Distribution of glucomannans and xylans in poplar xylem and their changes under tension stress

Present work investigated glucomannan (GM) and xylan distribution in poplar xylem cells of normal- (NW), opposite- (OW) and tension wood (TW) with immunolocalization methods. GM labeling was mostly detected in the middle- and inner S(2) (+S(3)) layer of NW and OW fibers, while xylan labeling was observed in the whole secondary cell wall. GM labeling in vessels of NW and OW was much weaker than in fibers and mostly detected in the S(2) layer, whereas slightly stronger xylan labeling than fibers was detected in the whole secondary cell wall of vessels. Ray cells in NW and OW showed no GM labeling, but strong xylan labeling. These results indicate that GMs and xylans are spatially distributed in poplar xylem cells with different concentrations present in different cell types. Surprisingly, TW showed significant decrease of GM labeling in the normal secondary cell wall of gelatinous (G) fibers compared to NW and OW, while xylan labeling was almost identical indicating that the GM and xylan synthetic pathways in fibers have different reaction mechanisms against tension stress. Unlike fibers, no notable changes in GM labeling were detected in vessels of TW, suggesting that GM synthesis in vessels may not be affected by tension stress. GM and xylan was also detected in the G-layer with slightly stronger and much weaker labeling than the normal secondary cell wall of G-fibers. Differences in GM and xylan distribution are also discussed for the same functional cells found in hardwoods and softwoods.     

Deposition and organisation of cell wall polymers during maturation of poplar tension wood by FTIR microspectroscopy

To advance our understanding of the formation of tension wood, we investigated the macromolecular arrangement in cell walls by Fourier transform infrared microspectroscopy (FTIR) during maturation of tension wood in poplar (Populus tremula x P. alba, clone INRA 717-1B4). The relation between changes in composition and the deposition of the G-layer in tension wood was analysed. Polarised FTIR measurements indicated that in tension wood, already before G-layer formation, a more ordered structure of carbohydrates at an angle more parallel to the fibre axis exists. This was clearly different from the behaviour of opposite wood. With the formation of the S₂ layer in opposite wood and the G-layer in tension wood, the orientation signals from the amorphous carbohydrates like hemicelluloses and pectins were different between opposite wood and tension wood. For tension wood, the orientation for these bands remains the same all along the cell wall maturation process, probably reflecting a continued deposition of xyloglucan or xylan, with an orientation different to that in the S₂ wall throughout the whole process. In tension wood, the lignin was more highly oriented in the S₂ layer than in opposite wood.     

Enzymatic accessibility of xylans in lignocellulosic materials

The hydrolysis of fibre-bound and isolated xylans from both birch and pine wood and kraft pulps was studied using purified xylanolytic enzymes of Trichoderma reesei. Despite high enzyme loading, the degree of hydrolysis of fibre-bound substrates did not exceed 20% of the theoretical value, apparently due to limited accessibility of the substrates. The fibre-bound xylans were as equally accessible in softwood as in hardwood pulps. The isolated xylans of wood and kraft pulps could be solubilized more extensively, with a hydrolysis yield of 50–65%. The substitution degree of the isolated xylan substrates was reflected in the different hydrolysis yields obtained by the two xylanases, with isoelectric point (pI) values of 9.0 and 5.5. On the more substituted substrates, i.e. pine kraft xylan and pine wood xylan, the two enzymes acted almost similarly, whereas on the less substituted xylan substrates, such as isolated birch kraft xylan, the pI-9.0 enzyme was more efficient. The side-group-cleaving enzymes increased only moderately the solubilization of the substrates.Correspondence to: L. Viikari     

Biochemical characterization of xylan xylosyltransferases involved in wood formation in poplar

The major polysaccharides in dicot wood biomass are cellulose and xylan. Although wood-associated cellulose synthase genes responsible for cellulose biosynthesis have been characterized, wood-associated xylan synthase genes have not been biochemically identified. A recent report by Lee et al. (2012) provides the first biochemical evidence that two functionally non-redundant Arabidopsis GT43 members are xylosyltransferases (XylTs) that function cooperatively in the elongation of the xylan backbone. We further extend this finding in the current report demonstrating that two poplar (Populus trichocarpa) GT43 glycosyltransferases, PtrGT43B and PtrGT43C, are xylan XylTs involved in wood formation. We show that microsomes from transgenic tobacco BY2 cells coexpressing PtrGT43B and PtrGT43C exhibited a high XylT activity capable of generating β-(1,4)-linked xylooligosaccharides, whereas little XylT activity was detected in microsomes with expression of PtrGT43B or PtrGT43C alone. These findings indicate that poplar GT43 members are XylTs that act cooperatively in catalyzing the successive transfer of xylosyl residues during xylan backbone biosynthesis, which provides further support of the hypothesis that the biochemical functions of GT43 members in vascular plants are evolutionarily conserved.     

