mutant. In contrast, CAD 1 complemented partly this mutant as revealed by the partial restoration of conventional lignin units and by the decrease in the frequency of beta-O-4 linked p-OH cinnamaldehydes.

Abstract: Drought is a major abiotic stress affecting all levels of plant organization and, in particular, leaf elongation. Several experiments were designed to study the effect of water deficits on maize (Zea mays) leaves at the protein level by taking into account the reduction of leaf elongation. Proteomic analyses of growing maize leaves allowed us to show that two isoforms of caffeic acid/5-hydroxyferulic 3-O-methyltransferase (COMT) accumulated mostly at 10 to 20 cm from the leaf point of insertion and that drought resulted in a shift of this region of maximal accumulation toward basal regions. We showed that this shift was due to the combined effect of reductions in growth and in total amounts of COMT. Several other enzymes involved in lignin and/or flavonoid synthesis (caffeoyl-CoA 3-O-methyltransferase, phenylalanine ammonia lyase, methylenetetrahydrofolate reductase, and several isoforms of S-adenosyl-l-methionine synthase and methionine synthase) were highly correlated with COMT, reinforcing the hypothesis that the zone of maximal accumulation corresponds to a zone of lignification. According to the accumulation profiles of the enzymes, lignification increases in leaves of control plants when their growth decreases before reaching their final size. Lignin levels analyzed by thioacidolysis confirmed that lignin is synthesized in the region where we observed the maximal accumulation of these enzymes. Consistent with the levels of these enzymes, we found that the lignin level was lower in leaves of plants subjected to water deficit than in those of well-watered plants.

Abstract: During lignin biosynthesis in angiosperms, coniferyl and sinapyl aldehydes are believed to be converted into their corresponding alcohols by cinnamyl alcohol dehydrogenase (CAD) and by sinapyl alcohol dehydrogenase (SAD), respectively. This work clearly shows that CAD-C and CAD-D act as the primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis thaliana by supplying both coniferyl and sinapyl alcohols. An Arabidopsis CAD double mutant (cad-c cad-d) resulted in a phenotype with a limp floral stem at maturity as well as modifications in the pattern of lignin staining. Lignin content of the mutant stem was reduced by 40%, with a 94% reduction, relative to the wild type, in conventional beta-O-4-linked guaiacyl and syringyl units and incorportion of coniferyl and sinapyl aldehydes. Fourier transform infrared spectroscopy demonstrated that both xylem vessels and fibers were affected. GeneChip data and real-time PCR analysis revealed that transcription of CAD homologs and other genes mainly involved in cell wall integrity were also altered in the double mutant. In addition, molecular complementation of the double mutant by tissue-specific expression of CAD derived from various species suggests different abilities of these genes/proteins to produce syringyl-lignin moieties but does not indicate a requirement for any specific SAD gene.

Abstract: Grass cell wall degradability is conventionally related to the lignin content and to the ferulic-mediated cross-linking of lignins to polysaccharides. To better understand the variations in degradability, 22 maize inbred lines were subjected to image analyses of Fasga- and Mäule-stained stem sections and to chemical analyses of lignins and p-hydroxycinnamic acids. For the first time, the nearness of biochemical and histological estimates of lignin levels was established. Combination of histological and biochemical traits could explain 89% of the variations for cell wall degradability and define a maize ideotype for cell wall degradability. In addition to a reduced lignin level, such an ideotype would contain lignins richer in syringyl than in guaiacyl units and preferentially localized in the cortical region rather than in the pith. Such enrichment in syringyl units would favor wall degradability in grasses, contrary to dicots, and could be related to the fact that grass syringyl units are noticeably p-coumaroylated. This might affect the interaction capabilities of lignins and polysaccharides.

Abstract: Summary EgMYB2, a member of a new subgroup of the R2R3 MYB family of transcription factors, was cloned from a library consisting of RNA from differentiating Eucalyptus xylem. EgMYB2 maps to a unique locus on the Eucalyptus grandis linkage map and co-localizes with a quantitative trait locus (QTL) for lignin content. Recombinant EgMYB2 protein was able to bind specifically the cis-regulatory regions of the promoters of two lignin biosynthetic genes, cinnamoyl-coenzyme A reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD), which contain MYB consensus binding sites. EgMYB2 was also able to regulate their transcription in both transient and stable expression assays. Transgenic tobacco plants over-expressing EgMYB2 displayed phenotypic changes relative to wild-type plants, among which were a dramatic increase in secondary cell wall thickness, and an alteration of the lignin profiles. Transcript abundance of genes encoding enzymes specific to lignin biosynthesis was increased to varying extents according to the position of individual genes in the pathway, whereas core phenylpropanoid genes were not significantly affected. Together these results suggest a role for EgMYB2 in the co-ordinated control of genes belonging to the monolignol-specific pathway, and therefore in the biosynthesis of lignin and the regulation of secondary cell wall formation.

Abstract: This study reports on the destructuration of Wheat straw and Spruce wood cell walls after maceration in potassium carbonate or sodium hydroxide at pH = 10 in the presence of copper acetate. The alkaline treatments had a predominant impact on the wheat straw cell wall components over copper acetate. Either K-carbonate or Na-hydroxide extracted from wheat straw a particular lignin fraction rich in condensed C-C linkages, leading to the unmasking of new ether-linked sub-structures in the cell wall. This unmasking was increased in the presence of copper salt but only in the nonextracted Wheat straw sample incubated in carbonate and not in the corresponding extractive-free sample. This difference was related to the leaching of compounds from the nonextracted cell wall, which could sustain oxidative activity of copper by hindering its precipitation into inactive hydroxide and/or carbonate species. In Spruce wood samples, copper salt was the principal factor impacting on the lignin structure over alkali alone. Its effect was, however, only detected at the level of C-C linked dimers. These results confirmed that unmasking of lignin sub-structures also occurred in Spruce wood, but probably through mechanisms different from that evidenced in Wheat straw.

