Hemicellulose Backbone Research Articles

Hemicellulose-rich paper-grade pulp as raw material for regenerated fibres in an ionic liquid-based process

Hemicellulose-rich pulp raw materials are avoided in the production of man-made cellulosic textile fibres due to hemicellulose reactivity with the currently used industrial solvent systems. Incorporation of hemicelluloses in regenerated fibres could, however, increase the share of used wood biomass and thus improve the environmental footprint of regenerated fibre products. Superbase ionic liquids have shown potential in dissolving and regenerating all the major wood polymers i.e. cellulose, hemicellulose and lignin into regenerated products. In this work, regenerated fibres were spun from hemicellulose-rich softwood and eucalyptus paper-grade pulps and eucalyptus dissolving pulp using a superbase ionic liquid [mTBNH][OAc]. Before dissolution and spinning, intrinsic viscosities of the paper-grade pulps were adjusted either enzymatically or by using a mild acid-treatment to improve dope rheology for dry-jet wet spinning. In fibre spinning, hemicellulose was found to regenerate in high yield and the obtained regenerated fibres had high dry tenacities (5.3 to 15 cN/dtex). The best mechanical properties were measured from fibres with high hemicellulose content (17.3% (w/w)). Pulp pretreatment was found to be critical for achieving good mechanical properties. Acid-pretreatment, which modified both cellulose and hemicellulose, yielded regenerated fibres with better mechanical properties compared to an enzyme-pretreatment which did not alter the hemicellulose structure. Removal of hemicellulose substituents and hydrolysis of hemicellulose backbone in acid-pretreatment may be the key to improved mechanical properties in hemicellulose-containing regenerated fibres. Enzymatic peeling and imaging with a xylan-specific monoclonal antibody (CCRC-M138) suggest that hemicelluloses were enriched to the outermost layers of the regenerated fibres.

Genome-Wide Identification of Banana Csl Gene Family and Their Different Responses to Low Temperature between Chilling-Sensitive and Tolerant Cultivars.

The cell wall plays an important role in responses to various stresses. The cellulose synthase-like gene (Csl) family has been reported to be involved in the biosynthesis of the hemicellulose backbone. However, little information is available on their involvement in plant tolerance to low-temperature (LT) stress. In this study, a total of 42 Csls were identified in Musa acuminata and clustered into six subfamilies (CslA, CslC, CslD, CslE, CslG, and CslH) according to phylogenetic relationships. The genomic features of MaCsl genes were characterized to identify gene structures, conserved motifs and the distribution among chromosomes. A phylogenetic tree was constructed to show the diversity in these genes. Different changes in hemicellulose content between chilling-tolerant and chilling-sensitive banana cultivars under LT were observed, suggesting that certain types of hemicellulose are involved in LT stress tolerance in banana. Thus, the expression patterns of MaCsl genes in both cultivars after LT treatment were investigated by RNA sequencing (RNA-Seq) technique followed by quantitative real-time PCR (qPCR) validation. The results indicated that MaCslA4/12, MaCslD4 and MaCslE2 are promising candidates determining the chilling tolerance of banana. Our results provide the first genome-wide characterization of the MaCsls in banana, and open the door for further functional studies.

Genome-wide identification of Cellulose-like synthase D gene family in Dendrobium catenatum

