Nativity of lignin carbohydrate bonds substantiated by biomimetic synthesis

The question of whether lignin is covalently linked to carbohydrates in native wood, forming what is referred to as lignin-carbohydrate complexes (LCCs), still lacks unequivocal proof. This is mainly due to the need to isolate lignin from woody materials prior to analysis, under conditions leading to partial chemical modification of the native wood polymers. Thus, the correlation between the structure of the isolated LCCs and LCCs in situ remains open. As a way to circumvent the problematic isolation, biomimicking lignin polymerization in vivo and in vitro is an interesting option. Herein, we report the detection of lignin-carbohydrate bonds in the extracellular lignin formed by tissue-cultured Norway spruce cells, and in modified biomimetic lignin synthesis (dehydrogenation polymers). Semi-quantitative 2D heteronuclear singular quantum coherence (HSQC)-, P-31 -, and C-13-NMR spectroscopy were applied as analytical tools. Combining results from these systems, four types of lignin-carbohydrate bonds were detected; benzyl ether, benzyl ester, gamma-ester, and phenyl glycoside linkages, providing direct evidence of lignin-carbohydrate bond formation in biomimicked lignin polymerization. Based on our findings, we propose a sequence for lignin-carbohydrate bond formation in plant cell walls.Peer reviewe

Peroxidases bound to the growing lignin polymer produce natural like extracellular lignin in a cell culture of Norway spruce.

Peer reviewe

Active fungal GH115 alpha-glucuronidase produced in Arabidopsis thaliana affects only the UX1-reactive glucuronate decorations on native glucuronoxylans

Background Expressing microbial polysaccharide-modifying enzymes in plants is an attractive approach to custom tailor plant lignocellulose and to study the importance of wall structures to plant development. Expression of α-glucuronidases in plants to modify the structures of glucuronoxylans has not been yet attempted. Glycoside hydrolase (GH) family 115 α-glucuronidases cleave the internal α-D-(4-O-methyl)glucopyranosyluronic acid ((Me)GlcA) from xylans or xylooligosaccharides. In this work, a GH115 α-glucuronidase from Schizophyllum commune, ScAGU115, was expressed in Arabidopsis thaliana and targeted to apoplast. The transgene effects on native xylans’ structures, plant development, and lignocellulose saccharification were evaluated and compared to those of knocked out glucuronyltransferases AtGUX1 and AtGUX2. Results The ScAGU115 extracted from cell walls of Arabidopsis was active on the internally substituted aldopentaouronic acid (XUXX). The transgenic plants did not show any change in growth or in lignocellulose saccharification. The cell wall (Me)GlcA and other non-cellulosic sugars, as well as the lignin content, remained unchanged. In contrast, the gux1gux2 double mutant showed a 70% decrease in (Me)GlcA to xylose molar ratio, and, interestingly, a 60% increase in the xylose content. Whereas ScAGU115-expressing plants exhibited a decreased signal in native secondary walls from the monoclonal antibody UX1 that recognizes (Me)GlcA on non-acetylated xylan, the signal was not affected after wall deacetylation. In contrast, gux1gux2 mutant was lacking UX1 signals in both native and deacetylated cell walls. This indicates that acetyl substitution on the xylopyranosyl residue carrying (Me)GlcA or on the neighboring xylopyranosyl residues may restrict post-synthetic modification of xylans by ScAGU115 in planta. Conclusions Active GH115 α-glucuronidase has been produced for the first time in plants. The cell wall–targeted ScAGU115 was shown to affect those glucuronate substitutions of xylan, which are accessible to UX1 antibody and constitute a small fraction in Arabidopsis, whereas majority of (Me)GlcA substitutions were resistant, most likely due to the shielding by acetyl groups. Plants expressing ScAGU115 did not show any defects under laboratory conditions indicating that the UX1 epitope of xylan is not essential under these conditions. Moreover the removal of the UX1 xylan epitope does not affect lignocellulose saccharification.Peer reviewe

Carbohydrate esterase family 16 contains fungal hemicellulose acetyl esterases (HAEs) with varying specificity

