Altering the substitution and cross-linking of glucuronoarabinoxylans affects cell wall architecture in Brachypodium distachyon.

The Poaceae family of plants provides cereal crops that are critical for human and animal nutrition, and also, they are an important source of biomass. Interacting plant cell wall components give rise to recalcitrance to digestion; thus, understanding the wall molecular architecture is important to improve biomass properties. Xylan is the main hemicellulose in grass cell walls. Recently, we reported structural variation in grass xylans, suggesting functional specialisation and distinct interactions with cellulose and lignin. Here, we investigated the functions of these xylans by perturbing the biosynthesis of specific xylan types. We generated CRISPR/Cas9 knockout mutants in Brachypodium distachyon XAX1 and GUX2 genes involved in xylan substitution. Using carbohydrate gel electrophoresis, we identified biochemical changes in different xylan types. Saccharification, cryo-SEM, subcritical water extraction and ssNMR were used to study wall architecture. BdXAX1A and BdGUX2 enzymes modify different types of grass xylan. Brachypodium mutant walls are likely more porous, suggesting the xylan substitutions directed by both BdXAX1A and GUX2 enzymes influence xylan-xylan and/or xylan-lignin interactions. Since xylan substitutions influence wall architecture and digestibility, our findings open new avenues to improve cereals for food and to use grass biomass for feed and the production of bioenergy and biomaterials.

Top five unanswered questions in plant cell surface research.

Plant cell wall researchers were asked their view on what the major unanswered questions are in their field. This article summarises the feedback that was received from them in five questions. In this issue you can find equivalent syntheses for researchers working on bacterial, unicellular parasite and fungal systems.

Silencing ScGUX2 reduces xylan glucuronidation and improves biomass saccharification in sugarcane.

There is an increasing need for renewable energy sources to replace part of our fossil fuel-based economy and reduce greenhouse gas emission. Sugarcane bagasse is a prominent feedstock to produce cellulosic bioethanol, but strategies are still needed to improve the cost-effective exploitation of this potential energy source. In model plants, it has been shown that GUX genes are involved in cell wall hemicellulose decoration, adding glucuronic acid substitutions on the xylan backbone. Mutation of GUX genes increases enzyme access to cell wall polysaccharides, reducing biomass recalcitrance in Arabidopsis thaliana. Here, we characterized the sugarcane GUX genes and silenced GUX2 in commercial hybrid sugarcane. The transgenic lines had no penalty in development under greenhouse conditions. The sugarcane GUX1 and GUX2 enzymes generated different patterns of xylan glucuronidation, suggesting they may differently influence the molecular interaction of xylan with cellulose and lignin. Studies using biomass without chemical or steam pretreatment showed that the cell wall polysaccharides, particularly xylan, were less recalcitrant in sugarcane with GUX2 silenced than in WT plants. Our findings suggest that manipulation of GUX in sugarcane can reduce the costs of second-generation ethanol production and enhance the contribution of biofuels to lowering the emission of greenhouse gases.

Differing structures of galactoglucomannan in eudicots and non-eudicot angiosperms.

The structures of cell wall mannan hemicelluloses have changed during plant evolution. Recently, a new structure called ß-galactoglucomannan (ß-GGM) was discovered in eudicot plants. This galactoglucomannan has ß-(1,2)-Gal-α-(1,6)-Gal disaccharide branches on some mannosyl residues of the strictly alternating Glc-Man backbone. Studies in Arabidopsis revealed ß-GGM is related in structure, biosynthesis and function to xyloglucan. However, when and how plants acquired ß-GGM remains elusive. Here, we studied mannan structures in many sister groups of eudicots. All glucomannan structures were distinct from ß-GGM. In addition, we searched for candidate mannan ß-galactosyltransferases (MBGT) in non-eudicot angiosperms. Candidate AtMBGT1 orthologues from rice (OsGT47A-VII) and Amborella (AtrGT47A-VII) did not show MBGT activity in vivo. However, the AtMBGT1 orthologue from rice showed MUR3-like xyloglucan galactosyltransferase activity in complementation analysis using Arabidopsis. Further, reverse genetic analysis revealed that the enzyme (OsGT47A-VII) contributes to proper root growth in rice. Together, gene duplication and diversification of GT47A-VII in eudicot evolution may have been involved in the acquisition of mannan ß-galactosyltransferase activity. Our results indicate that ß-GGM is likely to be a eudicot-specific mannan.

