Structure Of Xylan Research Articles

Unraveling the unique structural motifs of glucuronoxylan from hybrid aspen wood

Xylan is a fundamental structural polysaccharide in plant secondary cell walls and a valuable resource for biorefinery applications. Deciphering the molecular motifs of xylans that mediate their interaction with cellulose and lignin is fundamental to understand the structural integrity of plant cell walls and to design lignocellulosic materials. In the present study, we investigated the pattern of acetylation and glucuronidation substitution in hardwood glucuronoxylan (GX) extracted from aspen wood using subcritical water and alkaline conditions. Enzymatic digestions of GX with β-xylanases from glycosyl hydrolase (GH) families GH10, GH11 and GH30 generated xylo-oligosaccharides with controlled structures amenable for mass spectrometric glycan sequencing. We identified the occurrence of intramolecular motifs in aspen GX with block repeats of even glucuronidation (every 2 xylose units) and consecutive glucuronidation, which are unique features for hardwood xylans. The acetylation pattern of aspen GX shows major domains with evenly-spaced decorations, together with minor stretches of highly acetylated domains. These heterogenous patterns of GX can be correlated with its extractability and with its potential interaction with lignin and cellulose. Our study provides new insights into the molecular structure of xylan in hardwood species, which has fundamental implications for overcoming lignocellulose recalcitrance during biochemical conversion.

Modifying lignin composition and xylan O-acetylation induces changes in cell wall composition, extractability, and digestibility

BackgroundLignin and xylan are important determinants of cell wall structure and lignocellulosic biomass digestibility. Genetic manipulations that individually modify either lignin or xylan structure improve polysaccharide digestibility. However, the effects of their simultaneous modifications have not been explored in a similar context. Here, both individual and combinatorial modification in xylan and lignin was studied by analysing the effect on plant cell wall properties, biotic stress responses and integrity sensing.ResultsArabidopsis plant co-harbouring mutation in FERULATE 5-HYDROXYLASE (F5H) and overexpressing Aspergillus niger acetyl xylan esterase (35S:AnAXE1) were generated and displayed normal growth attributes with intact xylem architecture. This fah1-2/35S:AnAXE1 cross was named as hyper G lignin and hypoacetylated (HrGHypAc) line. The HrGHypAc plants showed increased crystalline cellulose content with enhanced digestibility after chemical and enzymatic pre-treatment. Moreover, both parents and HrGHypAc without and after pre-treating with glucuronyl esterase and alpha glucuronidase exhibited an increase in xylose release after xylanase digestion as compared to wild type. The de-pectinated fraction in HrGHypAc displayed elevated levels of xylan and cellulose. Furthermore, the transcriptomic analysis revealed differential expression in cell wall biosynthetic, transcription factors and wall-associated kinases genes implying the role of lignin and xylan modification on cellular regulatory processes.ConclusionsSimultaneous modification in xylan and lignin enhances cellulose content with improved saccharification efficiency. These modifications loosen cell wall complexity and hence resulted in enhanced xylose and xylobiose release with or without pretreatment after xylanase digestion in both parent and HrGHypAc. This study also revealed that the disruption of xylan and lignin structure is possible without compromising either growth and development or defense responses against Pseudomonas syringae infection.

Lignin's complex role in lignocellulosic biomass Recalcitrance: A case study on bamboo

Lignin is widely recognized as a key factor influencing the recalcitrance of lignocellulosic biomass, yet the specifics of this relationship remain complex and not fully understood due to the complex interplay between lignin content variation and cell wall structure. Bamboo, which exhibits greater recalcitrance compared to wood in biorefinery processing, was chosen as the focus of investigation in this study. Three variants of Dendrocalamus farinosus with high, middle and low lignin contents were systematically analyzed and compared, in terms of molecular structure of lignin, hemicelluloses and cellulose, cell wall polymer spatial orientation, lignin-carbohydrate complex (LCC) linkages, cell wall geometrical size, as well as deconstruction efficiency, including delignification, alkaline extraction, and cellulose enzymatic hydrolysis efficiency. Findings reveal that an increase in lignin content generally lowers bamboo's deconstruction efficiency, but the mechanisms are more nuanced than previously thought. While lignin content variations do not significantly affect the molecular structure of xylan, cellulose and the chemical linkage type of LCC linkages, they do impact cellulose crystallinity, xylan orientation and notably, the thickness of fiber cell wall in the free fiber stands of bamboo. These findings highlight that lignin's role extends beyond mere physical protection of the cell wall, but also induces subtle variations in polymer structure and cell wall morphologies, which collectively influence the deconstruction efficiency of bamboo cell walls.