Acetyl esterases are an important component of the enzymatic machinery fungi use to degrade plant biomass and are classified in several Carbohydrate Esterase families of the CAZy classification system. Carbohydrate Esterase family 16 (CE16) is one of the more recently discovered CAZy families, but only a small number of its enzyme members have been characterized so far, revealing activity on xylan-derived oligosaccharides, as well as activity related to galactoglucomannan. The number of CE16 genes differs significantly in the genomes of filamentous fungi. In this study, four CE16 members were identified in the genome of Aspergillus niger NRRL3 and it was shown that they belong to three of the four phylogenetic Clades of CE16. Significant differences in expression profiles of the genes and substrate specificity of the enzymes were revealed, demonstrating the diversity within this family of enzymes. Detailed characterization of one of these four A. niger enzymes (HaeA) demonstrated activity on oligosaccharides obtained from acetylated glucuronoxylan, galactoglucomannan and xyloglucan, thus establishing this enzyme as a general hemicellulose acetyl esterase. Their broad substrate specificity makes these enzymes highly interesting for biotechnological applications in which deacetylation of polysaccharides is required.Peer reviewe

Carbohydrate esterase family 16 contains fungal hemicellulose acetyl esterases (HAEs) with varying specificity

Acetyl esterases are an important component of the enzymatic machinery fungi use to degrade plant biomass and are classified in several Carbohydrate Esterase families of the CAZy classification system. Carbohydrate Esterase family 16 (CE16) is one of the more recently discovered CAZy families, but only a small number of its enzyme members have been characterized so far, revealing activity on xylan-derived oligosaccharides, as well as activity related to galactoglucomannan. The number of CE16 genes differs significantly in the genomes of filamentous fungi. In this study, four CE16 members were identified in the genome of Aspergillus niger NRRL3 and it was shown that they belong to three of the four phylogenetic Clades of CE16. Significant differences in expression profiles of the genes and substrate specificity of the enzymes were revealed, demonstrating the diversity within this family of enzymes. Detailed characterization of one of these four A. niger enzymes (HaeA) demonstrated activity on oligosaccharides obtained from acetylated glucuronoxylan, galactoglucomannan and xyloglucan, thus establishing this enzyme as a general hemicellulose acetyl esterase. Their broad substrate specificity makes these enzymes highly interesting for biotechnological applications in which deacetylation of polysaccharides is required

