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International audience; In order to mimic the first steps of lignification, pectin/synthetic lignin (dehydrogenation polymers (DHPs)) interactions were investigated using reconstructing approaches. In artificially lignified bacterial cellulose(BC)/pectin composites, DHP nodules display a more dispersed distribution than in BC mats. Occurrence of covalent linkages between pectin and DHP was investigated by solid state NMR. Non-covalent bonds seem to govern the associative behaviour and stability of the complexes. Studied by small angle neutron scattering, a compaction process of DHP/pectin clusters occurs during polymerization and phase separation of the two polymers was shown at the nanometer scale.

Mechanical and chemical properties as well as microfibril angles of wood tissues from different ontogenetic stages are determined for the neotropical lianas Bauhinia guianensis and Condylocarpon guianense. The mechanical properties include the elastic moduli under bending and under dynamic torsion. The chemical analyses cover (i) the content of cellulose, lignin and hemicelluloses fractions, (ii) the monomeric composition of the uncondensed lignin, and (iii) the composition of the hemicelluloses with respect to neutral monosaccharides. By comparing the wood properties of these lianas with the corresponding properties of wood from self-supporting deciduous trees, common characteristics and differences are revealed. Additionally, the changes in the lignin and polysaccharides fractions as well as the variations in microfibril orientation that occur during ontogeny of the two liana species are discussed with regard to their implications for the mechanical properties of wood.

Oxidants formed through the interaction between MnO2 and oxalate accumulated in rotten wood have been evaluated as abiotic agents possibly involved in wheat straw ligninolysis. The hemicellulose and cellulose content of straw remained unchanged, and no release of free soluble materials from lignin or polysaccharides could be evidenced. Structural analysis of oxidized lignin in situ has revealed up to 30% and 10% decrease of β-O-4 linked Guaiacyl and Syringyl units, respectively. Lignin phenolic moieties only were directly oxidized by MnO2/oxalate as no structural alteration was observed within extensively permethylated wheat straw. Modifications were also evidenced at the level of the cell wall linked cinnamic acids present in wheat straw. Esterified phenolic acids were more readily oxidized by Mn complexes than ethers analogues, and disappearance of ferulic moieties was always more pronounced than that of p-coumaric acids. The abiotic Mn oxidants generated from MnO2 and oxalate may therefore significantl...

International audience

Lignins definition, potential applications, (macro)molecular structures and properties, analytical characterisations and natural versus induced variability are reviewed with reference to both the biological diversity of lignification patterns in ecotypes, mutants and transgenic plants and the levels of organisation of the lignins polymeric networks. A discrimination between a common primary, guaiacyl type metabolism and a more specific secondary, syringyl type is suggested as a base of this variability and properties.

Contrairement a beaucoup d'arbres feuillus, le bois de reaction des Eucalyptus n'est pas caracterise par les fibres G. Il peut cependant etre caracterise par ses deformations de maturation. La mesure des deformations residuelles longitudinales de maturation (DRLIVI) par relaxation de contraintes en peripherie des troncs a permis un classement continu et quantitatif d'echantillons de bois de clones crEucaetus hybrides du Congo. Ce type de mesure permet d'identifier mecaniquement le bois de tension. Les relations entre structure et proprietes mecaniques de ces bois ont ete etudiees par une analyse ultrastructurale et chimique des echantillons, en particulier par l'examen de la composition monomerique des lignines par thioacidolyse. Les resultats presentes ici montrent qu'il existe : i) une forte variabilite des DRLM avec des valeurs elevees, mome sur nos arbres peu penches et peu excentres, et malgre l'absence de fibres G ; ii) une correlation negative entre le niveau de DRLM, le taux de lignines et l'angle des microfibrilles de cellulose, iii) une correlation positive entre le niveau de DRLM et le rapport des taux de monomeres de lignines: unites syringyles 1 unites guaiacyles. La signification de ces correlations est discutee (causalite mecanique directe ou correlations plus complexes entre variables structurales). (Resume d'auteur)