Carbohydrate-active enzymes identified by metagenomic analysis of deep-sea sediment bacteria

Subseafloor sediment samples derived from a sediment core of 60 m length were used to enrich psychrophilic aerobic bacteria on cellulose, xylan, chitin, and starch. A variety of species belonging to Alpha- and Gammaproteobacteria and to Flavobacteria were isolated from sediment depths between 12 and 42 mbsf. Metagenomic DNA purified from the pooled enrichments was sequenced and analyzed for phylogenetic composition and presence of genes encoding carbohydrate-active enzymes. More than 200 open reading frames coding for glycoside hydrolases were identified, and more than 60 of them relevant for enzymatic degradation of lignocellulose. Four genes encoding β-glucosidases with less than 52 % identities to characterized enzymes were chosen for recombinant expression in Escherichia coli. In addition one endomannanase, two endoxylanases, and three β-xylosidases were produced recombinantly. All genes could be actively expressed. Functional analysis revealed discrepancies and additional variability for the recombinant enzymes as compared to the sequence-based predictions.     

Activities of some enzymes of lignin formation in reaction wood of Thuja orientalis,Metasequoia glyptostroboides and Robinia pseudoacacia

The activities of the following five enzymes which are involved in the formation of lignin have been compared in reaction wood and in opposite wood: phenylalanine ammonia lyase (EC 4.3.1.5), caffeate 3-O-methyltransferase (EC 2.1.1.-), p-hydroxycinnamate: CoA ligase (EC 6.2.1.12), cinnamyl alcohol dehydrogenase (EC 1.1.1.-) and peroxidase (EC 1.11.1.7). The activities of the four first-named enzymes in the compression wood of Thuja orientalis L. and Metasequoia glyptostroboides Hu et Cheng were 2.8±1.4-fold and 2.6±1.5-fold higher than those in opposite wood, respectively, whereas peroxidase had the same level of activity in either type of wood. On the other hand, no differences were observed in the activities of the five enzymes between tension and opposite woods of Robinia pseudoacacia L. These findings are well in accord with the chemical structure of lignin in the compression and tension woods of the three species studied: high content of lignin rich in condensed units in compression wood, and little difference in lignin between tension and opposite woods.Abbreviations CAD cinnamyl alcohol dehydrogenase (EC 1.1.1.-) - OMT caffeate O-methyltransferase (EC 2.1.1-) - PAL phenylalanine ammonia lyase (EC 4.3.1.5) - PCL p-hydroxycinnamate: CoA ligase (EC 6.2.1.12) - PO peroxidase (EC 1.11.1.7)     

β-xylosidases and a nonspecific wall-bound β-glucosidase of the yeast cryptococcus albidus

Cryptococcus albidus grown on wood xylans possesses a soluble intracellular β-xylosidase (EC 3.2.1.37) as an additional constituent of the xylan-degrading enzyme system of this yeast. The enzyme attacks linear 1,4-β-xylooligosaccharides in an exo-fashion, liberating xylose from the non-reducing ends. The activity of the enzyme increases in the cells during growth on xylan and incubation with xylobiose or methyl β-D-xylopyranoside which are the best inducers of extracellular β-xylanase (EC 3.2.1.8). Various alkyl-, alkyl-1-thio- and aryl β-D-xylopyranosides were excellent of a different β-xylosidase of Cryptococcus albidus. This enzyme is localized outside the plasma membrane and is principally associated with cell walls. Unlike the soluble intracellular β-xylosidase, the wall-bound enzyme does not hydrolyze xylooligosaccharides. Evidence has been obtained that β-xylosidase activity in the cell walls is not due to the presence of a specific aryl β-xylosidase, but is exhibited by a nonspecific β-glucosidase (EC 3.2.1.21) inducible by β-D-xylopyranosides. The ratio of β-glucosidase and β-xylosidase activity in the cells and isolated cell walls from yeast induced by various β-xylopyranosides and β-glucopyranosides was very similar. Both wall-bound activities were inhibited in a similar pattern by inhibitors of β-glucosidases, 1,5-gluconolactone and nojirimycin. This bifunctional enzyme does not bear any relationship to the utilization of xylans in Cryptococcus albidus.     