Ligniinin biosynteesi kuusen solukkoviljelmässä ja kehittyvässä puusolukossa

Lignin is a hydrophobic polymer that is synthesised in the secondary cell walls of all vascular plants. It enables water conduction through the stem, supports the upright growth habit and protects against invading pathogens. In addition, lignin hinders the utilisation of the cellulosic cell walls of plants in pulp and paper industry and as forage. Lignin precursors are synthesised in the cytoplasm through the phenylpropanoid pathway, transported into the cell wall and oxidised by peroxidases or laccases to phenoxy radicals that couple to form the lignin polymer. This study was conducted to characterise the lignin biosynthetic pathway in Norway spruce (Picea abies (L.) Karst.). We focused on the less well-known polymerisation stage, to identify the enzymes and the regulatory mechanisms that are involved. Available data for lignin biosynthesis in gymnosperms is scarce and, for example, the latest improvements in precursor biosynthesis have only been verified in herbaceous plants. Therefore, we also wanted to study in detail the roles of individual gene family members during developmental and stress-induced lignification, using EST sequencing and real-time RT-PCR. We used, as a model, a Norway spruce tissue culture line that produces extracellular lignin into the culture medium, and showed that lignin polymerisation in the tissue culture depends on peroxidase activity. We identified in the culture medium a significant NADH oxidase activity that could generate H2O2 for peroxidases. Two basic culture medium peroxidases were shown to have high affinity to coniferyl alcohol. Conservation of the putative substrate-binding amino acids was observed when the spruce peroxidase sequences were compared with other peroxidases with high affinity to coniferyl alcohol. We also used different peroxidase fractions to produce synthetic in vitro lignins from coniferyl alcohol; however, the linkage pattern of the suspension culture lignin could not be reproduced in vitro with the purified peroxidases, nor with the full complement of culture medium proteins. This emphasised the importance of the precursor radical concentration in the reaction zone, which is controlled by the cells through the secretion of both the lignin precursors and the oxidative enzymes to the apoplast. In addition, we identified basic peroxidases that were reversibly bound to the lignin precipitate. They could be involved, for example, in the oxidation of polymeric lignin, which is required for polymer growth. The dibenzodioxocin substructure was used as a marker for polymer oxidation in the in vitro polymerisation studies, as it is a typical substructure in wood lignin and in the suspension culture lignin. Using immunolocalisation, we found the structure mainly in the S2+S3 layers of the secondary cell walls of Norway spruce tracheids. The structure was primarily formed during the late phases of lignification. Contrary to the earlier assumptions, it appears to be a terminal structure in the lignin macromolecule. Most lignin biosynthetic enzymes are encoded for by several genes, all of which may not participate in lignin biosynthesis. In order to identify the gene family members that are responsible for developmental lignification, ESTs were sequenced from the lignin-forming tissue culture and developing xylem of spruce. Expression of the identified lignin biosynthetic genes was studied using real-time RT-PCR. Candidate genes for developmental lignification were identified by a coordinated, high expression of certain genes within the gene families in all lignin-forming tissues. However, such coordinated expression was not found for peroxidase genes. We also studied stress-induced lignification either during compression wood formation by bending the stems or after Heterobasidion annosum infection. Based on gene expression profiles, stress-induced monolignol biosynthesis appeared similar to the developmental process, and only single PAL and C3H genes were specifically up-regulated by stress. On the contrary, the up-regulated peroxidase genes differed between developmental and stress-induced lignification, indicating specific responses.Ligniini on selluloosan jälkeen yleisin kasvien tuottama polymeeri. Se sijaitsee kasvien soluseinissä ja on välttämätön mm. vettä johtavien solukoiden toiminnan kannalta, tukirakenteena ja suojana tuhohyönteisiä ja tauteja vastaan. Suuri osa maapallon ligniinistä on puissa, sillä puusolukon, ksyleemin, painosta jopa kolmannes on ligniiniä. Ligniini heikentää kasvi- ja puukuitujen hyötykäyttöä, esimerkiksi eläinrehun sulavuutta ja paperinvalmistusprosessien tehokkuutta. Näin ollen ligniinillä on myös huomattava taloudellinen ja ympäristönsuojelullinen