The rate of oxidation of spruce milled wood lignin by Phanerochaete chrysosporium was investigated with precipitated lignin as well as lignin finely dispersed in water (colloidal lignin). H2O2 consumption rates in the course of lignin oxidation in the presence of purified lignin peroxidase were much higher with colloidal lignin compared with precipitated lignin. Characterization of reaction products and molecular-size distribution confirms that reaction rate of lignin peroxidase with 14C-lignin is strongly dependent on the physical state of lignin. The lignin peroxidase catalyse mainly repolymerization of colloidal lignin. However, in the presence of veratryl alcohol, both polymerization and depolymerization were observed.

International audience; Microscopic and chemical changes of hemp bast fibers were studied during the maturation from vegetative to grain maturity stages at both apical and basal regions of the stems. The content of protein was the main factor related to fiber maturation, whereas increased proportions of mannose and glucose and decreasing levels of galactose were also highly significant. Enhanced glucose deposition in apical fibers could be related to the gradual thickening of the fibers, whereas in basal regions the thickness of the fibers nearly reached the maximum at vegetative stages. In contrast, the extent of lignification remained close to 3-4% during plant growth. Hemp fiber lignins were rich in guaiacyl units and would be rather condensed in nature. In addition, the proportion of p-hydroxyphenyl units displayed a constant decline during maturation. A progressive chemical fractionation of hemp fibers provided further insights to the occurrence and nature of noncellulosic polysaccharides. Notably, these data pointed out that maturation is accompanied by a significant increase in water- and alkali-soluble components containing glucose- and mannose-related polymers and a decrease in arabinose and galactose components disrupted by diluted hydrochloric acid. Taken together, chemical features of the noncellulosic components suggest that the architecture of hemp fibers differs slightly from that of the more widely studied flax fibers.

International audience; The effects of heat treatments used to dry alfalfa stems were investigated. Heating at 70 or 100 °C caused no major change in the cell wall composition, but xylanase had lower activity on the cell wall of heated material and the amount of xylose released varied with the temperature used. Chemical fractionation of cell wall carbohydrates showed that the main changes occurring during stem dehydration concerned pectic polymers and probably hemicelluloses. There was less material soluble in ammonium oxalate from alfalfa heated at 100 °C than from fresh alfalfa. The results suggest that heat processing causes some changes in the cell wall network. Environmental scanning electron microscopy was used to examine fully hydrated tissues at high resolution. There was cell distortion without disruption of cell walls as water was lost.

International audience; Coniferyl alcohol was polymerised in the presence of pectin and a pectin-DHP complex was isolated. Characterisation of the complex has demonstrated that DHP (20% wt) was covalently linked by ester bonds to the pectin. The water sorption behaviour of the pectin-DHP complex was determined at several relative vapour pressures and compared with those obtained on pectin, DHP and a blend of both polymers in the same proportion as in the complex. The complex exhibited a lower hydrophilicity resulting from three associated phenomena: grafting, pectin-DHP interactions and the formation of a chemical network.

International audience; Dehydrogenation polymers (DHPs. lignin model compounds) were synthesized in the presence of increasing pectin concentrations using two different methods. The first method ('Zutropfverfahren'. ZT) consists in the slow adding of monomers whereas in the second method ('Zulaufverfahren', ZL) all the reactants are added simultaneously. DHPs solubility increases with the pectin concentration in the ZT experiments and remains stable in the ZL experiments. Covalent bonds between pectin and DHP are formed during ZT polymerization resulting in lignin carbohydrate complex (LCC) which keeps the unbound DHPs in solution by the formation of aggregate or micelle-like structures. In contrast LCC are not formed during the ZL process which behave like the DHP reference. The ZT DHP molar masses increase observed is attributed to the reactivity of the high molar mass polymer solubilized by the: LCC whereas ZL higher molar mass polymers are precipitated out of the solution and cannot react further.