Dendrobium catenatum, which grows on the semi humid rocks in the mountains, has been at the top of the ‘Nine Immortals of China’ since ancient times. It is a kind of yin tonic medicine and its main active component is polysaccharide. Cellulose synthase-like D(CslD) genes were predicted to catalyse the biosynthesis of 1,4-β-d-glycan backbone of hemicelluloses, which plays fundamental roles in plant development. To investigate the role of CslD in the development of D. catenatum, eight CslD genes (DcCslD1,2a,2b,3a,3b,4a,4b,5) were identified. The results of protein prediction and analysis showed that CslD2a/2b/4a/4b proteins were acidic proteins. All the proteins had obvious hydrophobic or hydrophilic regions, and had transmembrane structure. Phylogenetic analysis revealed that the DcCslD family could be divided into group I, II, III and IV. DcCslD proteins had a typical Cellulose synthase domain and similar protein structures to the CslDs of other plants. Their promoter regions contain cis regulatory elements related to stress and hormone response. The results of qRT-PCR showed that the identified DcCslDs were differentially expressed in roots, stems and leaves. Most of them were highly expressed in stems and leaves. The environmental stresses examination showed that the expression levels of DcCslD5 were closely associated with drought-recovery treatment; the expression levels of DcCslD1, DcCslD2a, DcCslD2b, DcCslD3a and DcCslD5 were significantly influenced by low temperature. This study systematically analyzed the sequence characteristics of CslD protein of D. catenatum, which can provide reference for further study on the function of CslD protein in the polysaccharide metabolism of D. catenatum. Supplemental data for this article is available online at https://doi.org/10.1080/13102818.2021.1941252.

Characterization of hemicelluloses in sugarcane (Saccharum spp. hybrids) culm during xylogenesis

Hemicelluloses are effective renewable biopolymers that can be used in many different industrial processes and preparations. In plants, the content of hemicellulose might change with different developmental stages and/or tissues. Thus, in here chemical and structural differences in hemicellulose isolated from the apical, middle and basal segments of sugarcane stem were characterized using chemical techniques. Further, difference in expression levels of genes related to synthesis of hemicelluloses from these three segments were studied by RNA-seq and qRT-PCR etc. The sugarcane hemicellulose backbone was xylose residues connected via β-1,4 glycosidic linkages which was further substituted with arabinose, acetyl and glucuronic acid side chains. Hemicellulose content was higher in the middle and basal segments with less backbone substitutions compared to apical segments. In terms of gene expression, hemicellulose synthesis and modification genes were intensely expressed in middle and basal segments. Taken together, our research describes differences in hemicellulose content and substitutions in sugarcane during xylogenesis, which will increase our knowledge for finding more refined use of sugarcane bagasse.

A molecular dynamics study of the effect of glycosidic linkage type in the hemicellulose backbone on the molecular chain flexibility.

The macromolecular conformation of the constituent polysaccharides in lignocellulosic biomass influences their supramolecular interactions, and therefore their function in plants and their performance in technical products. The flexibility of glycosidic linkages from the backbone of hemicelluloses was studied by evaluating the conformational freedom of the φ and ψ dihedral angles using molecular dynamic simulations, additionally selected molecules were correlated with experimental data by nuclear magnetic resonance spectroscopy. Three types of β-(1→4) glycosidic linkages involving the monosaccharides (Glcp, Xylp and Manp) present in the backbone of hemicelluloses were defined. Different di- and tetrasaccharides with combinations of such sugar monomers from hemicelluloses were simulated, and free energy maps of the φ - ψ space and hydrogen-bonding patterns were obtained. The glycosidic linkage between Glc-Glc or Glc-Man (C-type) was the stiffest with mainly one probable conformation; the linkage from Man-Man or Man-Glc (M-type) was similar but with an increased probability for an alternative conformation making it more flexible, and the linkage between two Xyl-units (X-type) was the most flexible with two almost equally populated conformations. Glycosidic linkages of the same type showed essentially the same conformational space in both disaccharides and in the central region of tetrasaccharides. Different probabilities of glycosidic linkage conformations in the backbone of hemicelluloses can be directly estimated from the free energy maps, which to a large degree affect the overall macromolecular conformations of these polymers. The information gained contributes to an increased understanding of the function of hemicelluloses both in the cell wall and in technical products.

Enzymatic hydrolysis of biomass from wood.