The biosynthesis, degradation, and function of cell wall ß-xylosylated xyloglucan mirrors that of arabinoxyloglucan.

Xyloglucan is an abundant polysaccharide in many primary cell walls and in the human diet. Decoration of its α-xylosyl sidechains with further sugars is critical for plant growth, even though the sugars themselves vary considerably between species. Plants in the Ericales order - prevalent in human diets - exhibit ß1,2-linked xylosyl decorations. The biosynthetic enzymes responsible for adding these xylosyl decorations, as well as the hydrolases that remove them in the human gut, are unidentified. GT47 xyloglucan glycosyltransferase candidates were expressed in Arabidopsis and endo-xyloglucanase products from transgenic wall material were analysed by electrophoresis, mass spectrometry, and nuclear magnetic resonance (NMR) spectroscopy. The activities of gut bacterial hydrolases BoGH43A and BoGH43B on synthetic glycosides and xyloglucan oligosaccharides were measured by colorimetry and electrophoresis. CcXBT1 is a xyloglucan ß-xylosyltransferase from coffee that can modify Arabidopsis xyloglucan and restore the growth of galactosyltransferase mutants. Related VmXST1 is a weakly active xyloglucan α-arabinofuranosyltransferase from cranberry. BoGH43A hydrolyses both α-arabinofuranosylated and ß-xylosylated oligosaccharides. CcXBT1's presence in coffee and BoGH43A's promiscuity suggest that ß-xylosylated xyloglucan is not only more widespread than thought, but might also nourish beneficial gut bacteria. The evolutionary instability of transferase specificity and lack of hydrolase specificity hint that, to enzymes, xylosides and arabinofuranosides are closely resemblant.

Ectopic callose deposition into woody biomass modulates the nano-architecture of macrofibrils.

Plant biomass plays an increasingly important role in the circular bioeconomy, replacing non-renewable fossil resources. Genetic engineering of this lignocellulosic biomass could benefit biorefinery transformation chains by lowering economic and technological barriers to industrial processing. However, previous efforts have mostly targeted the major constituents of woody biomass: cellulose, hemicellulose and lignin. Here we report the engineering of wood structure through the introduction of callose, a polysaccharide novel to most secondary cell walls. Our multiscale analysis of genetically engineered poplar trees shows that callose deposition modulates cell wall porosity, water and lignin contents and increases the lignin-cellulose distance, ultimately resulting in substantially decreased biomass recalcitrance. We provide a model of the wood cell wall nano-architecture engineered to accommodate the hydrated callose inclusions. Ectopic polymer introduction into biomass manifests in new physico-chemical properties and offers new avenues when considering lignocellulose engineering.

GoSAMTs are required for pectin methyl-esterification and mucilage release in seed coat epidermal cells.

Introduction: GoSAMTs play a role in the methylation of polysaccharides synthesized by the Golgi. Pectin homogalacturonan (HG) methyl-esterification is essential for the proper function of this polysaccharide in cell walls. In order to better understand the role of GoSAMTs in HG biosynthesis, we analyzed mucilage methyl-esterification in gosamt mutants. Methods: To determine the function of GoSAMT1 and GoSAMT2 in HG methyl-esterification we utilized epidermal cells of seed coats, as these structures produce mucilage, which is a pectic matrix. We evaluated differences in seed surface morphology and quantified mucilage release. We measured methanol release, and used antibodies and confocal microscopy to analyze HG methyl-esterification in mucilage. Results: We observed morphological differences on the seed surface and delayed, uneven mucilage release in gosamt1-1gosamt2-1 double mutants. We also found changes in the distal wall length indicating abnormal cell wall breakage in this double mutant. Using methanol release and immunolabeling, we confirmed that GoSAMT1 and GoSAMT2 are involved in HG methyl-esterification in mucilage. However, we did not find evidence of decreasing HG in the gosamt mutants. Confocal microscopy analyses detected different patterns in the adherent mucilage and a greater number of low-methyl-esterified domains near the seed coat surface, which correlates with a greater number of "egg-box" structures in this region. We also detected a shift in the partitioning between the Rhamnogalacturonan-I soluble and adherent layers of the double mutant, which correlated with increased amounts of arabinose and arabinogalactan-protein in the adherent mucilage. Discussion: The results show that the HG synthesized in gosamt mutant plants is less methyl esterified, resulting in more egg-box structures, which stiffen the cell walls in epidermal cells and change the rheological properties of the seed surface. The increased amounts of arabinose and arabinogalactan-protein in adherent mucilage, also suggests that compensation mechanisms were triggered in the gosamt mutants.