Abstract: Side chains of sugar beet (Beta vulgaris) pectins, which are mainly composed of arabinose (Ara) and galactose (Gal) residues, are esterified by ferulic acid units. Enzymatic hydrolysis of beet cell walls yielded several feruloylated oligosaccharides, which were separated by hydrophobic interaction chromatography. Two new oligomers were isolated in the fraction eluted by 25:75 (v/v) ethanol:water. An arabinotriose and an arabinotetraose esterified by two ferulic acid residues were obtained, and their structure was elucidated by mass spectrometry. It is shown that feruloyl groups are linked to O-5 of Ara residues, in addition to the known O-2 position. This work establishes for the first time, to our knowledge, that two neighboring Ara units may be esterified by two ferulic acid units. This close proximity may have important biochemical implications.

Abstract: A series of transgenic poplars down-regulated for cinnamyl alcohol dehydrogenase (CAD) was analyzed by thioacidolysis. Among the lignin-derived monomers, the indene compounds that were recently shown to originate from sinapaldehyde incorporated into lignins through 8-O-4-cross-coupling, were found to increase as a function of CAD deficiency level. While these syringyl markers were recovered in substantial amounts in the most severely depressed lines, the markers for coniferaldehyde incorporation were recovered in only low amounts. In conjunction with these additional sinapaldehyde units and relative to the control samples, lignins in CAD-deficient poplar lines had less conventional syringyl-units and beta-O-4-bonds and more free phenolic groups. We found that almost half of the polymers in the most deficient lines could be solubilized in alkali and at room temperature. This unusual behavior suggests that lignins in CAD-deficient poplars occur as small, alkali-leachable lignin domains. That mainly sinapaldehyde incorporates into the lignins of CAD-deficient poplars suggests that the recently identified sinapyl alcohol dehydrogenase (SAD), which is structurally distinct from the CAD enzyme targeted herein, does not play any substantial role in constitutive lignification in poplar.

Abstract: The hydroxyl group in the 3-position of the phenylpropanoid compounds is introduced at the level of coumarate shikimate/quinate esters, whose synthesis implicates an acyltransferase activity. Specific antibodies raised against the recombinant tobacco (Nicotiana tabacum) acyltransferase revealed the accumulation of the enzyme in stem vascular tissues of tobacco, in accordance with a putative role in lignification. For functional analysis, the acyltransferase gene was silenced in Arabidopsis thaliana and N. benthamiana by RNA-mediated posttranscriptional gene silencing. In Arabidopsis, gene silencing resulted in a dwarf phenotype and changes in lignin composition as indicated by histochemical staining. An in-depth study of silenced N. benthamiana plants by immunological, histochemical, and chemical methods revealed the impact of acyltransferase silencing on soluble phenylpropanoids and lignin content and composition. In particular, a decrease in syringyl units and an increase in p-hydroxyphenyl units were recorded. Enzyme immunolocalization by confocal microscopy showed a correlation between enzyme accumulation levels and lignin composition in vascular cells. These results demonstrate the function of the acyltransferase in phenylpropanoid biosynthesis.

Abstract: Poplar (Populus tremula x alba) trees (clone INRA 717-1-B4) were cultivated for 1 month in phytotronic chambers with two different levels of ozone (60 and 120 nL L(-1)). Foliar activities of shikimate dehydrogenase (EC 1.1.1.25), phenylalanine ammonia lyase (EC 4.3.1.5), and cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) were compared with control levels. In addition, we examined lignin content and structure in control and ozone-fumigated leaves. Under ozone exposure, CAD activity and CAD RNA levels were found to be rapidly and strongly increased whatever the foliar developmental stage. In contrast, shikimate dehydrogenase and phenylalanine ammonia lyase activities were increased in old and midaged leaves but not in the youngest ones. The increased activities of these enzymes involved in the late or early steps of the metabolic pathway leading to lignins were associated with a higher Klason lignin content in extract-free leaves. In addition, stress lignins synthesized in response to ozone displayed a distinct structure, relative to constitutive lignins. They were found substantially enriched in carbon-carbon interunit bonds and in p-hydroxyphenylpropane units, which is reminiscent of lignins formed at early developmental stages, in compression wood, or in response to fungal elicitor. The highest changes in lignification and in enzyme activities were obtained with the highest ozone dose (120 nL L(-1)). These results suggest that ozone-induced lignins might contribute to the poplar tolerance to ozone because of their barrier or antioxidant effect toward reactive oxygen species.

Abstract: The mammalian lignans enterolactone (ENL) and enterodiol, commonly found in human plasma and urine, are phytoestrogens that may contribute to the prevention of breast cancer and coronary heart disease. They are formed by the conversion of dietary precursors such as secoisolariciresinol and matairesinol lignans by the colonic microflora. The identification of lignins, cell-wall polymers structurally related to lignans, as precursors of mammalian lignans is reported here for the first time. In study 1, rats were fed rye or wheat bran (15% diet) for 5 d. Untreated brans and brans extracted with solvents to remove lignans were compared. ENL was estimated in urine samples collected for 24 h by time-resolved fluoroimmunoassay. ENL urinary excretion was reduced from 18.6 to 5.3 nmol/d (n=8; P<0.001) when lignans were removed from rye bran and from 30.5 to 6.2 nmol/d (P<0.001) when they were removed from wheat bran. These results suggest that lignins, embedded in the cell wall and retained in the bran during solvent extraction, account for 26-32% of the ENL formed from cereal brans. In study 2, rats were fed a deuterated synthetic lignin (0.2% diet) together with wheat bran (15%) for 3 d. The detection of deuterated ENL by LC-tandem MS in urine (20 nmol/d) clearly confirms the conversion of lignin into mammalian lignans. More research is warranted to determine the bioavailability of lignins in the human diet.