Current research and development in cellulosic ethanol production has been focused mainly on agricultural residues and dedicated energy crops such as corn stover and switchgrass; however, woody biomass remains a very important feedstock for ethanol production. The precise composition of hemicellulose in the wood is strongly dependent on the plant species, therefore different types of enzymes are needed based on hemicellulose complexity and type of pretreatment. In general, hardwood species have much lower recalcitrance to enzymes than softwood. For hardwood, xylanases, beta-xylosidases and xyloglucanases are the main hemicellulases involved in degradation of the hemicellulose backbone, while for softwood the effect of mannanases and beta-mannosidases is more relevant. Furthermore, there are different key accessory enzymes involved in removing the hemicellulosic fraction and increasing accessibility of cellulases to the cellulose fibres improving the hydrolysis process. A diversity of enzymatic cocktails has been tested using from low to high densities of biomass (2-20% total solids) and a broad range of results has been obtained. The performance of recently developed commercial cocktails on hardwoods and softwoods will enable a further step for the commercialization of fuel ethanol from wood.

Enzymatic Hydrolysis of Pretreated Kenaf using a Recombinant Xylanase: Effects of Reaction Conditions for Optimum Hemicellulose Hydrolysis

An efficient hemicellulose hydrolysis has now become a crucial step for xylooligosaccharides production for food additives and nutraceuticals industries. In this study, xylanase hydrolysis of pretreated hemicellulosic kenaf (Hibiscus cannabinus) for substantial hemicellulose hydrolysis was demonstrated. Experiments were performed primarily to investigate the effect of several critical reaction conditions towards the enzymatic hydrolysis of the pre-treated kenaf. The effects of various ranges of reaction conditions (substrate loading, xylanase loading, pH, temperature and time) were systematically and thoroughly studied. The study of hemicellulosic kenaf conversion using single enzyme was proven to effectively yield up to 59% of conversion after 48 h incubation using 3% (w/v) of substrate loading and 400 U of xylanase loading at 50°C in pH 4.0 buffer systems. The use of a single xylanase on the pre-treated kenaf was a key parameter in understanding the action of the main hemicellulose degrading enzyme towards hydrolysing the hemicellulose backbone of the pre-treated kenaf. Enzyme feeding strategy was also conducted and the results revealed that a batch feeding strategy of 400 U xylanase loading yielded the highest hemicellulose hydrolysis compared to the other fed batch enzyme feeding strategy. It was concluded that each reaction conditions and enzyme feeding strategy gave a significant impact towards the enzymatic hydrolysis and the use of optimum reaction conditions resulted in a high product yield at the end of the reaction. With the discovery of the importance of each reaction conditions involved in a hydrolysis, this study highlights the need for a comprehensive investigation on the reaction conditions using a mixture of enzymes towards achieving a complete hydrolysis with a higher product yield.

RNA-Seq Analysis of the Expression of Genes Encoding Cell Wall Degrading Enzymes during Infection of Lupin (Lupinus angustifolius) by Phytophthora parasitica.

RNA-Seq analysis has shown that over 60% (12,962) of the predicted transcripts in the Phytophthora parasitica genome are expressed during the first 60 h of lupin root infection. The infection transcriptomes included 278 of the 431 genes encoding P. parasitica cell wall degrading enzymes. The transcriptome data provide strong evidence of global transcriptional cascades of genes whose encoded proteins target the main categories of plant cell wall components. A major cohort of pectinases is predominantly expressed early but as infection progresses, the transcriptome becomes increasingly dominated by transcripts encoding cellulases, hemicellulases, β-1,3-glucanases and glycoproteins. The most highly expressed P. parasitica carbohydrate active enzyme gene contains two CBM1 cellulose binding modules and no catalytic domains. The top 200 differentially expressed genes include β-1,4-glucosidases, β-1,4-glucanases, β-1,4-galactanases, a β-1,3-glucanase, an α-1,4-polygalacturonase, a pectin deacetylase and a pectin methylesterase. Detailed analysis of gene expression profiles provides clues as to the order in which linkages within the complex carbohydrates may come under attack. The gene expression profiles suggest that (i) demethylation of pectic homogalacturonan occurs before its deacetylation; (ii) cleavage of the backbone of pectic rhamnogalacturonan I precedes digestion of its side chains; (iii) early attack on cellulose microfibrils by non-catalytic cellulose-binding proteins and enzymes with auxiliary activities may facilitate subsequent attack by glycosyl hydrolases and enzymes containing CBM1 cellulose-binding modules; (iv) terminal hemicellulose backbone residues are targeted after extensive internal backbone cleavage has occurred; and (v) the carbohydrate chains on glycoproteins are degraded late in infection. A notable feature of the P. parasitica infection transcriptome is the high level of transcription of genes encoding enzymes that degrade β-1,3-glucanases during middle and late stages of infection. The results suggest that high levels of β-1,3-glucanases may effectively degrade callose as it is produced by the plant during the defence response.