Study on the green extraction of corncob xylan by deep eutectic solvent

Corn as one of the world's major food crops, its by-product corn cob is also rich in resources. However, the unreasonable utilization of corn cob often causes the environmental pollution, waste of resources and other problems. As one of the most abundant polymers in nature, xylan is widely used in food, medicine, materials and other fields. Corn cob is rich in xylan, which is an ideal raw material for extracting xylan. However, the intractable lignin is covalently linked to xylan, which increases the difficulty of xylan extraction. It has been reported that the deep eutectic solvent (DES) could preferentially dissolve lignin in biomass, thereby dissolving the xylan. Then, the xylan in the extract was separated by ethanol precipitation method. The xylan precipitate was obtained after centrifugation, while the supernatant was retained. The components of the supernatant after ethanol precipitation were separated by the rotary evaporator. The ethanol, water and DES were collected for the subsequent extraction of corn cob xylan. In this study, a novel way was provided for the green production of corn cob xylan. The DES was used to extract xylan from corn cob which was used as the raw material. The effects of solid-liquid ratio, reaction time, reaction temperature and water content of DES on the extraction rate of corn cob xylan were investigated by the single factor test. Furthermore, the orthogonal test was designed to optimize the xylan extraction process. The structure of corn cob xylan was analyzed and verified. The results showed that the optimum extraction conditions of corn cob xylan were as follows: the ratio of corn cob to DES was 1 : 15 (g : mL), the extraction time was 3 h, the extraction temperature was 60 °C, and the water content of DES was 70%. Under these conditions, the extraction rate of xylan was 16.46%. The extracted corn cob xylan was distinctive triple helix of polysaccharide, which was similar to the structure of commercially available xylan. Xylan was effectively and workably extracted from corn cob by the DES method. This study provided a new approach for high value conversion of corn cob and the clean production of xylan.

The effects of holocellulose treatments on the yield and structure of dimethylsulfoxide-extracted birchwood xylans

Xylans with naturally occurring acetylation are vital to the plant cell wall functionalities and are also demonstrating excellent dispersing properties which can be applied in fine chemicals and advanced materials. The acetyl structure of xylan can be maintained by dimethyl sulfoxide (DMSO) extraction. A disadvantage of DMSO extraction is that its yield is much lower than that of alkali extraction, which is unfavorable to the research and application of xylans. In this study, sodium chlorite was used as the delignification method to produce holocellulose from birch wood. The effects of subsequent holocellulose solvent exchange, drying and ball-milling on xylan DMSO extraction were studied. The differences of three successive DMSO extractions from the same holocellulose sample and gradient xylan precipitation from DMSO were also evaluated. The majority of xylans are extracted in the first two extractions, while the xylans extracted in the third extraction usually had low yields and low molecular weights. Xylans extracted directly from aqueously wet holocellulose without drying had a very low yield of less than 1%, while xylans extracted from freeze or vacuum dried holocellulose have total yields of 4–9%. T-butanol or ether washed holocellulose shows slightly lower xylan yield than water washed holocellulose. Ball milled holocellulose has twice the xylan yield of holocellulose sample without ball milling. For gradient xylan precipitation, more than 95% of the xylans precipitate in 50% and 70% ethanol. Gradient ethanol precipitation fractionates xylans by molecular weight, but does not fractionate xylans by degree of acetylation. This study is significant for promoting the researches and applications of DMSO extraction of xylan.

