Fungi hijack a ubiquitous plant apoplastic endoglucanase to release a ROS scavenging ß-glucan decasaccharide to subvert immune responses.

Plant pathogenic and beneficial fungi have evolved several strategies to evade immunity and cope with host-derived hydrolytic enzymes and oxidative stress in the apoplast, the extracellular space of plant tissues. Fungal hyphae are surrounded by an inner insoluble cell wall layer and an outer soluble extracellular polysaccharide (EPS) matrix. Here, we show by proteomics and glycomics that these two layers have distinct protein and carbohydrate signatures, and hence likely have different biological functions. The barley (Hordeum vulgare) ß-1,3-endoglucanase HvBGLUII, which belongs to the widely distributed apoplastic glycoside hydrolase 17 family (GH17), releases a conserved ß-1,3;1,6-glucan decasaccharide (ß-GD) from the EPS matrices of fungi with different lifestyles and taxonomic positions. This low molecular weight ß-GD does not activate plant immunity, is resilient to further enzymatic hydrolysis by ß-1,3-endoglucanases due to the presence of three ß-1,6-linked glucose branches and can scavenge reactive oxygen species. Exogenous application of ß-GD leads to enhanced fungal colonization in barley, confirming its role in the fungal counter-defensive strategy to subvert host immunity. Our data highlight the hitherto undescribed capacity of this often-overlooked EPS matrix from plant-associated fungi to act as an outer protective barrier important for fungal accommodation within the hostile environment at the apoplastic plant-microbe interface.

Structural Characterisation and Assessment of the Novel Bacillus amyloliquefaciens RK3 Exopolysaccharide on the Improvement of Cognitive Function in Alzheimer's Disease Mice.

In this study Bacillus amyloliquefaciens RK3 was isolated from a sugar mill effluent-contaminated soil and utilised to generate a potential polysaccharide with anti-Alzheimer's activity. Traditional and molecular methods were used to validate the strain. The polysaccharide produced by B. amyloliquefaciens RK3 was purified, and the yield was estimated to be 10.35 gL-1. Following purification, the polysaccharide was structurally and chemically analysed. The structural analysis revealed the polysaccharide consists of α-d-mannopyranose (α-d-Manp) and ß-d-galactopyranose (ß-d-Galp) monosaccharide units connected through glycosidic linkages (i.e., ß-d-Galp(1→6)ß-d-Galp (1→6)ß-d-Galp(1→2)ß-d-Galp(1→2)[ß-d-Galp(1→6)]ß-d-Galp(1→2)α-d-Manp(1→6)α-d-Manp (1→6)α-d-Manp(1→6)α-d-Manp(1→6)α-d-Manp). The scanning electron microscopy and energy-dispersive X-ray spectroscopy imaging of polysaccharides emphasise their compactness and branching in the usual tubular heteropolysaccharide structure. The purified exopolysaccharide significantly impacted the plaques formed by the amyloid proteins during Alzheimer's disease. Further, the results also highlighted the potential applicability of exopolysaccharide in various industrial and pharmaceutical applications.

Microwave Assisted Pretreatment of Szarvasi (Agropyron elongatum) Biomass to Enhance Enzymatic Saccharification and Direct Glucose Production.

Biomass from perennial plants can be considered a carbon-neutral renewable resource. The tall wheatgrass hybrid Szarvasi-1 (Agropyron elongatum, hereafter referred to as "Szarvasi") belongs to the perennial Poaceae representing a species, which can grow on marginal soils and produce large amounts of biomass. Several conventional and advanced pretreatment methods have been developed to enhance the saccharification efficiency of plant biomass. Advanced pretreatment methods, such as microwave-assisted pretreatment methods are faster and use less energy compared to conventional pretreatment methods. In this study, we investigated the potential of Szarvasi biomass as a biorefinery feedstock. For this purpose, the lignocellulosic structure of Szarvasi biomass was investigated in detail. In addition, microwave-assisted pretreatments were applied to Szarvasi biomass using different reagents including weak acids and alkali. The produced pulp, hydrolysates, and extracted lignin were quantitatively characterized. In particular, the alkali pretreatment significantly enhanced the saccharification efficiency of the pulp 16-fold compared to untreated biomass of Szarvasi. The acid pretreatment directly converted 25% of the cellulose into glucose without the need of enzymatic digestion. In addition, based on lignin compositional and lignin linkage analysis a lignin chemical model structure present in Szarvasi biomass could be established.

Multiscale analysis of lignocellulose recalcitrance towards OrganoCat pretreatment and fractionation.