Solution Properties of Hemicellulose Polysaccharides with Four Common Carbohydrate Force Fields.

Hemicellulose polysaccharides play an important role in the swelling behavior of the primary plant cell wall, and molecular dynamics simulations provide the means of gaining a concise understanding of the interactions of hemicellulose polysaccharides with water. Here, we compare four of the main polysaccharide force fields (CHARMM36 TIP3P, GROMOS56A6(CARBO) SPC, GLYCAM06h TIP3P, and GLYCAM06h TIP5P) for the most abundant hemicellulose backbone components. In particular, we compare aggregation, diffusion coefficients, system density, and investigate the free energy of hydration of saccharides in water. We find that the saccharides show nonphysical aggregation at low concentrations with the GLYCAM06h TIP3P force field, which can be rectified by the use of the TIP5P water model. As a result of the aggregation, GLYCAM06h TIP3P does not lead to reasonable diffusion coefficients whereas the diffusion coefficients, as well as the system density, agrees best with experimental data for the GLYCAM06h TIP5P force field. Overall, GLYCAM06h TIP5P gives good agreement with experimental free energy of hydration data for small saccharides. In addition, the free energy of hydration for short polysaccharides calculated with the GLYCAM06h TIP5P force field is consistent with the radial distribution functions between the polysaccharides and water, the hydration number of the polysaccharides, and the hydrogen bonds formed in the system.

Thiolated hemicellulose as a versatile platform for one-pot click-type hydrogel synthesis.

A one-pot synthetic methodology for the thiolation of O-acetyl-galactoglucomannan (AcGGM) was developed to merge hemicellulose chemistry with "click" chemistry. This was realized by the AcGGM-mediated nucleophilic ring-opening of γ-thiobutyrolactone via the activation of the polysaccharide pendant hydroxyl groups. The incorporation of thiol functionalities onto the hemicellulose backbone was visualized by (1)H and (13)C NMR spectroscopy and was assessed by an Ellman's reagent assay of the thiol groups. The versatility of the thiolated AcGGM was elaborated and demonstrated by conducting several postmodification reactions together with hydrogel formation utilizing thiol-ene and thiol-Michael addition "click" reactions. The one-pot synthesis of thiolated AcGGM is a straightforward approach that can expand the applications of hemicelluloses derived from biomass by employing "click" chemistry.

Plant Biomass Recalcitrance: Effect of Hemicellulose Composition on Nanoscale Forces that Control Cell Wall Strength

Efficient conversion of lignocellulosic biomass to second-generation biofuels and valuable chemicals requires decomposition of resilient plant cell wall structure. Cell wall recalcitrance varies among plant species and even phenotypes, depending on the chemical composition of the noncellulosic matrix. Changing the amount and composition of branches attached to the hemicellulose backbone can significantly alter the cell wall strength and microstructure. We address the effect of hemicellulose composition on primary cell wall assembly forces by using the 3D-RISM-KH molecular theory of solvation, which provides statistical-mechanical sampling and molecular picture of hemicellulose arrangement around cellulose. We show that hemicellulose branches of arabinose, glucuronic acid, and especially glucuronate strengthen the primary cell wall by strongly coordinating to hydrogen bond donor sites on the cellulose surface. We reveal molecular forces maintaining the cell wall structure and provide directions for genetic modulation of plants and pretreatment design to render biomass more amenable to processing.