The Glycoside Hydrolase Family 35 ß-galactosidase from Trichoderma reesei debranches xyloglucan oligosaccharides from tamarind and jatobá.

Trichoderma reesei (anamorph Hypocrea jecorina) produces an extracellular beta-galactosidase from Glycoside Hydrolase Family 35 (TrBga1). Hydrolysis of xyloglucan oligosaccharides (XGOs) by TrBga1 has been studied by hydrolysis profile analysis of both tamarind (Tamarindus indica) and jatobá (Hymenaea courbaril) seed storage xyloglucans using PACE and MALDI-ToF-MS for separation, quantification and identification of the hydrolysis products. The TrBga1 substrate preference for galactosylated oligosaccharides from both the XXXG- and XXXXG-series of jatobá xyloglucan showed that the doubly galactosylated oligosaccharides were the first to be hydrolyzed. Furthermore, the TrBga1 showed more efficient hydrolysis against non-reducing end dexylosylated oligosaccharides (GLXG/GXLG and GLLG). This preference may play a key role in xyloglucan degradation, since galactosyl removal alleviates steric hindrance for other enzymes in the xyloglucanolytic complex resulting in complete xyloglucan mobilization. Indeed, mixtures of TrBga1 with the α-xylosidase from Escherichia coli (YicI), which shows a preference towards non-galactosylated xyloglucan oligosaccharides, reveals efficient depolymerization when either enzyme is applied first. This understanding of the synergistic depolymerization contributes to the knowledge of plant cell wall structure, and reveals possible evolutionary mechanisms directing the preferences of debranching enzymes acting on xyloglucan oligosaccharides.

Grass xylan structural variation suggests functional specialization and distinctive interaction with cellulose and lignin.

Xylan is the most abundant non-cellulosic polysaccharide in grass cell walls, and it has important structural roles. The name glucuronoarabinoxylan (GAX) is used to describe this variable hemicellulose. It has a linear backbone of ß-1,4-xylose (Xyl) residues that may be substituted with α-1,2-linked (4-O-methyl)-glucuronic acid (GlcA), α-1,3-linked arabinofuranose (Araf), and sometimes acetylation at the O-2 and/or O-3 positions. The role of these substitutions remains unclear, although there is increasing evidence that they affect the way xylan interacts with other cell wall components, particularly cellulose and lignin. Here, we used substitution-dependent endo-xylanase enzymes to investigate the variability of xylan substitution in grass culm cell walls. We show that there are at least three different types of xylan: (i) an arabinoxylan with evenly distributed Araf substitutions without GlcA (AXe); (ii) a glucuronoarabinoxylan with clustered GlcA modifications (GAXc); and (iii) a highly substituted glucuronoarabinoxylan (hsGAX). Immunolocalization of AXe and GAXc in Brachypodium distachyon culms revealed that these xylan types are not restricted to a few cell types but are instead widely detected in Brachypodium cell walls. We hypothesize that there are functionally specialized xylan types within the grass cell wall. The even substitutions of AXe may permit folding and binding on the surface of cellulose fibrils, whereas the more complex substitutions of the other xylans may support a role in the matrix and interaction with other cell wall components.

Glycoside hydrolase subfamily GH5_57 features a highly redesigned catalytic interface to process complex hetero-ß-mannans.