BACKGROUND: Biomass recalcitrance towards pretreatment and further processing can be related to the compositional and structural features of the biomass. However, the exact role and relative importance to those structural attributes has still to be further evaluated. Herein, ten different types of biomass currently considered to be important raw materials for biorefineries were chosen to be processed by the recently developed, acid-catalyzed OrganoCat pretreatment to produce cellulose-enriched pulp, sugars, and lignin with different amounts and qualities. Using wet chemistry analysis and NMR spectroscopy, the generic factors of lignocellulose recalcitrance towards OrganoCat were determined. RESULTS: The different materials were processed applying different conditions (e.g., type of acid catalyst and temperature), and fractions with different qualities were obtained. Raw materials and products were characterized in terms of their compositional and structural features. For the first time, generic correlation coefficients were calculated between the measured chemical and structural features and the different OrganoCat product yields and qualities. Especially lignin-related factors displayed a detrimental role for enzymatic pulp hydrolysis, as well as sugar and lignin yield exhibiting inverse correlation coefficients. Hemicellulose appeared to have less impact, not being as detrimental as lignin factors, but xylan-O-acetylation was inversely correlated with product yield and qualities. CONCLUSION: These results illustrate the role of generic features of lignocellulosic recalcitrance towards acidic pretreatments and fractionation, exemplified in the OrganoCat strategy. Discriminating between types of lignocellulosic biomass and highlighting important compositional variables, the improved understanding of how these parameters affect OrganoCat products will ameliorate bioeconomic concepts from agricultural production to chemical products. Herein, a methodological approach is proposed.

OrganoCat Fractionation of Empty Fruit Bunches from Palm Trees into Lignin, Sugars, and Cellulose-Enriched Pulp.

The palm oil industry produces large amounts of empty fruit bunches (EFB) as waste. EFB are very recalcitrant toward further processing, although their valorization could create novel incentives and bio-economic opportunities for the industries involved. Herein, EFB have been successfully subjected to the OrganoCat pretreatment-using 2,5-furandicarboxylic acid as the biogenic catalyst-to fractionate and separate this lignocellulosic material into its main components in a single step. The pretreatment of EFB leads to the deacetylation and depolymerization of noncellulosic polysaccharides and to the partial delignification of the cellulosic fiber. The OrganoCat processing of EFB yielded 45 ± 0.5 wt % cellulose-enriched pulp, 20 ± 0.7 wt % extracted lignin, 3.8 ± 0.2 wt % furfural, and 11 ± 0.6 wt % hydrolyzed sugars. The obtained EFB-pulp showed high accessibility to cellulases, resulting in a glucan conversion of 73 ± 2% after 72 h (15 ± 2% after 1 h) with commercial cellulase cocktail (Accellerase 1500). Overall, the results suggest that the treatment of the EFB material using OrganoCat may create promising paths for the full valorization of EFBs.

Deposition of lignin in four species of Saccharum.

We used primers designed on conserved gene regions of several species to isolate the most expressed genes of the lignin pathway in four Saccharum species. S. officinarum and S. barberi have more sucrose in the culms than S. spontaneum and S. robustum, but less polysaccharides and lignin in the cell wall. S. spontaneum, and S. robustum had the lowest S/G ratio and a lower rate of saccharification in mature internodes. Surprisingly, except for CAD, 4CL, and CCoAOMT for which we found three, two, and two genes, respectively, only one gene was found for the other enzymes and their sequences were highly similar among the species. S. spontaneum had the highest expression for most genes. CCR and CCoAOMT B presented the highest expression; 4CL and F5H showed increased expression in mature tissues; C3H and CCR had higher expression in S. spontaneum, and one of the CADs isolated (CAD B) had higher expression in S. officinarum. The similarity among the most expressed genes isolated from these species was unexpected and indicated that lignin biosynthesis is conserved in Saccharum including commercial varieties Thus the lignin biosynthesis control in sugarcane may be only fully understood with the knowledge of the promotor region of each gene.

Mechanistic insights from plant heteromannan synthesis in yeast.

Heteromannan (HM) is one of the most ancient cell wall polymers in the plant kingdom, consisting of ß-(1-4)-linked backbones of glucose (Glc) and mannose (Man) units. Despite the widespread distribution of HM polysaccharides, their biosynthesis remains mechanistically unclear. HM is elongated by glycosyltransferases (GTs) from the cellulose synthase-like A (CSLA) family. MANNAN-SYNTHESIS RELATED (MSR) putative GTs have also been implicated in (gluco)mannan synthesis, but their roles have been difficult to decipher in planta and in vitro. To further characterize the products of the HM synthases and accessory proteins, we chose a synthetic biology approach to synthesize plant HM in yeast. The expression of a CSLA protein in Pichia pastoris led to the abundant production of plant HM: up to 30% of glycans in the yeast cell wall. Based on sequential chemical and enzymatic extractions, followed by detailed structural analyses, the newly produced HM polymers were unbranched and could be larger than 270 kDa. Using CSLAs from different species, we programmed yeast cells to produce an HM backbone composed exclusively of Man or also incorporating Glc. We demonstrate that specific MSR cofactors were indispensable for mannan synthase activity of a coffee CSLA or modulated a functional CSLA enzyme to produce glucomannan instead of mannan. Therefore, this powerful platform yields functional insight into the molecular machinery required for HM biosynthesis in plants.