Transformations of glucuronoxylan during acid sulphite pulping of eucalyptus dissolving pulp

Xylan is the main contaminant of hardwood sulphite dissolving pulp used for the production of regenerated cellulose. The amount of xylan in pulp should not exceed the minimum allowable values and which is not easy to achieve. To control the pulping process, a basic knowledge of xylan removal is essential. In this study, Eucalyptus globulus glucuronoxylan (GX) conversion during acidic Mg-base acid sulphite pulping of dissolving pulp was assessed for the first time in pilot experiments simulating the industrial process. The kinetics of GX degradation and changes in its structure and molecular weight along all pulping steps were evaluated. According to pentosans analysis, more than 60% of wood xylan was removed already in the heating-up step. A pseudo-first-order reaction for xylan degradation at the final pulping temperature of 140–148 °C was obtained with an activation energy of 133.4 kJ/mol. Removal of residual GX from pulp was hampered by its entrapment in cellulose fibril aggregates. The dynamics of xylan deacetylation and depolymerization during pulping in isolated samples was revealed by 2D NMR and SEC techniques, respectively. Overall, the residual xylan structure in pulp is predetermined by its structural heterogeneity in the cell wall, rather than by structural changes occurring during pulping.

Synthesis of Birch Wood Xylan Sulfate in the Sulfamic Acid–Urea Melt

AbstractThe synthesis of polysaccharide sulfates without harmful solvents used is a serious environmental problem. A method for the synthesis of xylan sulfate using the sulfamic acid–urea melt as a sulfating agent without solvents is proposed. The melt is prepared by heating the sulfamic acid–urea mixture with a molar ratio of 1 : 1 at a temperature of 110 °C. This ratio allows significant reduction of side reactions, in particular, carbamation of xylan. Sulfation is carried out at temperatures of 110,115, 120, 125, and 130 °C. The maximum yield and degree of substitution (DSsulfate 1.40) are attained at atemperature of 115 °C. The proposed method makes it possible to synthesize xylan sulfate rapidly (for 0.5 h) and without contamination of the obtained product with toxic solvents, e. g., dimethyl sulfoxide, pyridine, etc. The composition and structure of initial and sulfated xylan are confirmed by FTIR, NMR, GC, and elemental analysis.

Hydrodynamic cavitation as a promising pretreatment technology to enhance the efficiency of cellulose nanocrystal production via enzymatic hydrolysis

Enzymatic hydrolysis to produce cellulose nanocrystals (CNCs) offers economical and sustainability advantages. However, since this is still an emerging technology, further improvements are needed to make the process feasible for scale-up. In our study, we propose a novel approach using hydrodynamic cavitation (HC) as a pretreatment technology to enhance the efficiency and yield of CNC production. HC treatments conducted under various scenarios induced structural modifications of the cellulose fibers that facilitated their defibrillation and subsequent enzymatic hydrolysis. Isolation through hydrolysis involved the synergistic action of specific enzymes, including xylanase and endoglucanase. Xylanase acted on the hemicellulosic fraction, breaking down the xylan structure and exposing the cellulose fibers. Subsequently, endoglucanase targeted the amorphous regions of cellulose, releasing CNCs. By integrating the HC pretreatment with enzymatic hydrolysis, we achieved remarkable results. High-yield CNCs, reaching approximately 60%, were obtained with a crystallinity index ranging from 81% to 85%. Moreover, this process demonstrated a significant reduction in total energy consumption of approximately 56%, contributing to its economic viability. Furthermore, CNCs with diverse morphologies, aspect ratios, and viscosity profiles were produced, enabling the tailoring of their properties for specific and new applications.

Fractionation of Aspen Wood to Produce Microcrystalline, Microfibrillated and Nanofibrillated Celluloses, Xylan and Ethanollignin.