Brown-midrib (bm) mutants of maize have modified lignin of reddish-brown colour. Although four independent bm loci are known, only one of the mutant genes has been previously identified. We report here that maize bm1, one of the less characterised mutants, shows severely reduced CAD activity in lignified tissues, resulting in the production of a modified lignin. Both the total lignin content and the structure of the polymer are altered by the mutation. We further describe the isolation and characterisation of the maize CAD cDNA and mapping of the CAD gene. CAD maps very closely to the known location of bm1 and co-segregates with the bm1 locus in two independent recombinant inbred populations. These data strongly support the premise that maize bm1 directly affects expression of the CAD gene.

In addition to morphological factors (height of the plant, proportion of stems), alfalfa quality is related to several biochemical factors, especially the content and monomeric composition of the cell wall components. This work was aimed at studying internode development in relation to the chemical composition and fractionation of cell walls. Analyses were performed on the fourth apical internodes (elongating), the two following ones (onset of cambial activity), and bottom internodes (mature). Deposition of guaiacyl type lignin occurred in the pectin rich cell walls of apical internodes. The onset of cambial activity corresponded to cell wall accumulation of syringyl-guaiacyl lignin, xylans and/or glucuronoxylans, and cellulose. Such events were related to the thickening of secondary cell walls which proportion increased in mature internodes. Chemical fractionation of the cell walls allowed the release of a high content of water soluble pectins from apical internodes, whereas alkalis were more efficient in extracting heteroxylans from bottom internodes. A partial release of lignin from the cell walls also occurred during the extraction steps. Lignin solubilization was enhanced by the cleavage of labile-ether lignin structures and appeared closely related to the extent of internode differentiation along the stem. Data gained from chemical fractionations evidenced that lignin-xylan-pectic complexes accumulated progressively in secondary wall rich tissues.

Peroxidases and laccases are key enzymes in the lignin biodegradation process. They oxidize phenolic and non-phenolic lignin model compounds into their phenoxy and cation radicals, respectively. Further non-enzymatic evolution lead then to various C-C and ether bond cleavages. Nevertheless, almost no information on the structural alterations undergone in vitro or in situ by lignin after enzymatic catalysis is available. We report here on the molecular structure of lignin oxidized by various (per)oxidasic systems. The oxidizability of phenolic and non-phenolic structures of the guaiacyl and syringyl type in the lignin network will be discussed as well as the modification of the macromolecular properties of the polymer oxidized in situ or in isolated state.

In the present study, we have performed chemical investigations of the stem cell walls during internode maturation in order to study the growth dynamics of alfalfa and the deposition of the main cell wall components (polysaccharides and lignins). Internode cell walls were analysed by chemical fractionation using a mild delignification step aiming at sequential removal of polysaccharides and lignins. Delignification facilitated the subsequent removal of the xylose-rich polysaccharides by NaOH extraction as previously shown. This trend was more pronounced in the case of older internodes which have a larger proportion of secondary tissues containing syringyl type lignins in contrast to younger ones which are mainly composed of primary tissues containing guaiacyl type lignins and pectin rich cell walls.

Cinnamyl alcohol dehydrogenase (CAD) catalyzes the last step in the biosynthesis of the lignin precursors, the monolignols. We have down-regulated CAD in transgenic poplar (Populus tremula X Populus alba) by both antisense and co-suppression strategies. Several antisense and sense CAD transgenic poplars had an approximately 70% reduced CAD activity that was associated with a red coloration of the xylem tissue. Neither the lignin amount nor the lignin monomeric composition (syringyl/guaiacyl) were significantly modified. However, phloroglucinol-HCl staining was different in the down-regulated CAD plants, suggesting changes in the number of aldehyde units in the lignin. Furthermore, the reactivity of the cell wall toward alkali treatment was altered: a lower amount of lignin was found in the insoluble, saponified residue and more lignin could be precipitated from the soluble alkali fraction. Moreover, large amounts of phenolic compounds, vanillin and especially syringaldehyde, were detected in the soluble alkali fraction of the CAD down-regulated poplars. Alkaline pulping experiments on 3-month-old trees showed a reduction of the kappa number without affecting the degree of cellulose degradation. These results indicate that reducing the CAD activity in trees might be a valuable strategy to optimize certain processes of the wood industry, especially those of the pulp and paper industry.