Identification of an arabinopyranosyltransferase from Physcomitrella patens involved in the synthesis of the hemicellulose xyloglucan.

The hemicellulose xyloglucan consists of a backbone of a ß-1,4 glucan substituted with xylosyl moieties and many other, diverse side chains that are important for its proper function. Many, but not all glycosyltransferases involved in the biosynthesis of xyloglucan have been identified. Here, we report the identification of an hitherto elusive xyloglucan:arabinopyranosyltransferase. This glycosyltransferase was isolated from the moss Physcomitrella patens, where it acts as a xyloglucan "D"-side chain transferase (XDT). Heterologous expression of PpXDT in the Arabidopsis thaliana double mutant mur3.1 xlt2, where xyloglucan consists of a xylosylated glucan without further glycosyl substituents, results in the production of the arabinopyranose-containing "D" side chain as characterized by oligosaccharide mass profiling, glycosidic linkage analysis, and NMR analysis. In addition, expression of a related Physcomitrella glycosyltransferase ortholog of PpXLT2 leads to the production of the galactose-containing "L" side chain. The presence of the "D" and "L" xyloglucan side chains in the Arabidopsis double mutant Atmur3.1 xlt2 expressing PpXDT and PpXLT2, respectively, rescues the dwarfed phenotype of untransformed Atmur3.1 xlt2 mutants to nearly wild-type height. Expression of PpXDT and PpXLT2 in the Atmur3.1 xlt2 mutant also enhanced root growth.

The role of the plant-specific ALTERED XYLOGLUCAN9 protein in Arabidopsis cell wall polysaccharide O-acetylation.

A mutation in the ALTERED XYLOGLUCAN9 (AXY9) gene was found to be causative for the decreased xyloglucan acetylation phenotype of the axy9.1 mutant, which was identified in a forward genetic screen for Arabidopsis (Arabidopsis thaliana) mutants. The axy9.1 mutant also exhibits decreased O-acetylation of xylan, implying that the AXY9 protein has a broad role in polysaccharide acetylation. An axy9 insertional mutant exhibits severe growth defects and collapsed xylem, demonstrating the importance of wall polysaccharide O-acetylation for normal plant growth and development. Localization and topological experiments indicate that the active site of the AXY9 protein resides within the Golgi lumen. The AXY9 protein appears to be a component of the plant cell wall polysaccharide acetylation pathway, which also includes the REDUCED WALL ACETYLATION and TRICHOME BIREFRINGENCE-LIKE proteins. The AXY9 protein is distinct from the TRICHOME BIREFRINGENCE-LIKE proteins, reported to be polysaccharide acetyltransferases, but does share homology with them and other acetyltransferases, suggesting that the AXY9 protein may act to produce an acetylated intermediate that is part of the O-acetylation pathway.

Ionic liquid crystals as alignment medium to measure residual dipolar couplings for carbohydrates.

Ionic liquids consisting of N-dodecyl-N-methyl pyrrolidinium bromide [C12MPB] in a mixture with D2O, decanol, and DMSO were for the first time found to give anisotropic molecular alignment in magnetic fields and are useful to measure residual dipolar couplings (RDCs) from polar analytes, for example, glucose. The system shows less quadrupolar splitting of the deuterated solvent signal compared with other liquid crystal systems and hence less undesired line broadening.

Study of an organogelator by diffusion-ordered NMR spectroscopy.

The low-molecular weight organogelator 2,3-di-n-decyloxyanthracene was synthezised and dissolved in dimethyl sulfoxide. With diffusion-ordered NMR spectroscopy, the temperature dependence of the diffusion coefficients was measured and a clear hysteresis of the gelation was observed between 320 and 330 K. This hysteresis was interpreted with respect to different entanglement behavior on heating and cooling. No alignment of the gelator with respect to the magnetic field was found despite its 14 π-electrons.

Polyisocyanides as a new alignment medium to measure residual dipolar couplings for small organic molecules.

Polyisocycanides were found to give anisotropic molecular alignment in the magnetic field and are useful to measure residual dipolar couplings (RDCs) from analytes, e.g. strychnine. They show less quadrupolar splitting of the deuterated solvent signal compared with other liquid crystal systems such as Poly-γ-benzyl-L-glutamate (PBLG) and hence less undesired line broadening.