Abstract: Lignification was investigated in wild-type (WT) and in transgenic poplar plantlets with a reduced caffeic acid O-methyl-transferase (COMT) activity. Coniferin and syringin, deuterated at their methoxyl, were incorporated into the culture medium of microcuttings. The gas chromatography-mass spectrometry (GC-MS) analysis of the thioacidolysis guaiacyl (G) and syringyl (S) lignin-derived monomers revealed that COMT deficiency altered stem lignification. GC-MS analysis proved that the deuterated precursors were incorporated into root lignins and, to a lower extent, in stem lignins without major effect on growth and lignification. Deuterium from coniferin was recovered in G and S lignin units, whereas deuterium from syringin was only found in S units, which further establishes that the conversion of G to S lignin precursors may occur at the level of p-OH cinnamyl alcohols.

Abstract: Hardwood trees are able to reorient their axes owing to tension wood differentiation. Tension wood is characterised by important ultrastructural modifications, such as the occurrence in a number of species, of an extra secondary wall layer, named gelatinous layer or G-layer, mainly constituted of cellulose microfibrils oriented nearly parallel to the fibre axis. This G-layer appears directly involved in the definition of tension wood mechanical properties. This review gathers the data available in the literature about lignification during tension wood formation. Potential roles for lignin in tension wood formation are inferred from biochemical, anatomical and mechanical studies, from the hypotheses proposed to describe tension wood function and from data coming from new research areas such as functional genomics.

Abstract: Lignification limits grass cell-wall digestion by herbivores. Lignification is spatially and temporally regulated, and lignin characteristics differ between cell walls, plant tissues, and plant parts. Grass lignins are anchored within walls by ferulate and diferulate cross-links, p-coumarate cyclodimers, and possibly benzyl ester and ether cross-links. Cell-wall degradability is regulated by lignin concentration, cross-linking, and hydrophobicity but not directly by most variations in lignin composition or structure. Genetic manipulation of lignification can improve grass cell-wall degradability, but the degree of success will depend on genetic background, plant modification techniques employed, and analytical methods used to characterize cell walls.

Abstract: The brown-midrib mutants of maize have a reddish-brown pigmentation of the leaf midrib and stalk pith, associated with lignified tissues. These mutants progressively became models for lignification genetics and biochemical studies in maize and grasses. Comparisons at silage maturity of bm1, bm2, bm3, bm4 plants highlighted their reduced lignin, but also illustrated the biochemical specificities of each mutant in p-coumarate, ferulate ester and etherified ferulate content, or syringyl/guaiacyl monomer ratio after thioacidolysis. Based on the current knowledge of the lignin pathway, and based on presently developed data and discussions, C3H and CCoAOMT activities are probably major hubs in controlling cell-wall lignification (and digestibility). It is also likely that ferulates arise via the CCoAOMT pathway.

Abstract: We investigated the potential of an electron-beam post-treatment to tailor the properties of 70/30 and 80/20 wt. extruded starch-lignin films. The effect of a 400 kGy radiation on films differing essentially by the kind of lignins incorporated (lignosulfonates/alkali lignins) was assessed both at the macroscopic and the molecular levels. Changes in the polymer molecular structure were studied by IR spectroscopy, by thioacidolysis as well as by model compound experiments. Electron beam-irradiation at 400 kGy, a rather high dose for processing natural polymers, alters to some extent the mechanical resistance of the starch-based materials. However this treatment substantially reduces the hydrophilic surface properties of the films, while not harming their biodegradability. Involved in radical cross-coupling reactions, lignin phenolic compounds are likely to play a primary role in the formation of a hydrophobic condensed network. This study suggests that lower irradiation doses might yield biomaterials with improved usage properties.

Abstract: Lignification of cell walls is the major factor controlling the digestibility of forage grasses. Thus far, from QTL analysis, about 15 locations involved in cell-wall lignification or digestibility have been identified in the maize genome, many of which colocalise with QTLs involved in corn borer susceptibility. Genetic diversity for enhancing cell-wall digestibility in maize must be identified in novel germplasm, but genetic engineering is also a relevant way both to design specific cell-wall characteristics for improved digestibility and to identify genes involved in these traits for further discovery of alleles of interest in grass germplasm.

Abstract: To investigate mechanisms involved in cell wall development, an Arabidopsis T-DNA insertion mutant collection was screened to identify mutants with beta-glucuronidase fusion gene expression in tissues undergoing secondary cell wall thickening. This promoter-trapping strategy allowed the isolation of a transformant containing the GUS coding sequence inserted 700 bp upstream of the ATG of a putative beta-xylosidase gene. The transformant has no phenotype as the expression of the gene was not disrupted by the insertion. The analysis of the predicted protein, AtBXL1, suggests its targeting to the extracellular matrix and its involvement in cell wall metabolism through a putative activity towards xylans. The 2-kb promoter sequence of AtBXL1 was fused to the GUS coding sequence and introduced into wild-type Arabidopsis thaliana. GUS expression was shown to be restricted to tissues undergoing secondary cell wall formation. Beta-xylosidase activity was associated with the cell wall-enriched fraction of different organs of wild-type plants. The level of activity correlates with transcript accumulation of AtBXL1 and other AtBXL1-related genes. Transgenic plants expressing the AtBXL1 cDNA in antisense orientation were generated. Lines exhibiting the highest decrease in AtBXL1 transcript accumulation and beta-xylosidase activity had phenotypic alterations. This newly identified gene is proposed to be involved in secondary cell wall hemicellulose metabolism and plant development.

Abstract: Capillary zone electrophoresis has been used to monitor the first steps of the dehydrogenative polymerization of coniferyl alcohol, sinapyl aldehyde, or a mixture of both, catalyzed by the horseradish peroxidase (HRP)-H(2)O(2) system. When coniferyl alcohol was the unique HRP substrate, three major dimers were observed (beta-5, beta-beta, and beta-O-4 interunit linkages) and their initial formation velocity as well as their relative abundance varied with pH. The beta-O-4 interunit linkage was thus slightly favored at lower pH values. In contrast, sinapyl aldehyde turned out to be a very poor substrate for HRP except in basic conditions (pH 8). The major dimer observed was the beta,beta'-di-sinapyl aldehyde, a red-brown exhibiting compound which might partly participate in the red coloration usually observed in cinnamyl alcohol dehydrogenase-deficient angiosperms. Finally, when a mixture of coniferyl alcohol and sinapyl aldehyde was used, it looked as if sinapyl aldehyde became a very good substrate for HRP. Indeed, coniferyl alcohol turned out to serve as a redox mediator (i.e. "shuttle oxidant") for the sinapyl aldehyde incorporation in the lignin-like polymer. This means that in particular conditions the specificity of oxidative enzymes might not hinder the incorporation of poor substrates into the growing lignin polymer.