The removal of lignin in paper manufacture is a toxic and energy-requiring process. For the paper industry and for the environment, it would be very beneficial to process trees with less lignin or with lignin that is more easily ex tractable. To achieve this goal, we are down-regulating the activity of a number of lignin biosynthesis enzymes in poplar by genetic engineering and studying their role in determining the quantity and quality of lignin. Here, our recent results obtained in this field are summarized with emphasis on bispecific caffeic acid/5-OH-ferulic acid-O-methyltransferase, cinnamoyl-CoA-reductase, caffeoyl-CoA-O-methyltransferase, and peroxidase.

Cinnamyl alcohol dehydrogenase (CAD) catalyses the last step in the biosynthesis of the lignin precursors, which is the conversion of cinnamaldehydes into cinnamyl alcohols. A poplar (P. trichocarpa x P. deltoides)CAD cDNA was introduced in sense and in antisense orientation into P. tremula x P. alba via an Agrobacterium- mediated transformation. The CAD activity was reduced by 70% in the xylem tissue of three antisense and two co-suppressed lines. A red coloration of the xylem was detected in all poplar lines with a residual CAD activity of less than 60 %. The lignin content and the lignin composition (syringyl/guaiacyl) were similar in co-suppressed, antisense CAD and control lines. However, chemical properties of the cell wall were different. First, a phloroglucinol staining showed a different coloration in the xylem of the down-regulated CAD poplars, being red-brown as compared to the typical pink staining of the control xylem. Second, after NaOH treatment, lignin could be precipitated by acidification of the alkali-soluble fraction of the cell wall of the red poplars but not of that of the white poplars. A spectral characterisation of the alkali fraction from red poplars showed an increased level of vanillin and syringaldehyde. Third, pulping experiments showed less residual lignin in the pulp of CAD down-regulated poplars.

International audience; The monomeric composition of tobacco lignin has been modified by genetic engineering. Sense or antisense expression of sequences encoding O-methyltransferase (OMT), a lignin biosynthetic enzyme, was shown to modulate enzyme activity. Ten constructs harboring the entire or a partial OMT cDNA were used. Populations of 20 trensgenic plants per construct were analyzed for OMT activity and compared with untransformed controls. As expected, expression of only the full-length sense construct led to an increase in OMT activity. An important reduction of activity was found in a variable number of plantlets from all other transgenic populations but the inhibition was sustained through the adult stage only in plants transformed with the complete cDNA. T-DNA genes were shown to be stably integrated into the tobacco genome and to be transmitted to the progeny. By using gene-specific probes, OMT inhibition in stems was correlated to a parallel disappearance of OMT transcripts originating from both the resident gene and the transgene. In contrast, transgene transcripts were detected in leaf tissues where the resident gene is poorly expressed, thus indicating that relative expression of the two OMT genes controls transcript turnover. In stems of inhibited plants, a marked decrease of syringyl units and the appearance of 5-hydroxy guaiacyl units were demonstrated. These two structural features are also characteristic of natural mutants of maize with an improved digestibility compared with wild lines. These data demonstrate the feasibility and the potential benefits of lignin manipulation.