Glycoside hydrolase family 5 (GH5) harbors diverse substrate specificities and modes of action, exhibiting notable molecular adaptations to cope with the stereochemical complexity imposed by glycosides and carbohydrates such as cellulose, xyloglucan, mixed-linkage ß-glucan, laminarin, (hetero)xylan, (hetero)mannan, galactan, chitosan, N-glycan, rutin and hesperidin. GH5 has been divided into subfamilies, many with higher functional specificity, several of which have not been characterized to date and some that have yet to be discovered with the exploration of sequence/taxonomic diversity. In this work, the current GH5 subfamily inventory is expanded with the discovery of the GH5_57 subfamily by describing an endo-ß-mannanase (CapGH5_57) from an uncultured Bacteroidales bacterium recovered from the capybara gut microbiota. Biochemical characterization showed that CapGH5_57 is active on glucomannan, releasing oligosaccharides with a degree of polymerization from 2 to 6, indicating it to be an endo-ß-mannanase. The crystal structure, which was solved using single-wavelength anomalous diffraction, revealed a massively redesigned catalytic interface compared with GH5 mannanases. The typical aromatic platforms and the characteristic α-helix-containing ß6-α6 loop in the positive-subsite region of GH5_7 mannanases are absent in CapGH5_57, generating a large and open catalytic interface that might favor the binding of branched substrates. Supporting this, CapGH5_57 contains a tryptophan residue adjacent and perpendicular to the cleavage site, indicative of an anchoring site for a substrate with a substitution at the -1 glycosyl moiety. Taken together, these results suggest that despite presenting endo activity on glucomannan, CapGH5_57 may have a new type of substituted heteromannan as its natural substrate. This work demonstrates the still great potential for discoveries regarding the mechanistic and functional diversity of this large and polyspecific GH family by unveiling a novel catalytic interface sculpted to recognize complex heteromannans, which led to the establishment of the GH5_57 subfamily.

Eudicot primary cell wall glucomannan is related in synthesis, structure, and function to xyloglucan.

Hemicellulose polysaccharides influence assembly and properties of the plant primary cell wall (PCW), perhaps by interacting with cellulose to affect the deposition and bundling of cellulose fibrils. However, the functional differences between plant cell wall hemicelluloses such as glucomannan, xylan, and xyloglucan (XyG) remain unclear. As the most abundant hemicellulose, XyG is considered important in eudicot PCWs, but plants devoid of XyG show relatively mild phenotypes. We report here that a patterned ß-galactoglucomannan (ß-GGM) is widespread in eudicot PCWs and shows remarkable similarities to XyG. The sugar linkages forming the backbone and side chains of ß-GGM are analogous to those that make up XyG, and moreover, these linkages are formed by glycosyltransferases from the same CAZy families. Solid-state nuclear magnetic resonance indicated that ß-GGM shows low mobility in the cell wall, consistent with interaction with cellulose. Although Arabidopsis ß-GGM synthesis mutants show no obvious growth defects, genetic crosses between ß-GGM and XyG mutants produce exacerbated phenotypes compared with XyG mutants. These findings demonstrate a related role of these two similar but distinct classes of hemicelluloses in PCWs. This work opens avenues to study the roles of ß-GGM and XyG in PCWs.

Acetylated Xylan Degradation by Glycoside Hydrolase Family 10 and 11 Xylanases from the White-rot Fungus Phanerochaete chrysosporium.

Endo-type xylanases are key enzymes in microbial xylanolytic systems, and xylanases belonging to glycoside hydrolase (GH) families 10 or 11 are the major enzymes degrading xylan in nature. These enzymes have typically been characterized using xylan prepared by alkaline extraction, which removes acetyl sidechains from the substrate, and thus the effect of acetyl groups on xylan degradation remains unclear. Here, we compare the ability of GH10 and 11 xylanases, PcXyn10A and PcXyn11B, from the white-rot basidiomycete Phanerochaete chrysosporium to degrade acetylated and deacetylated xylan from various plants. Product quantification revealed that PcXyn10A effectively degraded both acetylated xylan extracted from Arabidopsis thaliana and the deacetylated xylan obtained by alkaline treatment, generating xylooligosaccharides. In contrast, PcXyn11B showed limited activity towards acetyl xylan, but showed significantly increased activity after deacetylation of the xylan. Polysaccharide analysis using carbohydrate gel electrophoresis showed that PcXyn11B generated a broad range of products from native acetylated xylans extracted from birch wood and rice straw, including large residual xylooligosaccharides, while non-acetylated xylan from Japanese cedar was readily degraded into xylooligosaccharides. These results suggest that the degradability of native xylan by GH11 xylanases is highly dependent on the extent of acetyl group substitution. Analysis of 31 fungal genomes in the Carbohydrate-Active enZymes database indicated that the presence of GH11 xylanases is correlated to that of carbohydrate esterase (CE) family 1 acetyl xylan esterases (AXEs), while this is not the case for GH10 xylanases. These findings may imply co-evolution of GH11 xylanases and CE1 AXEs.

Functional metagenomic screening identifies an unexpected ß-glucuronidase.