A new method for extractive-catalytic fractionation of aspen wood to produce microcrystalline (MCC), microfibrillated (MFC), nanofibrilllated (NFC) celluloses, xylan, and ethanollignin is suggested in order to utilize all of the main components of wood biomass. Xylan is obtained with a yield of 10.2 wt.% via aqueous alkali extraction at room temperature. Ethanollignin was obtained with a yield of 11.2 wt.% via extraction with 60% ethanol from the xylan-free wood at 190 °C. The lignocellulose residue formed after the extraction of xylan and ethanollignin was subjected to catalytic peroxide delignification in the acetic acid-water medium at 100 °C in order to obtain microcrystalline cellulose. MCC is hydrolyzed with 56% sulfuric acid and treated with ultrasound to produce microfibrillated cellulose and nanofibrillated cellulose. The yields of MFC and NFC were 14.4 and 19.0 wt.%, respectively. The average hydrodynamic diameter of NFC particles was 36.6 nm, the crystallinity index was 0.86, and the average zeta-potential was 41.5 mV. The composition and structure of xylan, ethanollignin, cellulose product, MCC, MFC, and NFC obtained from aspen wood were characterized using elemental and chemical analysis, Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) analyses, Gas chromatography (GC), Gel permeation-chromatography (GPC), Scanning electron microscopy (SEM), Atomic force microscopy (AFM), Dynamic light scattering (DLS), Thermal gravimetric analysis (TGA).

Hydrothermal combined alkali pretreatment for fractionation the xylan from cotton stalk

Hydrothermal combined alkaline method was conducted to improve the yield of xylan from cotton stalk in this study. The extraction process was optimized by single factor and orthogonal experiments. The maximum yield of recovered xylan was 67.75 ± 0.27% obtaining from hydrothermal extraction of 170 ºC, 60 min, 1:10 and alkaline extraction with 4% alkali at 65 ºC for 2 h with S/L of 1:10. The physic-chemical properties of water-soluble xylan (Xwater) and alkali-soluble xylan (Xalkali) were determined by Fourier transform infrared spectroscopy, Gel permeation chromatography, thermogravimetric analysis and 2D nuclear magnetic resonance (2D NMR). The observed xylan was composed of (1→4)-β-d-xylp as main chain, β-glycosidic bond, and 4-O-methyl-α-D-glucuronic acid as branch chain. Xalkali also contains α-L-arabinose side chains. And the molecular weight of Xalkali was higher than that of Xwater, indicating that alkali pretreatment had a positive protective effect on xylan structure compared with hydrothermal pretreatment. The proposed combined pretreatment method has established a reliable xylan separation method, which has high industrial application feasibility.

Monoclonal antibodies against methyl glucuronoxylan epitopes for understanding xylan structure and modification

Lignocellulosic biomass is an abundant and renewable source that could be utilized for production of many bioproducts. It mainly consists of cellulose, hemicellulose, and lignin, all of which come together to form a recalcitrant wall that impedes production of bioproducts. In order to overcome the recalcitrant nature of the biomass, it is important to fully understand how cell wall components are synthesized and assembled. Xylan is one of the hemicellulosic components of the wall that contributes to recalcitrance. Xylan has substitutions on the backbone that are formed by the orchestration of many enzymes. For example, glucuronoxylan methyltransferases are responsible for 4-O-methylation of glucuronic acid (GlcA) side chains on xylan. Mutant lines are valuable sources to understand the structure and importance of xylan. Here, we investigated the wild-type and gxm1gxm2gxm3 triple mutant of Arabidopsis by employing a set of four xylan antibodies that specifically recognize methyl glucuronoxylan epitopes on xylan to prove their epitope. The triple mutant cell walls were observed with methyl glucuronoxylan specific monoclonal antibodies for the first time. Cell-wall-directed antibodies could help further our understanding on the synthesis and structure of plant cell walls.