Abstract: The health effects of dietary polyphenols might be explained by both intact compounds and their metabolites formed either in the tissues or in the colon by the microflora. The quantitative importance and biological activities of the microbial metabolites have seldom been examined in vivo. We measured the microbial metabolites formed in four groups of rats (n = 8) fed for 8 d a diet supplemented with 0.12 g/100 g catechin, 0.25 or 0.50 g/100 g red wine powder containing proanthocyanidins, phenolic acids, flavanols, anthocyanins and flavonols or an unsupplemented diet. Fourteen aromatic acid metabolites were assayed in urine collected for 24 h by an HPLC-electrospray ionization (ESI)-mass spectrometry (MS)-MS method. The three main metabolites formed from the catechin diet were 3-hydroxyphenylpropionic acid, 3-hydroxybenzoic acid and 3-hydroxyhippuric acid. Their total urinary excretion accounted for 4.7 g/100 g of the catechin ingested and that of intact catechins for 45.3 g/100 g. For wine polyphenols, the same microbial metabolites as observed for the catechin diet were identified in urine along with hippuric, p-coumaric, vanillic, 4-hydroxybenzoic and 3-hydroxyphenylacetic acids. All together, these aromatic acids accounted for 9.2 g/100 g of the total wine polyphenols ingested and intact catechins for only 1.2 g/100 g. The higher excretion of aromatic acids by rats fed wine polyphenols is likely due to their poor absorption in the proximal part of the gut. Some of the microbial metabolites still bear a reducing phenolic group and should also prevent oxidative stress in inner tissues. More attention should be given in the future to these microbial metabolites and their biological properties to help explain the health effects of polyphenols that are not easily absorbed through the gut barrier.

Abstract: The mechanical properties of wheat bran and the contribution of each constitutive tissue on overall bran properties were determined on a hard wheat (cv. Baroudeur) and a soft wheat (cv. Scipion). Manual dissection allowed three different layers to be separated from wheat bran, according to radial and longitudinal grain orientations, which were identified by confocal laser scanning microscopy as outer pericarp, an intermediate strip (comprising inner pericarp, testa, and nucellar tissue), and aleurone layer, respectively. Tissue microstructure and cell wall composition were determined. Submitted to traction tests, whole bran, intermediate, and aleurone layers demonstrated elastoplastic behavior, whereas pericarp exhibited elastic behavior. By longitudinal orientation, pericarp governed 50% bran elasticity (elastic strength and rigidity), whereas, in the opposite orientation, bran elastic properties were mostly influenced by the other tissues. Regardless of test orientation, the linear force required to bran rupture corresponded to the sum of intermediate and aleurone layer strengths. According to radial orientation, the intermediate strip governed bran extensibility, but according to longitudinal orientation, all tissues contributed until bran disruption. Tissues from both wheat cultivars behaved similarly. A structural model of wheat bran layers illustrated the detachment of pericarp from intermediate layer within radial bran strips.

Abstract: Studying Arabidopsis mutants of the phenylpropanoid pathway has unraveled several biosynthetic steps of monolignol synthesis. Most of the genes leading to monolignol synthesis have been characterized recently in this herbaceous plant, except those encoding cinnamyl alcohol dehydrogenase (CAD). We have used the complete sequencing of the Arabidopsis genome to highlight a new view of the complete CAD gene family. Among nine AtCAD genes, we have identified the two distinct paralogs AtCAD-C and AtCAD-D, which share 75% identity and are likely to be involved in lignin biosynthesis in other plants. Northern, semiquantitative restriction fragment-length polymorphism-reverse transcriptase-polymerase chain reaction and western analysis revealed that AtCAD-C and AtCAD-D mRNA and protein ratios were organ dependent. Promoter activities of both genes are high in fibers and in xylem bundles. However, AtCAD-C displayed a larger range of sites of expression than AtCAD-D. Arabidopsis null mutants (Atcad-D and Atcad-C) corresponding to both genes were isolated. CAD activities were drastically reduced in both mutants, with a higher impact on sinapyl alcohol dehydrogenase activity (6% and 38% of residual sinapyl alcohol dehydrogenase activities for Atcad-D and Atcad-C, respectively). Only Atcad-D showed a slight reduction in Klason lignin content and displayed modifications of lignin structure with a significant reduced proportion of conventional S lignin units in both stems and roots, together with the incorporation of sinapaldehyde structures ether linked at Cbeta. These results argue for a substantial role of AtCAD-D in lignification, and more specifically in the biosynthesis of sinapyl alcohol, the precursor of S lignin units.

Abstract: Cinnamoyl CoA reductase (CCR; EC 1.2.1.44) is the first enzyme specific to the biosynthetic pathway leading to monolignols. Arabidopsis thaliana (L.) Heynh. plants transformed with a vector containing a full-length AtCCR1 cDNA in an antisense orientation were obtained and characterized. The most severely down-regulated homozygous plants showed drastic alterations to their phenotypical features. These plants had a 50% decrease in lignin content accompanied by changes in lignin composition and structure, with incorporation of ferulic acid into the cell wall. Microscopic analyses coupled with immunolabelling revealed a decrease in lignin deposition in normally lignified tissues and a dramatic loosening of the secondary cell wall of interfascicular fibers and vessels. Evaluation of in vitro digestibility demonstrated an increase in the enzymatic degradability of these transgenic lines. In addition, culture conditions were shown to play a substantial role in lignin level and structure in the wild type and in the effects of AtCCR1 repression efficiency.

