Molecular architecture of softwood revealed by solid-state NMR

Economically important softwood from conifers is mainly composed of the polysaccharides cellulose, galactoglucomannan and xylan, and the phenolic polymer, lignin. The interactions between these polymers lead to wood mechanical strength and must be overcome in biorefining. Here, we use 13C multidimensional solid-state NMR to analyse the polymer interactions in never-dried cell walls of the softwood, spruce. In contrast to some earlier softwood cell wall models, most of the xylan binds to cellulose in the two-fold screw conformation. Moreover, galactoglucomannan alters its conformation by intimately binding to the surface of cellulose microfibrils in a semi-crystalline fashion. Some galactoglucomannan and xylan bind to the same cellulose microfibrils, and lignin is associated with both of these cellulose-bound polysaccharides. We propose a model of softwood molecular architecture which explains the origin of the different cellulose environments observed in the NMR experiments. Our model will assist strategies for improving wood usage in a sustainable bioeconomy

Covalent interactions between lignin and hemicelluloses in plant secondary cell walls.

The plant secondary cell wall is a complex structure composed of polysaccharides and lignin, and is a key evolutionary innovation of vascular land plants. Although cell wall composition is well understood, the cross-linking of the different polymers is only now yielding to investigation. Cross-linking between hemicelluloses and lignin occurs via two different mechanisms: incorporation into lignin by radical coupling of ferulate substitutions on xylan in commelinid monocots, and incorporation of hemicellulosic glycosyl residues by re-aromatisation of lignification intermediates. Recent genetic evidence indicates that hemicellulose:lignin cross-linking has a substantial impact on plant cell wall recalcitrance. Engineering plant biomass with modified frequencies of cross-links will have significant impacts on biomass utilisation.O.M.T was a recipient of an iCASE studentship from the BBSRC and Novozymes (Reference BB/M015432/1). P.D. was supported by the Leverhulme Trust Centre for Natural Material Innovation and the OpenPlant Synthetic Biology Research Centre BB/L014130/1

A Transcriptomic Analysis of Xylan Mutants Does Not Support the Existence of a Secondary Cell Wall Integrity System in Arabidopsis.

Yeast have long been known to possess a cell wall integrity (CWI) system, and recently an analogous system has been described for the primary walls of plants (PCWI) that leads to changes in plant growth and cell wall composition. A similar system has been proposed to exist for secondary cell walls (SCWI). However, there is little data to support this. Here, we analyzed the stem transcriptome of a set of cell wall biosynthetic mutants in order to investigate whether cell wall damage, in this case caused by aberrant xylan synthesis, activates a signaling cascade or changes in cell wall synthesis gene expression. Our data revealed remarkably few changes to the transcriptome. We hypothesize that this is because cells undergoing secondary cell wall thickening have entered a committed programme leading to cell death, and therefore a SCWI system would have limited impact. The absence of transcriptomic responses to secondary cell wall alterations may facilitate engineering of the secondary cell wall of plants

Probing the molecular architecture of Arabidopsis thaliana secondary cell walls using two- and three-dimensional (13)C solid state nuclear magnetic resonance spectroscopy.

The plant secondary cell wall is a thickened polysaccharide and phenolic structure, providing mechanical strength to cells, particularly in woody tissues. It is the main feedstock for the developing bioenergy and green chemistry industries. Despite the role that molecular architecture (the arrangement of biopolymers relative to each other, and their conformations) plays in dictating biomass properties, such as recalcitrance to breakdown, it is poorly understood. Here, unprocessed dry (13)C-labeled stems from the model plant Arabidopsis thaliana were analyzed by a variety of (13)C solid state magic angle spinning nuclear magnetic resonance methods, such as one-dimensional cross-polarization and direct polarization, two-dimensional refocused INADEQUATE, RFDR, PDSD, and three-dimensional DARR, demonstrating their viability for the study of native polymer arrangements in intact secondary cell walls. All carbon sites of the two main glucose environments in cellulose (previously assigned to microfibril surface and interior residues) are clearly resolved, as are carbon sites of the other major components of the secondary cell wall: xylan and lignin. The xylan carbon 4 chemical shift is markedly different from that reported previously for solution or primary cell wall xylan, indicating significant changes in the helical conformation in these dried stems. Furthermore, the shift span indicates that xylan adopts a wide range of conformations in this material, with very little in the 31 conformation typical of xylan in solution. Additionally, spatial connections of noncarbohydrate species were observed with both cellulose peaks conventionally assigned as "surface" and as "interior" cellulose environments, raising questions about the origin of these two cellulose signals.This work was supported by BBSRC Grant BB/G016240/1, via The BBSRC Sustainable Bioenergy Cell Wall Sugars Programme. The UK 850 MHz solid state NMR Facility was funded by EPSRC Grant EP/F017901/1 and the BBSRC, as well as the University of Warwick, including via partial funding through Birmingham Science City Advanced Materials Projects 1 and 2, by Advantage West Midlands (AWM) and the European Regional Development Fund (ERDF).This is the final published version. It first appeared at http://pubs.acs.org/doi/abs/10.1021/bi501552k

BdGT43B2 functions in xylan biosynthesis and is essential for seedling survival in Brachypodium distachyon.