Impact of structure and composition of different sorghum xylans as substrates on production of xylanase enzyme by Aspergillus fumigatus RSP-8

Xylan composition from six different bmr sorghum (Sorghum bicolor L.) biomasses (Atlas 12, BN 612, Atlas 3986, IS 27333, Atlas N598 & IS 27206) with variable lignin content were analyzed for their structure and evaluated as a substrate for xylanase complex production using Aspergillus fumigatus RSP-8. Chemical compositional analysis of xylans revealed that all the xylans belong to arabinoxylan (AX) type and are composed of xylose, glucose, and arabinose in varying concentrations. The relative content of xylose varied from 90 % (of total sugars) in the variety IS 27206 to 72 % in Atlas N598. Atlas N598 xylan contained the highest relative glucose content of 22 % and the least content was observed in IS 27206 xylan (2.9 %). The relative content of L-arabinose was constant with fewer variations (5.1–6.7 %). FT-IR analysis showed that the xylans extracted from different sorghum substrates varied in structure. The spectra of all the xylans exhibited absorption maxima of ∼895 cm-1 which is characteristic for the β-(1−4) xylans. The strongest absorption bands in the IR spectra were observed at 3700–3000 and 1200–1000 cm-1 with varying intensities. Xylanase production revealed that the source of xylan significantly affected the fungal growth and xylanase productivity. The highest xylanase production (1248 U/mL) and fungal biomass dry weight (5.94 g/L) was observed with IS 27206 xylan and the least productivity of 964 U/mL and fungal biomass dry weight (3.74 g/L) with Atlas N598 xylan noticed as substrate.

Spatial correlation of water distribution and fine structure of arabinoxylans in the developing wheat grain

This study was to investigate the distribution of water and arabinoxylan structures in growing wheat grain using two complementary imaging techniques, magnetic resonance microimaging (μMRI) and mass spectrometry imaging (MSI). μMRI showed an inhomogeneous water distribution, particularly at early stages. This heterogeneity revealed histological differences that corresponded, within the limits of resolution of μMRI, to tissues with specific physiological functions, including the vascular bundles, the cavity and the endosperm periphery. All of these tissues had a higher water content than the central endosperm. MSI revealed distinct xylan structures in these regions with high levels of Araf substitution around the cavity and acetylated xylans concentrated at the endosperm periphery. For the first time, acetylation and Araf substitution of arabinoxylans were found by image processing to spatially correlate with water distribution in planta. Acetylation and Araf substitution of xylans, which alter chain-chain interactions and increase wall porosity, decreased as the grain matured.

Comparison of glycoside hydrolase family 3 β-xylosidases from basidiomycetes and ascomycetes reveals evolutionarily distinct xylan degradation systems

Xylan is the most common hemicellulose in plant cell walls, though the structure of xylan polymers differs between plant species. Here, to gain a better understanding of fungal xylan degradation systems, which can enhance enzymatic saccharification of plant cell walls in industrial processes, we conducted a comparative study of two glycoside hydrolase family 3 (GH3) β-xylosidases (Bxls), one from the basidiomycete Phanerochaete chrysosporium (PcBxl3), and the other from the ascomycete Trichoderma reesei (TrXyl3A). A comparison of the crystal structures of the two enzymes, both with saccharide bound at the catalytic center, provided insight into the basis of substrate binding at each subsite. PcBxl3 has a substrate-binding pocket at subsite -1, while TrXyl3A has an extra loop that contains additional binding subsites. Furthermore, kinetic experiments revealed that PcBxl3 degraded xylooligosaccharides faster than TrXyl3A, while the KM values of TrXyl3A were lower than those of PcBxl3. The relationship between substrate specificity and degree of polymerization of substrates suggested that PcBxl3 preferentially degrades xylobiose (X2), while TrXyl3A degrades longer xylooligosaccharides. Moreover, docking simulation supported the existence of extended positive subsites of TrXyl3A in the extra loop located at the N-terminus of the protein. Finally, phylogenetic analysis suggests that wood-decaying basidiomycetes use Bxls such as PcBxl3 that act efficiently on xylan structures from woody plants, whereas molds use instead Bxls that efficiently degrade xylan from grass. Our results provide added insights into fungal efficient xylan degradation systems.