Abstract: BACKGROUND: Proanthocyanidins, the most abundant polyphenols in chocolate, are not depolymerized in the stomach and reach the small intestine intact, where they are hardly absorbed because of their high molecular weight. In vitro and in vivo studies using pure compounds as substrates suggest that proanthocyanidins and the related catechin monomers may be degraded into more bioavailable low-molecular-weight phenolic acids by the microflora in the colon. OBJECTIVE: The aim of the study was to estimate the amounts of phenolic acids formed by the microflora and excreted in the urine of human subjects after consumption of polyphenol-rich chocolate. DESIGN: After consumption of a polyphenol-free diet for 2 d and a subsequent overnight fast, 11 healthy subjects (7 men and 4 women) consumed 80 g chocolate containing 439 mg proanthocyanidins and 147 mg catechin monomers. All urine was collected during the 24 h before chocolate consumption and at 3, 6, 9, 24, and 48 h after chocolate consumption. Aromatic acids were identified in urine by gas chromatography-mass spectrometry and were quantified by HPLC-electrospray ionization tandem mass spectrometry. RESULTS: Chocolate intake increased the urinary excretion of the 6 following phenolic acids: m-hydroxyphenylpropionic acid, ferulic acid, 3,4-dihydroxyphenylacetic acid, m-hydroxyphenylacetic acid, vanillic acid, and m-hydroxybenzoic acid. CONCLUSION: The antioxidant and biological effects of chocolate may be explained not solely by the established absorption of catechin monomers but also by the absorption of microbial phenolic acid metabolites.

Abstract: A promoter-trap screen allowed us to identify an Arabidopsis line expressing GUS in the root vascular tissues. T-DNA border sequencing showed that the line was mutated in the caffeic acid O-methyltransferase 1 gene (AtOMT1) and therefore deficient in OMT1 activity. Atomt1 is a knockout mutant and the expression profile of the AtOMT1 gene has been determined as well as the consequences of the mutation on lignins, on soluble phenolics, on cell wall digestibility, and on the expression of the genes involved in monolignol biosynthesis. In this mutant and relative to the wild type, lignins lack syringyl (S) units and contain more 5-hydroxyguaiacyl units (5-OH-G), the precursors of S-units. The sinapoyl ester pool is modified with a two-fold reduction of sinapoyl-malate in the leaves and stems of mature plants as well as in seedlings. In addition, LC-MS analysis of the soluble phenolics extracted from the seedlings reveals the occurrence of unusual derivatives assigned to 5-OH-feruloyl malate and to 5-OH-feruloyl glucose. Therefore, AtOMT1 enzymatic activity appears to be involved not only in lignin formation but also in the biosynthesis of sinapate esters. In addition, a deregulation of other monolignol biosynthetic gene expression can be observed in the Atomt1 mutant. A poplar cDNA encoding a caffeic acid OMT (PtOMT1) was successfully used to complement the Atomt1 mutant and restored both the level of S units and of sinapate esters to the control level. However, the over-expression of PtOMT1 in wild-type Arabidopsis did not increase the S-lignin content, suggesting that OMT is not a limiting enzyme for S-unit biosynthesis.

Abstract: In an attempt to draw relationships between the molecular structure and the thermal behavior of lignins, thermomechanical analyses were run on six milled wood and enzyme poplar lignin fractions prepared from genetically modified and control woods. All the lignin samples displayed similar thermal profiles with a clear inflection point assigned to the glass transition point. The temperature (T(g)) at which this transition occurs showed large variations from 170 to 190 degrees C, depending both on the genetic modification and on the age of the tree. These variations were found to be closely related to the condensation degree of lignins evaluated by thioacidolysis.

Abstract: Many reports now describe the manipulation of plant metabolism by suppressing the expression of single genes. The potential of such work could be greatly expanded if multiple genes could be coordinately suppressed. In the work presented here, we test a novel method for achieving this by using single chimeric constructs incorporating partial sense sequences for multiple genes to target suppression of two or three lignin biosynthetic enzymes. We compare this method with a more conventional approach to achieving the same end by crossing plants harboring different antisense transgenes. Our results indicate that crossing antisense plants is less straightforward and predictable in outcome than anticipated. Most progeny had higher levels of target enzyme activity than predicted and had lost the expected modifications to lignin structure. In comparison, plants transformed with the chimeric partial sense constructs had more consistent high level suppression of target enzymes and had significant changes to lignin content, structure, and composition. It was possible to suppress three target genes coordinately using a single chimeric construct. Our results indicate that chimeric silencing constructs offer great potential for the rapid and coordinate suppression of multiple genes on diverse biochemical pathways and that the technique therefore deserves to be adopted by other researchers.

Abstract: The physiological role of the Norway spruce [ Picea abies (L.) Karst.] spi 2 gene, encoding a defense-related cationic peroxidase was examined in transgenic tobacco (Nicotiana tabacum L.). Expression of spi 2, under control of the 35S promoter, in tobacco plants resulted in higher total peroxidase activities. The phenotype of the spi 2-transformed lines was normal. The spi 2-transformed lines displayed lignin levels similar to levels in the control line, but with some alteration in lignin histochemistry and structure. These changes were associated with reduced flexibility of the tobacco stems. The defense against pathogenic microorganisms was altered in the transgenic tobacco plants compared with control plants. High peroxidase activities increased the susceptibility to the pathogenic oomycete Phytophthora parasitica var. nicotianae, but increased the ability of the tobacco plants to suppress growth of the pathogenic bacterium Erwinia carotovora.

Abstract: The agronomic and pulping performance of transgenic trees with altered lignin has been evaluated in duplicated, long-term field trials. Poplars expressing cinnamyl alcohol dehydrogenase (CAD) or caffeate/5-hydroxy-ferulate O-methyltransferase (COMT) antisense transgenes were grown for four years at two sites, in France and England. The trees remained healthy throughout the trial. Growth indicators and interactions with insects were normal. No changes in soil microbial communities were detected beneath the transgenic trees. The expected modifications to lignin were maintained in the transgenics over four years, at both sites. Kraft pulping of tree trunks showed that the reduced-CAD lines had improved characteristics, allowing easier delignification, using smaller amounts of chemicals, while yielding more high-quality pulp. This work highlights the potential of engineering wood quality for more environmentally benign papermaking without interfering with tree growth or fitness.