The effects of the maize brown-midrib genes bm3 and bm1 on feeding value and agronomic traits were investigated by animal studies and biochemical approaches. When sheep were fed bm3 silage, an increase in digestibility was observed, equal to 2.4 points for digestibility of organic matter (DOM), and 8.1 points for digestibility of crude fiber (DCF). Phenotypic variation was reduced for all observed traits of bm3 hybrids. This allowed the assumption that the bm3 gene acted by levelling to a similar value of almost 74% DOM, quite independently of the original value of the normal counterpart. The bm1 hybrids were intermediate between normal and bm3 ones for DOM, DCF and intake, but the dry matter yield was not reduced for bm1 hybrids. From experiments with young bulls, feed efficiency of INRA260 bm1 was not significantly improved, compared with normal INRA260, but INRA260 bm3 was a lot more efficient. Despite its lower adverse effect on agronomic characteristics of hybrids, the bm1 allele did not appear to be of any interest in improving the silage maize feed efficiency. Variations for lignin content due to the bm3 gene was similar for the 3 hybrids studied, despite their variation for susceptibility to root and stalk lodging. Ratios and content of alkali-labile hydroxycinnamic acids behaved similarly for the 3 hybrids, with a low yield of para-coumaric acid. Structural studies of lignin, after thioacidolysis, strengthened the results previously obtained, with a clear decrease of syringyl units, and the occurrence of the 5-hydroxyguaiacyl residue. The lignin of bm3 genotypes also appeared as a more condensed polymer than that of the normal types. The decrease of lignin content was observed in only 1 bm1 hybrid and appeared the same between normal and bm1 hybrids as between normal and bm3 hybrids. The lower yield of para-coumaric acid, and the equal yield of ferulic acid after alkaline hydrolysis of bm1 material were in good agreement with previous work. After thioacidolysis, the release of monomeric syringyl units was similar between bm1 and bm3, about 30% of the normal counterpart. However, it was very different for guaiacyl units, with a large decrease of the release of these units from the bm1 hybrid. The ratio between syringyl and guaiacyl units was about 0.3 for bm3 hybrids and about 1.3 for the bm1 hybrid. The lignin of this bm1 hybrid appeared as a very condensed polymer compared with the normal type. A large variation for digestibility was also found among normal hybrids. However, inbred lines inducing high digestibility often included unfavorable agronomic traits, such as poor yield, susceptibility to lodging or stalk rot. Second or third cycle lines following pedigree breeding might combine all traits, even if stalk standability and stalk digestibility are partly antagonistic. This method may be considered as easier than the bm3 method, but at present, the 2 ways are probably complementary, and worth investigating for improving silage digestibility, and simultaneously increasing knowledge on silage maize digestibility., Les effets des gènes brown-midrib bm1 et bm3 sur la valeur alimentaire et les caractéristiques agronomiques d’hybrides de maïs ont été étudiés à partir d’essais sur animaux et de mesures de composition biochimique de la lignine. Sur moutons, les hybrides bm3 conduisent à une amélioration moyenne de la digestibilité de la matière organique (DOM) voisine de 2,4 et de celle de la cellulose brute (DCF) voisine de 8,1 points. La variabilité phénotypique est nettement réduite entre hybrides bm3, comparée à celle observée pour les quasi isogéniques normaux, pour la quasi-totalité des caractères étudiés. En particulier, il semble que la présence du gène bm3 conduise à augmenter la DOM des hybrides à une valeur proche de 74%, quel que soit le niveau de digestibilité de l’hybride normal de départ. Les hybrides bm1 se situent de façon intermédiaire entre les formes normales et bm3, sauf pour la productivité en biomasse qui est équivalente à celle des normaux. Mais des essais de croissance comparée de taurillons nourris avec des ensilages de maïs INRA260, INRA260 bm1 et INRA260 bm3 montrent que la forme bm1 ne permet pas, contrairement à la forme bm3, une amélioration notable des performances animales. La réduction de teneur en lignine a été semblable pour les 3 hybrides bm3 étudiés, alors que ces hybrides présentaient des différences importantes de sensibilité à la verse ou à la casse. Les teneurs et rapports entre acides hydroxycinnamiques libérés après hydrolyse alcaline étaient semblables chez les 3 hybrides, avec une libération faible d’acide para-coumarique caractéristique des génotypes bm3. La thioacydolyse confirme que la lignine des plantes bm3 est plus pauvre en unités syringyl, avec, de plus, la présence d’une unité 5-hydroxyguaiacyl. La lignine des plantes bm3 est un polymère plus condensé que la lignine des plantes normales. Observée à partir d’un seul hybride bm1, la diminution de la teneur en lignine a été semblable chez les plantes bm1 et bm3. Après thioacidolyse, la libération d’unités syringyl était équivalente entre bm1 et bm3. Mais la libération d’unité guaiacyl était beaucoup plus faible pour la lignine des plantes bm 1, avec des rapports syringyl/guaiacyl voisins de 0,3 pour les bm3 et de 1,3 pour les bm1. La lignine des plantes bm1 est également un polymère hautement condensé. Une variabilité importante pour la DOM et la DCF existe aussi entre hybrides normaux. Toutefois, les hybrides les plus digestibles présentent en général des défauts agronomiques importants, qu’il sera sans doute possible de corriger au niveau de lignées de second ou troisième cycle. Actuellement, on peut considérer que les voies normales et bm3 sont deux approches complémentaires de l’amélioration et de la compréhension de la digestibilité du maïs ensilage.