Xylan is the predominant hemicellulose in the primary cell walls of grasses, but its synthesis and interactions with other wall polysaccharides are complex and incompletely understood. To probe xylan biosynthesis, we generated CRISPR/Cas9 knockout and amiRNA knockdown lines of BdGT43B2, an ortholog of the wheat TaGT43-4 xylan synthase scaffolding protein in the IRX14 clade, in Brachypodium distachyon. Knockout of BdGT43B2 caused stunting and premature death in Brachypodium seedlings. Immunofluorescence labeling of xylans was greatly reduced in homozygous knockout BdGT43B2 mutants, whereas cellulose labeling was unchanged or slightly increased. Biochemical analysis showed reductions in digestible xylan in knockout mutant walls, and cell size was smaller in knockout leaves. BdGT43B2 knockdown plants appeared morphologically normal as adults, but showed slight reductions in seedling growth and small decreases in xylose content in isolated cell walls. Immunofluorescence labeling of xylan and cellulose staining was both reduced in BdGT43B2 knockdown plants. Together, these data indicate that BdGT43B2 functions in the synthesis of a form of xylan that is required for seedling growth and survival in Brachypodium distachyon

Plant cell wall architecture guided design of CBM3-GH11 chimeras with enhanced xylanase activity using a tandem repeat left-handed β-3-prism scaffold.

Effective use of plant biomass as an abundant and renewable feedstock for biofuel production and biorefinery requires efficient enzymatic mobilization of cell wall polymers. Knowledge of plant cell wall composition and architecture has been exploited to develop novel multifunctional enzymes with improved activity against lignocellulose, where a left-handed β-3-prism synthetic scaffold (BeSS) was designed for insertion of multiple protein domains at the prism vertices. This allowed construction of a series of chimeras fusing variable numbers of a GH11 β-endo-1,4-xylanase and the CipA-CBM3 with defined distances and constrained relative orientations between catalytic domains. The cellulose binding and endoxylanase activities of all chimeras were maintained. Activity against lignocellulose substrates revealed a rapid 1.6- to 3-fold increase in total reducing saccharide release and increased levels of all major oligosaccharides as measured by polysaccharide analysis using carbohydrate gel electrophoresis (PACE). A construct with CBM3 and GH11 domains inserted in the same prism vertex showed highest activity, demonstrating interdomain geometry rather than number of catalytic sites is important for optimized chimera design. These results confirm that the BeSS concept is robust and can be successfully applied to the construction of multifunctional chimeras, which expands the possibilities for knowledge-based protein design

Three Decades of Advances in Arabinogalactan-Protein Biosynthesis.

Arabinogalactan-proteins (AGPs) are a large, complex, and highly diverse class of heavily glycosylated proteins that belong to the family of cell wall hydroxyproline-rich glycoproteins. Approximately 90% of the molecules consist of arabinogalactan polysaccharides, which are composed of arabinose and galactose as major sugars and minor sugars such as glucuronic acid, fucose, and rhamnose. About half of the AGP family members contain a glycosylphosphatidylinositol (GPI) lipid anchor, which allows for an association with the outer leaflet of the plasma membrane. The mysterious AGP family has captivated the attention of plant biologists for several decades. This diverse family of glycoproteins is widely distributed in the plant kingdom, including many algae, where they play fundamental roles in growth and development processes. The journey of AGP biosynthesis begins with the assembly of amino acids into peptide chains of proteins. An N-terminal signal peptide directs AGPs toward the endoplasmic reticulum, where proline hydroxylation occurs and a GPI anchor may be added. GPI-anchored AGPs, as well as unanchored AGPs, are then transferred to the Golgi apparatus, where extensive glycosylation occurs by the action of a variety glycosyltransferase enzymes. Following glycosylation, AGPs are transported by secretory vesicles to the cell wall or to the extracellular face of the plasma membrane (in the case of GPI-anchored AGPs). GPI-anchored proteins can be released from the plasma membrane into the cell wall by phospholipases. In this review, we present an overview of the accumulated knowledge on AGP biosynthesis over the past three decades. Particular emphasis is placed on the glycosylation of AGPs as the sugar moiety is essential to their function. Recent genetics and genomics approaches have significantly contributed to a broader knowledge of AGP biosynthesis. However, many questions remain to be elucidated in the decades ahead

Characterisation of FUT4 and FUT6 α-(1 → 2)-fucosyltransferases reveals that absence of root arabinogalactan fucosylation increases Arabidopsis root growth salt sensitivity.