aguA, the Gene Encoding an Extracellular α-Glucuronidase from Aspergillus tubingensis, Is Specifically Induced on Xylose and Not on Glucuronic Acid

An extracellular α-glucuronidase was purified and characterized from a commercial Aspergillus preparation and from culture filtrate of Aspergillus tubingensis. The enzyme has a molecular mass of 107 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 112 kDa as determined by mass spectrometry, has a determined pI just below 5.2, and is stable at pH 6.0 for prolonged times. The pH optimum for the enzyme is between 4.5 and 6.0, and the temperature optimum is 70°C. The α-glucuronidase is active mainly on small substituted xylo-oligomers but is also able to release a small amount of 4-O-methylglucuronic acid from birchwood xylan. The enzyme acts synergistically with endoxylanases and β-xylosidase in the hydrolysis of xylan. The enzyme is N glycosylated and contains 14 putative N-glycosylation sites. The gene encoding this α-glucuronidase (aguA) was cloned from A. tubingensis. It consists of an open reading frame of 2,523 bp and contains no introns. The gene codes for a protein of 841 amino acids, containing a eukaryotic signal sequence of 20 amino acids. The mature protein has a predicted molecular mass of 91,790 Da and a calculated pI of 5.13. Multiple copies of the gene were introduced in A. tubingensis, and expression was studied in a highly overproducing transformant. The aguA gene was expressed on xylose, xylobiose, and xylan, similarly to genes encoding endoxylanases, suggesting a coordinate regulation of expression of xylanases and α-glucuronidase. Glucuronic acid did not induce the expression of aguA and also did not modulate the expression on xylose. Addition of glucose prevented expression of aguA on xylan but only reduced the expression on xylose.     

Structural diversity of xylans in the cell walls of monocots

Main conclusion

Xylans in the cell walls of monocots are structurally diverse. Arabinofuranose-containing glucuronoxylans are characteristic of commelinids. However, other structural features are not correlated with the major transitions in monocot evolution. Most studies of xylan structure in monocot cell walls have emphasized members of the Poaceae (grasses). Thus, there is a paucity of information regarding xylan structure in other commelinid and in non-commelinid monocot walls. Here, we describe the major structural features of the xylans produced by plants selected from ten of the twelve monocot orders. Glucuronoxylans comparable to eudicot secondary wall glucuronoxylans are abundant in non-commelinid walls. However, the α-d-glucuronic acid/4-O-methyl-α-d-glucuronic acid is often substituted at O-2 by an α-l-arabinopyranose residue in Alismatales and Asparagales glucuronoxylans. Glucuronoarabinoxylans were the only xylans detected in the cell walls of five different members of the Poaceae family (grasses). By contrast, both glucuronoxylan and glucuronoarabinoxylan are formed by the Zingiberales and Commelinales (commelinids). At least one species of each monocot order, including the Poales, forms xylan with the reducing end sequence -4)-β-d-Xylp-(1,3)-α-l-Rhap-(1,2)-α-d-GalpA-(1,4)-d-Xyl first identified in eudicot and gymnosperm glucuronoxylans. This sequence was not discernible in the arabinopyranose-containing glucuronoxylans of the Alismatales and Asparagales or the glucuronoarabinoxylans of the Poaceae. Rather, our data provide additional evidence that in Poaceae glucuronoarabinoxylan, the reducing end xylose residue is often substituted at O-2 with 4-O-methyl glucuronic acid or at O-3 with arabinofuranose. The variations in xylan structure and their implications for the evolution and biosynthesis of monocot cell walls are discussed.

     

Gibberellin mediates the development of gelatinous fibres in the tension wood of inclined Acacia mangium seedlings

Background and Aims

Gibberellin stimulates negative gravitropism and the formation of tension wood in tilted Acacia mangium seedlings, while inhibitors of gibberellin synthesis strongly inhibit the return to vertical growth and suppress the formation of tension wood. To characterize the role of gibberellin in tension wood formation and gravitropism, this study investigated the role of gibberellin in the development of gelatinous fibres and in the changes in anatomical characteristics of woody elements in Acacia mangium seedlings exposed to a gravitational stimulus.

Methods

Gibberellin, paclobutrazol and uniconazole-P were applied to the soil in which seedlings were growing, using distilled water as the control. Three days after the start of treatment, seedlings were inclined at 45 ° to the vertical and samples were harvested 2 months later. The effects of the treatments on wood fibres, vessel elements and ray parenchyma cells were analysed in tension wood in the upper part of inclined stems and in the opposite wood on the lower side of inclined stems.