Effect of temperature on simultaneous separation and extraction of hemicellulose using p-toluenesulfonic acid treatment at atmospheric pressure

Hemicelluloses were effectively separated using p-toluenesulfonic acid (p-TsOH) treatment at high temperature. High temperature and pressure promoted hydrolysis of hemicellulose, which limited its value upon recovery. In this study, bagasse hemicellulose was separated and extracted by p-TsOH treatment at atmospheric pressure. The effects of temperature, p-TsOH concentration, and time on hemicellulose separation and extraction were investigated. The optimal conditions were 80 °C, 3.0% p-TsOH, and 120 min. The separation and extraction yield of hemicellulose was 73.23% and 36.02%, respectively. Extraction hemicellulose with 95.60% purity was obtained. In addition, the dissolution mechanism of hemicellulose was analyzed. Degradation of β-glycosidic bonds was inhibited. Benzyl ether bond between carbohydrates and lignin was selectively cleaved. The skeleton structure of xylan in hemicellulose was protected while the functional groups of branch chain were severely damaged. It provides a valuable theoretical basis for the efficient separation and extraction of hemicellulose.

Sylfation of Birch Wood Xylan with Sulfamic Acid in 1,4-dioxane

Sulfation of birch wood xylan in 1,4-dioxane by sulfamic acid in the presence of urea at 90 and 100 °C was studied for the first time. The effect of the duration of xylan sulfation on the yield of xylan sulfates and the sulfur content in them was studied. It was found that the sulfur content in the obtained xylan sulfates increases from 12.5 to 17.5 wt% with an increase in the duration of sulfation from 2 to 4 hours. The structure of initial and sulfated xylan was studied by FTIR and NMR spectroscopy. The ease of purification of the obtained xylan sulfates from 1,4-dioxane in comparison with the purification of xylan sulfates obtained by sulfation in pyridine and N, N‑dimethylformamide is an advantage of the proposed method of xylan sulfation

Characterization of glycoside hydrolase family 11 xylanase from Streptomyces sp. strain J103; its synergetic effect with acetyl xylan esterase and enhancement of enzymatic hydrolysis of lignocellulosic biomass

BackgroundXylanase-containing enzyme cocktails are used on an industrial scale to convert xylan into value-added products, as they hydrolyse the β-1,4-glycosidic linkages between xylopyranosyl residues. In the present study, we focused on xynS1, the glycoside hydrolase (GH) 11 xylanase gene derived from the Streptomyces sp. strain J103, which can mediate XynS1 protein synthesis and lignocellulosic material hydrolysis.ResultsxynS1 has an open reading frame with 693 base pairs that encodes a protein with 230 amino acids. The predicted molecular weight and isoelectric point of the protein were 24.47 kDa and 7.92, respectively. The gene was cloned into the pET-11a expression vector and expressed in Escherichia coli BL21(DE3). Recombinant XynS1 (rXynS1) was purified via His-tag affinity column chromatography. rXynS1 exhibited optimal activity at a pH of 5.0 and temperature of 55 °C. Thermal stability was in the temperature range of 50–55 °C. The estimated Km and Vmax values were 51.4 mg/mL and 898.2 U/mg, respectively. One millimolar of Mn2+ and Na+ ions stimulated the activity of rXynS1 by up to 209% and 122.4%, respectively, and 1 mM Co2+ and Ni2+ acted as inhibitors of the enzyme. The mixture of rXynS1, originates from Streptomyces sp. strain J103 and acetyl xylan esterase (AXE), originating from the marine bacterium Ochrovirga pacifica, enhanced the xylan degradation by 2.27-fold, compared to the activity of rXynS1 alone when Mn2+ was used in the reaction mixture; this reflected the ability of both enzymes to hydrolyse the xylan structure. The use of an enzyme cocktail of rXynS1, AXE, and commercial cellulase (Celluclast® 1.5 L) for the hydrolysis of lignocellulosic biomass was more effective than that of commercial cellulase alone, thereby increasing the relative activity 2.3 fold.ConclusionThe supplementation of rXynS1 with AXE enhanced the xylan degradation process via the de-esterification of acetyl groups in the xylan structure. Synergetic action of rXynS1 with commercial cellulase improved the hydrolysis of pre-treated lignocellulosic biomass; thus, rXynS1 could potentially be used in several industrial applications.

Xylan Structure and Dynamics in Native Brachypodium Grass Cell Walls Investigated by Solid-State NMR Spectroscopy.