Oxidation of spruce milled-wood lignins in colloidal state catalyzed by horseradish peroxidase (HRP) and lignin peroxidase (LiP) in the presence of H 2 O 2 was compared in nonbuffered dimethyl-formamide/water. Lignins were characterized for their hydrodynamic properties by size-exclusion chromatography and structural bonding pattern by analysis after thioacidolysis. In contrast to LiP, HRP did not induce modification of lignin hydrodynamic properties. The lignin content in β-O-4-linked guaiacyl monomers and dimeric structures, however, decreased after oxidation by the two enzymes, indicating large structural changes in the polymer. Preferential degradation by both LiP and HRP of β-5 and β-1 lignin dimeric units was also observed. This suggests a greater susceptibility to enzyme oxidation, as compared to the 5-5′ and 4-O-5 lignin substructures. In conclusion, the action of HRP on the lignin polymer is similar in many respects to that of fungal LiP, but is distinctly different in its inability to cause a net destructuring of the macromolecular three-dimensional network.

Cell wall-linked phenolics were investigated in maize internodes located at three positions of the stem (top-middle-bottom). While the lignin content did not change drastically with position, the amount of cell wall-ester linked p-coumaric acid sharply increased from the top to the bottom internodes of the stem. Conversely, the saponified ferulic acid content remained relatively unchanged along the stem. Moreover, the highest syringyl content of the β-O-4-lignin structures was found in the basal (most mature) internode. Therefore, enhanced p-coumaric esterification of the cell wall and preferential deposition of syringyl units in the lignin polymer might indicate an extended maturity stage of the cell wall of maize internodes. The bm3 mutation in the three maize hybrids is expressed by lignin reduction and ester-bound p-coumaric decrease in the mutant lines. Furthermore, all bm3 hybrids synthesised lignin polymers which were characterised by a very low S/G molar ratio (0.16-0.43). This ratio originates from the substantial reduction of the syringyl unit content in β-O-4-lignin structures compared with the normal lignin. The occurrence in the same range of the 5-hydroxyguaiacyl unit in bm3 lignin from each type of hybrids was noteworthy, demonstrating the high heritability of the bm3 mutation at the molecular level. The alkaline solubility of lignin was greater for the three mutant lines compared to the normal cultivars. Furthermore, the alkali-labile fraction of lignin of both normal and mutant lines had a monomeric composition which was consistent with the non-condensed structures of in-situ lignin.