Rice SLENDER LEAF 1 gene encodes cellulose synthase-like D4 and is specifically expressed in M-phase cells to regulate cell proliferation

Cellulose synthase-like (CSL) genes are predicted to catalyse the biosynthesis of non-cellulosic polysaccharides such as the β-d-glycan backbone of hemicelluloses and are classified into nine subfamilies (CSLA–CSLH and CSLJ). The CSLD subfamily is conserved in all land plants, and among the nine CSL subfamilies, it shows the highest sequence similarity to the cellulose synthase genes, suggesting that it plays fundamental roles in plant development. This study presents a detailed analysis of slender leaf 1 (sle1) mutants of rice that showed rolled and narrow leaf blades and a reduction in plant height. The narrow leaf blade of sle1 was caused by reduced cell proliferation beginning at the P3 primordial stage. In addition to the size reduction of organs, sle1 mutants exhibited serious developmental defects in pollen formation, anther dehiscence, stomata formation, and cell arrangement in various tissues. Map-based cloning revealed that SLE1 encodes the OsCSLD4 protein, which was identified previously from a narrow leaf and dwarf 1 mutant. In situ hybridization experiments showed that OsCSLD4 was expressed in a patchy pattern in developing organs. Double-target in situ hybridization and quantitative RT-PCR analyses revealed that SLE1 was expressed specifically during the M-phase of the cell cycle, and suggested that the cell-cycle regulation was altered in sle1 mutants. These results suggest that the OsCSLD4 protein plays a pivotal role in the M phase to regulate cell proliferation. Further study of OsCSLD4 is expected to yield new insight into the role of hemicelluloses in plant development.

Cleavage of acetyl groups from northeast hardwood for acetic acid production in kraft pulp mills

This study identified the optimum conditions for cleavage of acetyl groups from wood hemicelluloses for possible conversion to salts of acetate or to acetic acid in kraft pulp mills. Acetyl groups in wood hemicelluloses can be hydrolyzed by either OH- (hydroxide) or H+ (hydronium) ions. Experimental data are presented for the extraction of industrial northeast hardwood chips using alkali streams that are available in the kraft pulp mills: caustic, green liquor, and white liquor. The effects of extraction time, chip soaking temperature, alkali concentration, and extraction temperature on cleavage of acetyl groups were investigated. Soaking at elevated temperature was found to be more effective than no soaking or soaking at room temperature. The rate of cleavage of acetyl groups from wood hemicelluloses was proportional to the initial hydroxide ion concentration in the liquor. Both white liquor and 0.5 N sodium hydroxide had higher rates of hydrolysis of acetyl groups compared to green liquor, which contained fewer hydroxide ions. The initial hydroxide ion concentration in the liquor also determined the mechanism by which acetyl groups were hydrolyzed from the hemicellulose backbone. If the extraction liquor contained excess hydroxide ions, then most acetyl groups were directly hydrolyzed from the xylan polymer to form sodium acetate, and the xylan remained in the wood, provided the temperature was low. The extraction temperature had a negligible effect on rate of cleavage of acetyl groups if the liquor contained excess hydroxide ions.

Novel hydrophobic hemicelluloses: Synthesis and characteristic

Novel hydrophobic hemicelluloses possessing hydrophobic groups were prepared by the benzylation of wheat straw hemicelluloses with benzyl chloride under the presence of catalyst in an ethanol/water system. In particular, the progress of the benzylation reaction was studied as a function of the volume ratio of ethanol/water from 4:1 to 6:4, the molar ratio of NaOH/anhydroxylose unit in hemicelluloses from 0.6:1 to 1.5:1, the molar ratio of benzyl chloride/anhydroxylose unit in hemicelluloses from 0.5:1 to 2.0:1, reaction temperature 50–80°C, and reaction time 4–20h. Benzylated hemicelluloses with the low degree of substitution from 0.09 to 0.35 were obtained depending on the experimental conditions. The incorporation of benzyl groups into the backbone of hemicelluloses was confirmed by FT-IR and 13C NMR spectroscopies. The thermal stability increased after the modification of hemicelluloses due to the introduction of benzyl groups. The introduction of benzyl groups endows hemicelluloses with the hydrophobicity, which could be potentially applied in plastic industries.