merkitys. Ligniini on osa laajaa fenyylipropanoidien ryhmää, johon kuuluu erilaisia, mm. kasvien väreihin, rakenteisiin ja puolustukseen liittyviä aineita. Ligniini rakentuu kolmesta alayksiköstä, joiden muodostukseen osallistuvat geeni- ja entsyymiperheet osallistuvat myös muiden fenyylipropanoidien tuottoon. Selvitimme ligniinin alayksikköjen syntyyn liittyvien geeniperheiden roolia ligniinin tuotossa tutkimalla geenien ilmentymistä ligniiniä muodostavissa solukoissa. Jokaisesta geeniperheestä yksi geeni ilmentyi muita voimakkaammin kehittyvässä puusolukossa, niin nuorissa taimissa kuin vanhemmissa puissa. Nämä geenit todennäköisesti vastaavat ligniinin tuotosta kuusessa, ja ne ovat mahdollisia kohteita esimerkiksi metsäpuiden jalostuksessa ligniinin määrän tai laadun suhteen. Työssä selvitettiin myös stressiperäisen ja luonnollisen ligniinin muodostuksen välistä suhdetta geenitasolla. Stressiligniinin muodostus saatiin aikaan joko sieni-infektiolla (juurikääpä) tai taivuttamalla taimia. Molempien stressien seurauksena muodostuu enemmän ja osin rakenteeltaan erilaista ligniiniä. Geenien ilmenemisen tasolla stressiligniinin muodostus oli samankaltaista kuin normaalisti kehittyvässä puussa. Ligniinin muodostuksen alkuvaiheessa aktivoitui kaksi uutta geeniä, jotka todennäköisesti lisäävät ligniinin kokonaismäärää sekä ohjaavat eri alayksiköiden määräsuhteita. Ligniinin alayksiköistä muodostetaan verkkomainen polymeeri peroksidaasi- ja/tai lakkaasientsyymien toimesta. Molemmat ovat suurten geeniperheiden tuotteita, joten ligniinin polymerisaatiosta vastaavia yksittäisiä geenejä ei täysin tunneta, kuten ei myöskään polymerisaation säätelymekanismeja. Tutkimme näitä vaiheita entsyymitasolla kuusen solukkoviljelmässä, joka tuottaa luonnollisen kaltaista ligniiniä kasvualustaansa. Osoitimme, että ligniinin tuotto on peroksidaaseista riippuvaista, ja löysimme kaksi peroksidaasia, jotka olivat erikoistuneet ligniinin alayksiköiden hapetukseen. Tuotimme näillä peroksidaaseilla keinotekoista ligniiniä koeputkessa ja havaitsimme, että se sisälsi vain vähän luonnollisen kaltaisen ligniinin merkkirakenteita. Samaan tulokseen päädyttiin käytettäessä kaikkia kasvualustasta eristettyjä proteiineja yhdessä. Tämä osoittaa että peroksidaasien lisäksi tai niiden sijasta jokin muu tekijä säätelee ligniinin kokonaisrakennetta solukkoviljelmässä. Eräs mahdollinen tekijä on ligniinin rakenneyksiköiden pitoisuus kasvatusalustassa. Elävät solut kontrolloivat pitoisuutta tarkemmin kuin mihin koeputkituoton aikana kyetään. Havaitsimme myös pienempiä eroja solukkoviljelmäligniinin ja puusta eristetyn ligniinin välillä. Nämä erot vahvistavat teoriaa, jonka mukaan puusolukon soluseinät vaikuttavat oleellisesti syntyvän ligniinin rakenteeseen. Osa solukkoviljelmän peroksidaasientsyymeistä pystyi sitoutumaan erityyppisiin ligniineihin. Sitoutumiskyvyn perusteella voidaan olettaa, että nämä peroksidaasit ovat ligniinin tärkeitä ligniinin polymerisaation kannalta. Lisäksi työssä tuotettiin vasta-aineita erästä luonnollisen ligniinin merkkirakennetta vastaan. Vasta-aineiden avulla paikallistimme rakenteen soluseinän pintakerroksiin. Tämä rakenne on todennäköisesti ligniinin pääterakenne, joka mahdollisesti vaikuttaa ligniinin käyttäytymiseen esimerkiksi sellun keittoprosesseissa

Lignin biosynthesis studies in plant tissue cultures

v2010o

Acetylation of woody lignocellulose: significance and regulation

Non-cellulosic cell wall polysaccharides constitute approximately one quarter of usable biomass for human exploitation. In contrast to cellulose, these components are usually substituted by O-acetyl groups, which affect their properties and interactions with other polymers, thus affecting their solubility and extractability. However, details of these interactions are still largely obscure. Moreover, polysaccharide hydrolysis to constituent monosaccharides, is hampered by the presence of O-acetyl groups, necessitating either enzymatic (esterase) or chemical de-acetylation, increasing the costs and chemical consumption. Reduction of polysaccharide acetyl content in planta is a way to modify lignocellulose towards improved saccharification. In this review we: 1) summarize literature on lignocellulose acetylation in different tree species, 2) present data and current hypotheses concerning the role of O-acetylation in determining woody lignocellulose properties, 3) describe plant proteins involved in lignocellulose O-acetylation, 4) give examples of microbial enzymes capable to de-acetylate lignocellulose, and 5) discuss prospects for exploiting these enzymes in planta to modify xylan acetylation

Immunolabelling of xylan side groups - a novel 4-O-methylglucuronic acid specific antibody

International audienc

Cell wall antibody collection

National audienc