Abstract: Molecular marker compounds, derived from lignin by the thioacidolysis degradative method, for cinnamyl alcohol dehydrogenase (CAD) deficiency in angiosperms have been structurally identified as indene derivatives. They are shown to derive from hydroxycinnamyl aldehydes that have undergone 8-O-4-cross-coupling during lignification. As such, they are valuable markers for ascertaining plant responses to various levels of CAD down-regulation. Their derivation illustrates that hydroxycinnamyl aldehydes incorporate into angiosperm lignins by endwise coupling reactions in much the same way as normal monolignols do, suggesting that the hydroxycinnamyl aldehydes should be considered authentic lignin precursors.

Abstract: A capillary zone electrophoretic method has been developed for the quantitative determination of syringyl (S) and guaiacyl (G) monomers resulting from lignin thioacidolysis. The effects on the separation of altering a number of parameters have been investigated, resulting in an efficient and rapid separation. Analysis times were about 10 min instead of 50 min for the conventional GC methods, with no requirement for a derivatisation step prior to analysis. Relative standard deviations ranged between 8 and 12% for the absolute determination of S and G monomers, and between 1.4 and 3.5% for the S/G ratio.

Abstract: Transgenic maize (Zea mays) plants were generated with a construct harboring a maize caffeic acid O-methyltransferase (COMT) cDNA in the antisense (AS) orientation under the control of the maize Adh1 (alcohol dehydrogenase) promoter. Adh1-driven beta-glucuronidase expression was localized in vascular tissues and lignifying sclerenchyma, indicating its suitability in transgenic experiments aimed at modifying lignin content and composition. One line of AS plants, COMT-AS, displayed a significant reduction in COMT activity (15%-30% residual activity) and barely detectable amounts of COMT protein as determined by western-blot analysis. In this line, transgenes were shown to be stably integrated in the genome and transmitted to the progeny. Biochemical analysis of COMT-AS showed: (a) a strong decrease in Klason lignin content at the flowering stage, (b) a decrease in syringyl units, (c) a lower p-coumaric acid content, and (d) the occurrence of unusual 5-OH guaiacyl units. These results are reminiscent of some characteristics already observed for the maize bm3 (brown-midrib3) mutant, as well as for COMT down-regulated dicots. However, as compared with bm3, COMT down-regulation in the COMT-AS line is less severe in that it is restricted to sclerenchyma cells. To our knowledge, this is the first time that an AS strategy has been applied to modify lignin biosynthesis in a grass species.

Abstract: Transgenic plants severely suppressed in the activity of cinnamoyl-CoA reductase were produced by introduction of a partial sense CCR transgene into tobacco. Five transgenic lines with CCR activities ranging from 2 to 48% of wild-type values were selected for further study. Some lines showed a range of aberrant phenotypes including reduced growth, and all had changes to lignin structure making the polymer more susceptible to alkali extraction. The most severely CCR-suppressed line also had significantly decreased lignin content and an increased proportion of free phenolic groups in non-condensed lignin. These changes are likely to make the lignin easier to extract during chemical pulping. Direct Kraft pulping trials confirmed this. More lignin could be removed from the transgenic wood than from wild-type wood at the same alkali charge. A similar improvement in pulping efficiency was recently shown for poplar trees expressing an antisense cinnamyl alcohol dehydrogenase gene. Pulping experiments performed here on CAD-antisense tobacco plants produced near-identical results--the modified lignin was more easily removed during pulping without any adverse effects on the quality of the pulp or paper produced. These results suggest that pulping experiments performed in tobacco can be predictive of the results that will be obtained in trees such as poplar, extending the utility of the tobacco model. On the basis of our results on CCR manipulation in tobacco, we predict that CCR-suppressed trees may show pulping benefits. However, it is likely that CCR-suppression will not be the optimal target for genetic manipulation of pulping character due to the potential associated growth defects.

Abstract: Inhibition of specific lignin biosynthetic steps by antisense strategy has previously been shown to alter lignin content and/or structure. In this work, homozygous tobacco (Nicotiana tabacum) lines transformed with cinnamoyl-coenzyme A reductase (CCR) or caffeic acid/5-hydroxy ferulic acid-O-methyltransferase I (COMT I) antisense sequences have been crossed and enzyme activities, lignin synthesis, and cell wall structure of the progeny have been analyzed. In single transformed parents, CCR inhibition did not affect COMT I expression, whereas marked increases in CCR activity were observed in COMT I antisense plants, suggesting potential cross talk between some genes of the pathway. In the progeny, both CCR and COMT I activities were shown to be markedly decreased due to the simultaneous repression of the two genes. In these double transformants, the lignin profiles were dependent on the relative extent of down-regulation of each individual enzyme. For the siblings issued from a strongly repressed antisense CCR parent, the lignin patterns mimicked the patterns obtained in single transformants with a reduced CCR activity. In contrast, the specific lignin profile of COMT I repression could not be detected in double transformed siblings. By transmission electron microscopy some cell wall loosening was detected in the antisense CCR parent but not in the antisense COMT I parent. In double transformants, immunolabeling of non-condensed guaiacyl-syringyl units was weaker and revealed changes in epitope distribution that specifically affected vessels. Our results more widely highlight the impact of culture conditions on phenotypes and gene expression of transformed plants.

Abstract: 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.