The polysaccharide composition and dynamics of the intact stem and leaf cell walls of the model grass Brachypodium distachyon are investigated to understand how developmental stage affects the polysaccharide structure of grass cell walls. 13C enrichment of the entire plant allowed detailed analysis of the xylan structure, side-chain functionalization, dynamics, and interaction with cellulose using magic-angle-spinning solid-state NMR spectroscopy. Quantitative one-dimensional 13C NMR spectra and two-dimensional 13C–13C correlation spectra indicate that stem and leaf cell walls contain less pectic polysaccharides compared to previously studied seedling primary cell walls. Between the stem and the leaf, the secondary cell wall-rich stem contains more xylan and more cellulose compared to the leaf. Moreover, the xylan chains are about twofold more acetylated and about 60% more ferulated in the stem. These highly acetylated and ferulated xylan chains adopt a twofold conformation more prevalently and interact more extensively with cellulose. These results support the notion that acetylated xylan is found more in the twofold screw conformation, which preferentially binds cellulose. This in turn promotes cellulose–lignin interactions that are essential for the formation of the secondary cell wall.

Method for the Production and Purification of Plant Immuno-Active Xylanase from Trichoderma.

Plants lack a circulating adaptive immune system to protect themselves against pathogens. Therefore, they have evolved an innate immune system based upon complicated and efficient defense mechanisms, either constitutive or inducible. Plant defense responses are triggered by elicitors such as microbe-associated molecular patterns (MAMPs). These components are recognized by pattern recognition receptors (PRRs) which include plant cell surface receptors. Upon recognition, PRRs trigger pattern-triggered immunity (PTI). Ethylene Inducing Xylanase (EIX) is a fungal MAMP protein from the plant-growth-promoting fungi (PGPF)–Trichoderma. It elicits plant defense responses in tobacco (Nicotiana tabacum) and tomato (Solanum lycopersicum), making it an excellent tool in the studies of plant immunity. Xylanases such as EIX are hydrolytic enzymes that act on xylan in hemicellulose. There are two types of xylanases: the endo-1, 4-β-xylanases that hydrolyze within the xylan structure, and the β-d-xylosidases that hydrolyze the ends of the xylan chain. Xylanases are mainly synthesized by fungi and bacteria. Filamentous fungi produce xylanases in high amounts and secrete them in liquid cultures, making them an ideal system for xylanase purification. Here, we describe a method for cost- and yield-effective xylanase production from Trichoderma using wheat bran as a growth substrate. Xylanase produced by this method possessed xylanase activity and immunogenic activity, effectively inducing a hypersensitive response, ethylene biosynthesis, and ROS burst.

Solid-state NMR of unlabeled plant cell walls: high-resolution structural analysis without isotopic enrichment

BackgroundMultidimensional solid-state nuclear magnetic resonance (ssNMR) spectroscopy has emerged as an indispensable technique for resolving polymer structure and intermolecular packing in primary and secondary plant cell walls. Isotope (13C) enrichment provides feasible sensitivity for measuring 2D/3D correlation spectra, but this time-consuming procedure and its associated expenses have restricted the application of ssNMR in lignocellulose analysis.ResultsHere, we present a method that relies on the sensitivity-enhancing technique Dynamic Nuclear Polarization (DNP) to eliminate the need for 13C-labeling. With a 26-fold sensitivity enhancement, a series of 2D 13C–13C correlation spectra were successfully collected using the unlabeled stems of wild-type Oryza sativa (rice). The atomic resolution allows us to observe a large number of intramolecular cross peaks for fully revealing the polymorphic structure of cellulose and xylan. NMR relaxation and dipolar order parameters further suggest a sophisticated change of molecular motions in a ctl1 ctl2 double mutant: both cellulose and xylan have become more dynamic on the nanosecond and microsecond timescale, but the motional amplitudes are uniformly small for both polysaccharides.ConclusionsBy skipping isotopic labeling, the DNP strategy demonstrated here is universally extendable to all lignocellulose materials. This time-efficient method has landed the technical foundation for understanding polysaccharide structure and cell wall assembly in a large variety of plant tissues and species.