Key Results

Application of paclobutrazol or uniconazole-P inhibited the increase in the thickness of gelatinous layers and prevented the elongation of gelatinous fibres in the tension wood of inclined stems. By contrast, gibberellin stimulated the elongation of these fibres. Application of gibberellin and inhibitors of gibberellin biosynthesis had only minor effects on the anatomical characteristics of vessel and ray parenchyma cells.

Conclusions

The results suggest that gibberellin is important for the development of gelatinous fibres in the tension wood of A. mangium seedlings and therefore in gravitropism.     

Xylem-specific and tension stress-responsive coexpression of KORRIGAN endoglucanase and three secondary wall-associated cellulose synthase genes in aspen trees

In nature, angiosperm trees develop tension wood on the upper side of their leaning trunks and drooping branches. Development of tension wood is one of the straightening mechanisms by which trees counteract leaning or bending of stem and resume upward growth. Tension wood is characterized by the development of a highly crystalline cellulose-enriched gelatinous layer next to the lumen of the tension wood fibers. Thus experimental induction of tension wood provides a system to understand the process of cellulose biosynthesis in trees. Since KORRIGAN endoglucanases (KOR) appear to play an important role in cellulose biosynthesis in Arabidopsis, we cloned PtrKOR, a full-length KOR cDNA from aspen xylem. Using RT-PCR, in situ hybridization, and tissue-print assays, we show that PtrKOR gene expression is significantly elevated on the upper side of the bent aspen stem in response to tension stress while KOR expression is significantly suppressed on the opposite side experiencing compression stress. Moreover, three previously reported aspen cellulose synthase genes, namely, PtrCesA1, PtrCesA2, and PtrCesA3 that are closely associated with secondary cell wall development in the xylem cells exhibited similar tension stress-responsive behavior. Our results suggest that coexpression of these four proteins is important for the biosynthesis of highly crystalline cellulose typically present in tension wood fibers. Their simultaneous genetic manipulation may lead to industrially relevant improvement of cellulose in transgenic crops and trees.Suchita Bhandari and Takeshi Fujino contributed equally to this research.     

Heterologous expression,purification and characterization of a highly thermolabile endoxylanase from the Antarctic fungus Cladosporium sp.

Numerous endoxylanases from mesophilic fungi have been purified and characterized. However, endoxylanases from cold-adapted fungi, especially those from Antarctica, have been less studied. In this work, a cDNA from the Antarctic fungus Cladosporium sp. with similarity to endoxylanases from glycosyl hydrolase family 10, was cloned and expressed in Pichia pastoris. The pure recombinant enzyme (named XynA) showed optimal activity on xylan at 50 °C and pH 6–7. The enzyme releases xylooligosaccharides but not xylose, indicating that XynA is a classical endoxylanase. The enzyme was most active on xylans with high content of arabinose (rye arabinoylan and wheat arabinoxylan) than on xylans with low content of arabinose (oat spelts xylan, birchwood xylan and beechwood xylan). Finally, XynA showed a very low thermostability. After 20–30 min of incubation at 40 °C, the enzyme was completely inactivated, suggesting that XynA would be the most thermolabile endoxylanase described so far in filamentous fungi. This is one of the few reports describing the heterologous expression and characterization of a xylanase from a fungus isolated from Antarctica.     

Identification and characterisation of xylanolytic yeasts isolated from decaying wood and sugarcane bagasse in Brazil

In this study, yeasts associated with lignocellulosic materials in Brazil, including decaying wood and sugarcane bagasse, were isolated, and their ability to produce xylanolytic enzymes was investigated. A total of 358 yeast isolates were obtained, with 198 strains isolated from decaying wood and 160 strains isolated from decaying sugarcane bagasse samples. Seventy-five isolates possessed xylanase activity in solid medium and were identified as belonging to nine species: Candida intermedia, C. tropicalis, Meyerozyma guilliermondii, Scheffersomyces shehatae, Sugiyamaella smithiae, Cryptococcus diffluens, Cr. heveanensis, Cr. laurentii and Trichosporon mycotoxinivorans. Twenty-one isolates were further screened for total xylanase activity in liquid medium with xylan, and five xylanolytic yeasts were selected for further characterization, which included quantitative analysis of growth in xylan and xylose and xylanase and β-d-xylosidase activities. The yeasts showing the highest growth rate and cell density in xylan, Cr. laurentii UFMG-HB-48, Su. smithiae UFMG-HM-80.1 and Sc. shehatae UFMG-HM-9.1a, were, simultaneously, those exhibiting higher xylanase activity. Xylan induced the highest level of (extracellular) xylanase activity in Cr. laurentii UFMG-HB-48 and the highest level of (intracellular, extracellular and membrane-associated) β-d-xylosidase activity in Su. smithiae UFMG-HM-80.1. Also, significant β-d-xylosidase levels were detected in xylan-induced cultures of Cr. laurentii UFMG-HB-48 and Sc. shehatae UFMG-HM-9.1a, mainly in extracellular and intracellular spaces, respectively. Under xylose induction, Cr. laurentii UFMG-HB-48 showed the highest intracellular β-d-xylosidase activity among all the yeast tested. C. tropicalis UFMG-HB 93a showed its higher (intracellular) β-d-xylosidase activity under xylose induction and higher at 30 °C than at 50 °C. This study revealed different xylanolytic abilities and strategies in yeasts to metabolise xylan and/or its hydrolysis products (xylo-oligosaccharides and xylose). Xylanolytic yeasts are able to secrete xylanolytic enzymes mainly when induced by xylan and present different strategies (intra- and/or extracellular hydrolysis) for the metabolism of xylo-oligosaccharides. Some of the unique xylanolytic traits identified here should be further explored for their applicability in specific biotechnological processes.     