Abstract: Maize bran heteroxylan samples were extracted in various conditions of severity. Their ferulate and diferulate content was investigated by GC-MS of methyl ester-TMSi derivatives. When extracted by 0.5 M NaOH in mild conditions, the heteroxylan sample contained a low level of ferulic acid (0.032% by wt.) and the main diferulate surviving alkaline extraction was found to be the 8-8' diferulate. On peroxidase treatment, this sample nevertheless produced a firm and brittle gel without any change in the diferulate profile. Typical lignin structures, mainly comprising syringyl units interconnected through beta-O-4, beta-1 and beta-beta interunit bonds, were evidenced in the maize bran sample. More importantly, these lignin structures were found to be tightly associated with the alkali-extracted heteroxylans. Thioacidolysis revealed the occurrence of 0.1-0.5% (by wt.) lignin structures in heteroxylan fractions extracted in mild or severe conditions, before and after purification of the polysaccharides. The gelling potential of the heteroxylan fractions was not only dependent on their ferulate level, but also influenced by associated lignin structures. These results argue for the occurrence of covalent linkages between heteroxylan chains and lignin structures which could participate in the peroxidase-driven gelation of feruloylated polysaccharides. They demonstrate the role of low lignin levels in the organization of native or reconstructed polysaccharide networks.

Abstract: Different transgenic tobacco lines down-regulated for either one or two enzymes of the monolignol pathway were compared for their lignin content and composition, and developmental patterns. The comparison concerned CCR and CAD down-regulated lines (homozygous or heterozygous for the transgene) and the hybrids resulting from the crossing of transgenic lines individually altered for CCR or CAD activities. Surprisingly, the crosses containing only one allele of each antisense transgene, exhibit a dramatic reduction of lignin content similar to the CCR down-regulated parent but, in contrast to this transgenic line, display a normal phenotype and only slight alterations of the shape of the vessels. Qualitatively the lignin of the double transformant displays characteristics more like the wild type control than either of the other transgenics. In the transgenics with a low lignin content, the transformations induced other biochemical changes involving polysaccharides, phenolic components of the cell wall and also soluble phenolics. These results show that the ectopic expression of a specific transgene may have a different impact depending on the genetic background and suggest that the two transgenes present in the crosses may operate synergistically to reduce the lignin content. In addition, these data confirm that plants with a severe reduction in lignin content may undergo normal development at least in controlled conditions.

Abstract: 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.

Abstract: Capillary zone electrophoresis (CZE) was developed for the quantitative determination of dimers obtained by horseradish peroxidase-catalyzed oxidation of coniferyl alcohol. The influence of pH, electrolyte concentration, applied voltage, and temperature on CZE performance was investigated, resulting in an efficient and rapid separation. Coniferyl alcohol-derived dimers were directly analyzed from their reaction mixtures, without any extraction or derivatization step. In addition, these dimers were analyzed within 14 min, a substantially shorter time than is required for the HPLC method or the conventional capillary gas chromatography of their silylated derivatives. Standard deviations between injection replicates were in the 0.4-0.7% range for migration times and in the 1.8-5.1% range for relative normalized peak areas. The method could therefore be successfully applied to follow the peroxidase-catalyzed oxidation of coniferyl alcohol.

Abstract: Among the different enzymatic steps leading to lignin biosynthesis, two methylation reactions introduce the methyl groups borne by guaiacyl (G) and syringyl (S) units. Tobacco possesses a complex system of methylation comprising three classes of CCoAOMTs (caffeoyl-CoA-O-methyltransferases) and two classes of COMTs (caffeic acid OMTs). Antisense plants transformed with the CCoAOMT sequence alone or fused to COMT I sequence have been produced and compared to ASCOMT I plants in order to study the specific role of each OMT isoform in lignin biosynthesis, plant development and resistance to pathogens. Tobacco plants strongly inhibited in OMT activities have been selected and analyzed. Whereas antisense COMT I plants exhibited no visual phenotype, CCoAOMT repression was shown to strongly affect the development of both single and double transformants: a reduction of plant growth and the alteration of flower development were observed in the most inhibited plants. Lignin analysis performed by Klason and thioacidolysis methods, showed a decrease in the lignin quantity and changes in the lignin structure of ASCCoAOMT and ASCCoAOMT/ASCOMT I transgenics but not in ASCOMT I plants. Inhibition of COMT I in single as well as in double transformed tobacco was demonstrated to decrease S unit synthesis and to provoke the accumulation of 5-hydroxyguaiacyl lignin units. ASCCoAOMT/ASCOMT I tobacco was affected in lignin amount and composition, thus demonstrating additive effects of inhibition of both enzymes. The changes of lignin profiles and the phenotypical and molecular alterations observed in the different transgenic lines were particularly prominent at the later stages of plant development.

Abstract: Caffeoyl-coenzyme A O-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 of O(3)-beta-d-glucopyranosyl-caffeic acid, O(4)-beta-d-glucopyranosyl-vanillic acid, and O(4)-beta-d-glucopyranosyl-sinapic acid (GSA), as authenticated by (1)H NMR. Feeding experiments showed that O(3)-beta-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 vitro enzymatic data that suggest that the route from caffeic acid to sinapic acid is not used for lignin biosynthesis.

Abstract: Cinnamyl alcohol dehydrogenase (CAD) activity is deficient in loblolly pine (Pinus taeda L.) harboring a mutated allele of the cad gene (cad-n1). We compared lignin structure of CAD-deficient and wild-type pines, both types segregating within full-sib families obtained by controlled crosses. The type and frequency of lignin building units and distribution of interunit bonds were determined from the GC-MS analysis of thioacidolysis monomers and dimers. While the lignin content was only slightly reduced, the lignin structure was dramatically modified by the mutation in both mature and juvenile trees. Lignins from CAD-deficient pine displayed unusually high levels of coniferaldehyde and dihydroconiferyl alcohol. In addition, biphenyl and biphenyl ether bonds were in large excess in these abnormal lignins. These results suggest that the CAD-deficient pines efficiently compensate for the shortage in normal lignin precursors by utilizing nontraditional wall phenolics to construct unusual lignins particularly enriched in resistant interunit bonds.