The Preparation and Application of the Cationic Biopolymer Based on Xylan-Rich Hemicelluloses from Agricultural Biomass

During the last few years increasing attention has been paid to the exploitation of hemicelluloses as biopolymer resources because they are available in very large amounts in organic wasters from renewable forest and agricultural biomass. The efficient method to prepare the cationic biopolymers based on xylan-rich hemicelluloses from agricultural biomass has been developed in this paper. These biopolymers with cationic functional groups, having many good properties such as solubility, bio-degradation, cationic or ampholytic polymers, were synthesized by the chemical modification of sugarcane bagasse xylan-rich hemicelluloses with CHMAC and preferably with ETA in different media. These biopolymers based on xylan-rich hemicelluloses with the degree of substitution (DS) up to 0.55 could be obtained by varying the molar ratio of ETA or CHMAC to anhydroxylose units in hemicelluloses from 0.5 to 3.0. 13C NMR confirmed that cationic groups were grafted onto the backbone of hemicelluloses. In addition, their applications in papermaking as dry strengthen agent were investigated. It was found that these biopolymers could obviously improve the physical properties of hand sheets formed from old corrugated container pulp fibres. These results mentioned above highlight the remarkable potential for using the hemicelluloses-based biopolymers as wet end additives in papermaking.

Membrane extraction for removal of acetic acid from biomass hydrolysates

Production of bioethanol from lignocellulosic biomass requires pretreatment of the biomass in order to improve the susceptibility of the cellulose to enzymatic hydrolysis to glucose. When dilute acid is used to perform this process, the hemicellulose is also hydrolyzed to its component sugars while simultaneously releasing acetyl groups attached to the hemicellulose backbone. Other compounds from the lignin and sugar degradation products are also produced that inhibit subsequent bioconversion of the solubilized sugars to the desired products. In this work we focused on removal of acetic acid from a dilute sulphuric acid pretreated corn stover hydrolysate. Acetic acid has been extracted into an organic phase at pH values below its p K a. The organic phase diluent consisted of octanol. Alamine 336, a tertiary amine and Aliquat 336 a quaternary amine were used as the aliphatic amine extractants. Our results indicate more than 60% removal of acetic acid using Alamine 336. Extraction rates were much slower for Aliquat 336 probably due to the higher viscosity of the Aliquat 336/octanol phase. The presence of sulphate anions, as a result of dilute sulphuric acid pretreatment, results in the co-extraction of bisulphate anion. Bisulphate anion is preferentially extracted at pH values below its p K a. Consequently the pH of the hydrolysate increases from between 1 and 2 to above 4.0 during extraction. In addition, extraction of low molecular weight lignins and phenolics is also observed. Thus the membrane extraction process developed here may be used not only for removal of acetic acid but also to adjust the pH of the hydrolysate to values that are more compatible for fermentation and to remove other inhibitory compounds.