Plant type II arabinogalactan (AG) polysaccharides are attached to arabinogalactan proteins (AGPs) at hydroxyproline residues, and they are very diverse and heterogeneous structures. The AG consists of a β-(1 → 3)-linked galactan backbone with β-(1 → 6)-galactan side chains that are modified mainly with arabinose, but they may also contain glucuronic acid, rhamnose or other sugars. Here, we studied the positions of fucose substitutions in AGPs, and we investigated the functions of this fucosylation. Monosaccharide analysis of Arabidopsis leaf AGP extracts revealed a significant reduction in L-Fucose content in the fut4 mutant, but not in the fut6 mutant. In addition, Fucose was reduced in the fut4 mutant in root AGP extracts and was absent in the fut4/fut6 mutant. Curiously, in all cases reduction of fucose was accompanied with a reduction in xylose levels. The fucosylated AGP structures in leaves and roots in wild type and fut mutant plants were characterised by sequential digestion with AG specific enzymes, analysis by Polysaccharide Analysis using Carbohydrate gel Electrophoresis, and Matrix Assisted Laser Desorption/Ionisation (MALDI)-Time of Flight Mass spectrometry (MS). We found that FUT4 is solely responsible for the fucosylation of AGPs in leaves. The Arabidopsis thaliana FUT4 and FUT6 genes have been previously proposed to be non-redundant AG-specific fucosyltransferases. Unexpectedly, FUT4 and FUT6 enzymes both fucosylate the same AGP structures in roots, suggesting partial redundancy to each other. Detailed structural characterisation of root AGPs with high energy MALDI-Collision Induced Dissociation MS and NMR revealed an abundant unique AG oligosaccharide structure consisting of terminal xylose attached to fucose. The loss of this structure in fut4/fut6 mutants explains the reduction of both fucose and xylose in AGP extracts. Under salt-stress growth conditions the fut4/fut6 mutant lacking AGP fucosylation exhibited a shorter root phenotype than wild type plants, implicating fucosylation of AGPs in maintaining proper cell expansion under these conditions

Xylan decoration patterns and the plant secondary cell wall molecular architecture.

The molecular architecture of plant secondary cell walls is still not resolved. There are several proposed structures for cellulose fibrils, the main component of plant cell walls and the conformation of other molecules is even less well known. Glucuronic acid (GlcA) substitution of xylan (GUX) enzymes, in CAZy family glycosyl transferase (GT)8, decorate the xylan backbone with various specific patterns of GlcA. It was recently discovered that dicot xylan has a domain with the side chain decorations distributed on every second unit of the backbone (xylose). If the xylan backbone folds in a similar way to glucan chains in cellulose (2-fold helix), this kind of arrangement may allow the undecorated side of the xylan chain to hydrogen bond with the hydrophilic surface of cellulose microfibrils. MD simulations suggest that such interactions are energetically stable. We discuss the possible role of this xylan decoration pattern in building of the plant cell wall.We thank Nadine Anders for helpful comments on the manuscript. The work was supported by a Leverhulme Trust Programme Grant : The Centre for Natural Material Innovation and the Biotechnology and Biological Sciences Research Council grant numbers [BB/K005537/1] and [BB/G016240/1].This is the author accepted manuscript. The final version is available from Portland Press via http://dx.doi.org/10.1042/BST2015018

Meritor brake assembly bench part setup process improvement

In partnership with Meritor\u27s Manning, South Carolina facility, this capstone project focuses on the changeover process of a drum brake assembly for industrial trucks. The changeover process is the downtime an operator spends preparing the workstation for the next set of brake orders. This team\u27s objective is to use process improvement techniques to reduce downtime in between brake orders. Once the customer needs were determined, the team developed concepts to increase the efficiency of the changeover process time. The concepts consisted of real world applications and the utilization of a discrete event simulation software called Arena. In addition to the Arena model, the team has begun testing individual concepts. With the results from these tested concepts, the team will be able to effectively develop solutions to reduce downtime of operators during the changeover process