L'addition d'eau a une solution de lignine dans le DMF entraine la precipitation d'une partie de la lignine et le passage d'une autre fraction dans un etat colloidal. Toute la lignite presente peut etre transformee dans cet etat par des cycles successifs de solubilisation precpitation de la lignine non-colloidale. Cette lignine colloidale ne presente pas de difference significative de structure moleculaire ni de distribution de masse moleculaire par rapport a la lignine native, mais sa reactivite dans l'oxydation par la peroxydase de la lignine est beaucoup plus elevee

In the case of proanthocyanidins, reaction with vanillin in the presence of sulfuric acid is more sensitive than estimation by anthocyanidin formation. It is more specific than the assay by formaldehyde precipitation. Reaction with nitrous acid affords a selective estimation of hexahydroxydiphenoyl esters. These methods are applied to tannin estimation in wood extracts of 5 gymnosperms and 12 angiosperms and the values obtained compared to total phenols amounts

Un protocole de dosage de la lignine insoluble dans l’acide sulfurique concentré, lignine Klason, est décrit : il comporte une étape de préhydrolyse par l’acide sulfurique 5 % et un lavage, en milieu chlorhydrique concentré, du résidu de préhydrolyse. Ce lavage permet d’améliorer la reproductibilité du dosage de la lignine après préhydrolyse et d’atteindre une reproductibilité comparable à celle obtenue par le dosage Klason standard. L’analyse élémentaire des préparations de lignines isolées au cours de ces dosages indique des variations complexes qui dépendent de la nature du produit analysé., A procedure for estimating lignin insoluble in concentrated acids, Klason lignin, is described ; it includes a 5 % sulfuric acid prehydrolysis step and washing in concentrated hydrochloric acid. This procedure allowed better reproducibility than without washing, of the same order as that given by the standard Klason procedure. Elemental analysis showed complex changes depending on the plant analysed.

To mimic the lignin polymerization process, mats of bacterial cellulose and of a pectin/cellulose composite were used as a host matrix for in vitro polymerization of coniferyl alcohol. A diffusion cell was used to allow the diffusion of both hydrogen peroxide and coniferyl alcohol into the peroxidase impregnated cellulose mats through dialysis membranes. The results indicate that significant polymerization occurs within the mats. The resulting binary and ternary blends were imaged by scanning electron microscopy (SEM) and characterized by chemical means. The presence of pectin induces a better dispersion of the synthetic lignin in the cellulose network and enhances the proportion of alkyl-aryl-ether in the polymer.

The cell wall phenolic components in the internodes of three maize genotypes, namely normal, bm2 and bm3 maize, were determined. The bm2 and bm3 brown midrib mutations lowered the lignin content of the bottom, middle and top internodes to a similar extent. However, unlike bm3, the bm2 trait did not induce a sharp reduction of the level of ester-bound p-coumaric acid in maize internodes. The other main alkali labile phenolic acid, ferulic acid, reached similar levels in the three genotypes. The main difference between bm2 and bm3 mutations occurred in the alkyl aryl ether linked structures of the lignin component. In contrast to bm3 lignins, which are characterised by a low syringyl content, the bm2 lignin had a lower content of guaiacyl units than lignin of normal maize internode. Consequently, the syringyl/guaiacyl molar ratio of bm2 lignin gave higher values (2.7–3.2) than those from either normal (0.9–1.5) or bm3 lignins (0.3). The alkali solubility of lignin was also compared between the three genotypes. Incorporation of the bm3 trait in maize led to a high recovery of alkali soluble lignin whereas the bm2 lignin had a similar solubility to the normal one in 2 M NaOH. The monomeric composition of the alkali soluble lignins was consistent with the non-condensed structures of the in-situ polymer. Although the bm3 and bm2 mutations had different effects on lignification, the modification of the cell wall phenolic level was also found in the bm2 maize stem as previously studied.

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.