Abstract: Transgenic poplars (Populus tremula x Populus alba) were obtained by introduction of a sense homologous transgene encoding caffeic acid O-methyltransferase (COMT) under the control either of the cauliflower mosaic virus double 35S promoter or of the eucalyptus cinnamyl alcohol dehydrogenase promoter. Although these constructs conferred a moderate overexpression of COMT in some lines, a transgenic line with the double 35S promoter was found where COMT activity in woody tissues was close to zero due to a gene-silencing phenomenon. For the first time in COMT down-regulated trees, this alteration substantially reduced lignin level in 6-month-old trees (17% decrease). Lignin structure was found to be strongly altered, with a two times higher content in condensed bonds, an almost complete lack of syringyl units, and the incorporation of 5-hydroxyguaiacyl units to the most remarkable extent reported so far. Consistent with the higher cellulose content and with the higher condensation degree of the lignin, the impact of the transformation on the kraft-pulping performances of the poplar trees positively affected the pulp yield (10% relative increase), but made lignins less amenable to industrial degradations.

Abstract: Polymeric proanthocyanidins are common constituents of many foods and beverages. Their fate in the human body remains largely unknown. Their metabolism by human colonic microflora incubated in vitro in anoxic conditions has been investigated using nonlabeled and (14)C-labeled purified proanthocyanidin polymers. Polymers were almost totally degraded after 48 h of incubation. Phenylacetic, phenylpropionic and phenylvaleric acids, monohydroxylated mainly in the meta or para position, were identified as metabolites by gas chromatography coupled to mass spectrometry (GC-MS). Yields were similar to those previously reported for flavonoid monomers. These results provide the first evidence of degradation of dietary phenolic polymers into low-molecular-weight aromatic compounds. To understand the nutritional properties of proanthocyanidins, it is therefore essential to consider the biological properties of these metabolites.

Abstract: The action of an abiotic lignin oxidant and a diffusible xylanase on wheat straw was studied and characterized at the levels of the molecular structures by chemical analysis and of the cell wall ultrastructure by transmission electron microscopy. While distinct chemical changes in the target polymers were observed when each system was used separately, a combination of the two types of catalysts did not significantly increase either lignin oxidation or hemicellulose hydrolysis. Microscopic observations however revealed that the supramolecular organization of the cell wall polymers was significantly altered. This suggests that the abiotic Mn-oxalate complex and the xylanase cooperate in modifying the cell wall architecture, without noticeably enhancing the degradation of the constitutive polymers.

Abstract: We evaluated lignin profiles and pulping performances of 2-year-old transgenic poplar (Populus tremula x Populus alba) lines severely altered in the expression of caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT) or cinnamyl alcohol dehydrogenase (CAD). Transgenic poplars with CAD or COMT antisense constructs showed growth similar to control trees. CAD down-regulated poplars displayed a red coloration mainly in the outer xylem. A 90% lower COMT activity did not change lignin content but dramatically increased the frequency of guaiacyl units and resistant biphenyl linkages in lignin. This alteration severely lowered the efficiency of kraft pulping. The Klason lignin level of CAD-transformed poplars was slightly lower than that of the control. Whereas CAD down-regulation did not change the frequency of labile ether bonds or guaiacyl units in lignin, it increased the proportion of syringaldehyde and diarylpropane structures and, more importantly with regard to kraft pulping, of free phenolic groups in lignin. In the most depressed line, ASCAD21, a substantially higher content in free phenolic units facilitated lignin solubilization and fragmentation during kraft pulping. These results point the way to genetic modification of lignin structure to improve wood quality for the pulp industry.

Abstract: Homologous antisense constructs were used to down-regulate tobacco cinnamyl-alcohol dehydrogenase (CAD; EC 1.1.1.195) and cinnamoyl-CoA reductase (CCR; EC 1.2.1.44) activities in the lignin monomer biosynthetic pathway. CCR converts activated cinnamic acids (hydroxycinnamoyl-SCoAs) to cinnamaldehydes; cinnamaldehydes are then reduced to cinnamyl alcohols by CAD. The transformations caused the incorporation of nontraditional components into the extractable tobacco lignins, as evidenced by NMR. Isolated lignin of antisense-CAD tobacco contained fewer coniferyl and sinapyl alcohol-derived units that were compensated for by elevated levels of benzaldehydes and cinnamaldehydes. Products from radical coupling of cinnamaldehydes, particularly sinapaldehyde, which were barely discernible in normal tobacco, were major components of the antisense-CAD tobacco lignin. Lignin content was reduced in antisense-CCR tobacco, which displayed a markedly reduced vigor. That lignin contained fewer coniferyl alcohol-derived units and significant levels of tyramine ferulate. Tyramine ferulate is a sink for the anticipated build-up of feruloyl-SCoA, and may be up-regulated in response to a deficit of coniferyl alcohol. Although it is not yet clear whether the modified lignins are true structural components of the cell wall, the findings provide further indications of the metabolic plasticity of plant lignification. An ability to produce lignin from alternative monomers would open new avenues for manipulation of lignin by genetic biotechnologies.

Abstract: Suspension cultures of Picea abies (L.) Karst released polymeric material into the culture medium when treated with an elicitor preparation from the spruce needle pathogen Rhizosphaera kalkhoffii. The presence of lignin (about 35%, w/w) was demonstrated by phloroglucinol/HCI reactivity and quantitation with thioglycolic acid. Carbohydrate (about 14%, w/w) and protein (about 32%, w/w) were also detected. Amino acid analysis revealed that hydroxyproline and proline predominated. Thioacidolysis and subsequent Raney nickel desulfurization allowed the analysis of lignin-building units and interunit bonds. Compared with spruce wood lignin, an approximately 20-fold higher relative amount of p-hydroxyphenyl units was determined. A high content of p-hydroxyphenyl units is typical for certain developmental lignins, such as conifer compression wood and middle lamella lignins, as well as all induced cell culture lignins so far analyzed. Cross-linkages of the pinoresinol type ([beta]-[beta]) in the excreted cell culture lignin were markedly increased, whereas [beta]-1 interunit linkages were decreased relative to spruce wood lignin. The amount and nature of cross-linkages were shown to be intermediate between those in wood lignin and in enzymatically prepared lignins. In summary, the elicitor-induced stress lignin was excreted as a lignin-extensin complex that closely resembled early developmental lignins.