Cloning,Sequencing, and Expression of the Gene Encoding an Intracellular β-D-Xylosidase from Streptomyces thermoviolaceus OPC-520

The intracellular β-xylosidase was induced when Streptomyces thermoviolaceus OPC-520 was grown at 50°C in a minimal medium containing xylan or xylooligosaccharides. The 82-kDa protein with β-xylosidase activity was partially purified and its N-terminal amino acid sequence was analyzed. The gene encoding the enzyme was cloned, sequenced, and expressed in Escherichia coli. The bxlA gene consists of a 2,100-bp open reading frame encoding 770 amino acids. The deduced amino acid sequence of the bxlA gene product had significant similarity with β-xylosidases classified into family 3 of glycosyl hydrolases. The bxlA gene was expressed in E. coli, and the recombinant protein was purified to homogeneity. The enzyme was a monomer with a molecular mass of 82 kDa. The purified enzyme showed hydrolytic activity towards only p-nitrophenyl-β-D-xylopyranoside among the synthetic glycosides tested. Thin-layer chromatography analysis showed that the enzyme is an exo-type enzyme that hydrolyze xylooligosaccharides, but had no activity toward xylan. High activity against pNPX occurred in the pH range 6.0-7.0 and temperature range 40-50°C.     

Rhamnogalacturonan‐I is a determinant of cell–cell adhesion in poplar wood

The molecular basis of cell–cell adhesion in woody tissues is not known. Xylem cells in wood particles of hybrid poplar (Populus tremula × P. alba cv. INRA 717‐1B4) were separated by oxidation of lignin with acidic sodium chlorite when combined with extraction of xylan and rhamnogalacturonan‐I (RG‐I) using either dilute alkali or a combination of xylanase and RG‐lyase. Acidic chlorite followed by dilute alkali treatment enables cell–cell separation by removing material from the compound middle lamellae between the primary walls. Although lignin is known to contribute to adhesion between wood cells, we found that removing lignin is a necessary but not sufficient condition to effect complete cell–cell separation in poplar lines with various ratios of syringyl:guaiacyl lignin. Transgenic poplar lines expressing an Arabidopsis thaliana gene encoding an RG‐lyase (AtRGIL6) showed enhanced cell–cell separation, increased accessibility of cellulose and xylan to hydrolytic enzyme activities, and increased fragmentation of intact wood particles into small cell clusters and single cells under mechanical stress. Our results indicate a novel function for RG‐I, and also for xylan, as determinants of cell–cell adhesion in poplar wood cell walls. Genetic control of RG‐I content provides a new strategy to increase catalyst accessibility and saccharification yields from woody biomass for biofuels and industrial chemicals.     

Xylans from the tropical grass Panicum maximum

Two xylans have been isolated from the mature tissues of the tropical grass Panicum maximum—an arabino(4-O-methylglucurono)xylan and an acidic galactoarabinoxylan. Both consist of a main chain of β(1 → 4) linked d-xylopyranosyl residues. The former has average of ca 46 such residues to which are attached ca 7 l-arabinofuranosyl and (ca 2 4-O-methyl-d-glucopyranuronosyl residues at C3 and C2 positions respectively. The acidic galactoarabinoxylan has a $\text{DP}$_n of ca 90 and contains arabinose, galactose, xylose and uronic acid residues in the molar ratio 10:5:22:4. Methylation analysis and periodate oxidation indicated the highly branched nature of this polysaccharide.