Synthesis of cationic hemicellulosic derivatives with a low degree of substitution in dimethyl sulfoxide media

AbstractA series of water‐soluble cationic 2‐hydroxylpropyltrimethylammonium hemicellulosic derivatives with low average degrees of substitution (DS's) were prepared by the incorporation of the cationic moiety 2,3‐epoxypropyltrimethylammonium chloride (ETA) onto the backbone of hemicelluloses in the presence of NaOH as a nucleophilic catalyst in homogeneous dimethyl sulfoxide (DMSO) media. The dependence of the homogeneous reaction on the different affecting factors was investigated. The average DS was calculated from the N/C ratio in the products and from the weight gain. The degree of substitution determined by the nitrogen content (DSN) values up to 0.25 in a one‐step synthesis of the etherified hemicelluloses could be controlled by the adjustment of the amount of solvent used and the molar ratio of NaOH or ETA to the anhydromonomer units in the hemicelluloses. The structure of the cationic hemicellulosic derivatives formed was determined by Fourier transform infrared spectroscopy and further confirmed with solution‐state 13C‐NMR spectroscopy. In comparison, no significant degradation of the hemicellulosic derivatives occurred during the etherification of the polymers in the homogeneous DMSO system. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008

Maize Pollen Coat Xylanase Facilitates Pollen Tube Penetration into Silk during Sexual Reproduction

Cell wall hydrolases are well documented to be present on pollen, but their roles on the stigma during sexual reproduction have not been previously demonstrated. We explored the function of the tapetum-synthesized xylanase, ZmXYN1, on maize (Zea mays L.) pollen. Transgenic lines (xyl-less) containing little or no xylanase in the pollen coat were generated with use of an antisense construct of the xylanase gene-coding region driven by the XYN1 gene promoter. Xyl-less and wild-type plants had similar vegetative growth. Electron microscopy revealed no appreciable morphological difference in anther cells and pollen between xyl-less lines and the wild type, whereas immunofluorescence microscopy and biochemical analyses indicated an absence of xylanase on xyl-less pollen. Xyl-less pollen germinated as efficiently as wild-type pollen in vitro in a liquid medium but less so on gel media of increasing solidity or on silk, which is indicative of partial impaired water uptake. Once germinated in vitro or on silk, the xyl-less and wild-type pollen tubes elongated at comparable rates. Tubes of germinated xyl-less pollen on silk did not penetrate into the silk as efficiently as tubes of wild-type pollen, and this lower efficiency could be overcome by the addition of xylanase to the silk. For wild-type pollen, coat xylanase activity on oat spelled xylan in vitro and tube penetration into silk were inhibited by xylose but not glucose. The overall findings indicate that maize pollen coat xylanase facilitates pollen tube penetration into silk via enzymatic xylan hydrolysis.

Carbon catabolite repression of xylanase I (xyn1) gene expression in Trichoderma reesei.

The filamentous fungus Trichoderma reesei forms two specific, xylan-inducible xylanases encoded by xyn1 and xyn2 to degrade the beta-1,4-D-xylan backbone of hemicelluloses. This enzyme system is formed in the presence of xylan, but not glucose. The molecular basis of the absence of xylanase I formation on glucose was the purpose of this study. Northern blotting of the xyn1 transcript as well as the use of the Escherichia coli hygromycin B phosphotransferase-encoding gene (hph) as a reporter consistently showed that the basal expression of xyn1 was affected by glucose, whereas its induction by xylan remained uninfluenced. The repression of basal xyn1 transcription is mediated by the carbon catabolite repressor protein Cre1, which in vivo binds to two of four consensus sites (5'-SYG-GRG-3') in the xyn1 promoter, which occurred in the form of an inverted repeat. T. reesei strains, bearing a xyn1::hph reporter construct, in which four nucleotides from the middle of the inverted repeat had been removed, expressed hph on glucose at a level comparable to that observed during growth on a carbon catabolite derepressing carbon source. Northern analysis of xyn1 expression in a T. reesei mutant strain (RUT C-30), which contains a truncated, non-functional cre1 gene, also confirmed basal transcription of xyn1. In this strain, xyn1 transcription was still inducible by xylose or xylan to an even higher degree than in the wild-type strain, suggesting that induction overcomes glucose repression at the level of xyn1 expression. Based on these data, we postulate that basal transcription of xyn1 is repressed by glucose and mediated by an inverted repeat of the consensus motif for Cre1-mediated carbon catabolite repression.