The abundance of recorded protein sequence data stands in contrast to the small number of experimentally verified functional annotation. Here we screened a million-membered metagenomic library at ultrahigh throughput in microfluidic droplets for ß-glucuronidase activity. We identified SN243, a genuine ß-glucuronidase with little homology to previously studied enzymes of this type, as a glycoside hydrolase 3 family member. This glycoside hydrolase family contains only one recently added ß-glucuronidase, showing that a functional metagenomic approach can shed light on assignments that are currently 'unpredictable' by bioinformatics. Kinetic analyses of SN243 characterized it as a promiscuous catalyst and structural analysis suggests regions of divergence from homologous glycoside hydrolase 3 members creating a wide-open active site. With a screening throughput of >107 library members per day, picolitre-volume microfluidic droplets enable functional assignments that complement current enzyme database dictionaries and provide bridgeheads for the annotation of unexplored sequence space.

Biogeographic implication of temperature-induced plant cell wall lignification.

More than 200 years after von Humboldt's pioneering work on the treeline, our understanding of the cold distribution limit of upright plant growth is still incomplete. Here, we use wood anatomical techniques to estimate the degree of stem cell wall lignification in 1770 plant species from six continents. Contrary to the frequent belief that small plants are less lignified, we show that cell wall lignification in 'woody' herbs varies considerably. Although trees and shrubs always exhibit lignified cell walls in their upright stems, small plants above the treeline may contain less lignin. Our findings suggest that extremely cold growing season temperatures can reduce the ability of plants to lignify their secondary cell walls. Corroborating experimental and observational evidence, this study proposes to revisit existing theories about the thermal distribution limit of upright plant growth and to consider biochemical and biomechanical factors for explaining the global treeline position.

Golgi-localized putative S-adenosyl methionine transporters required for plant cell wall polysaccharide methylation.

Polysaccharide methylation, especially that of pectin, is a common and important feature of land plant cell walls. Polysaccharide methylation takes place in the Golgi apparatus and therefore relies on the import of S-adenosyl methionine (SAM) from the cytosol into the Golgi. However, so far, no Golgi SAM transporter has been identified in plants. Here we studied major facilitator superfamily members in Arabidopsis that we identified as putative Golgi SAM transporters (GoSAMTs). Knockout of the two most highly expressed GoSAMTs led to a strong reduction in Golgi-synthesized polysaccharide methylation. Furthermore, solid-state NMR experiments revealed that reduced methylation changed cell wall polysaccharide conformations, interactions and mobilities. Notably, NMR revealed the existence of pectin 'egg-box' structures in intact cell walls and showed that their formation is enhanced by reduced methyl esterification. These changes in wall architecture were linked to substantial growth and developmental phenotypes. In particular, anisotropic growth was strongly impaired in the double mutant. The identification of putative transporters involved in import of SAM into the Golgi lumen in plants provides new insights into the paramount importance of polysaccharide methylation for plant cell wall structure and function.

Hydroxycinnamic acid-modified xylan side chains and their cross-linking products in rice cell walls are reduced in the Xylosyl arabinosyl substitution of xylan 1 mutant.

The intricate architecture of cell walls and the complex cross-linking of their components hinders some industrial and agricultural applications of plant biomass. Xylan is a key structural element of grass cell walls, closely interacting with other cell wall components such as cellulose and lignin. The main branching points of grass xylan, 3-linked l-arabinosyl substitutions, can be modified by ferulic acid (a hydroxycinnamic acid), which cross-links xylan to other xylan chains and lignin. XAX1 (Xylosyl arabinosyl substitution of xylan 1), a rice (Oryza sativa) member of the glycosyltransferase family GT61, has been described to add xylosyl residues to arabinosyl substitutions modified by ferulic acid. In this study, we characterize hydroxycinnamic acid-decorated arabinosyl substitutions present on rice xylan and their cross-linking, in order to decipher the role of XAX1 in xylan synthesis. Our results show a general reduction of hydroxycinnamic acid-modified 3-linked arabinosyl substitutions in xax1 mutant rice regardless of their modification with a xylosyl residue. Moreover, structures resembling the direct cross-link between xylan and lignin (ferulated arabinosyl substitutions bound to lignin monomers and dimers), together with diferulates known to cross-link xylan, are strongly reduced in xax1. Interestingly, apart from feruloyl and p-coumaroyl modifications on arabinose, putative caffeoyl and oxalyl modifications were characterized, which were also reduced in xax1. Our results suggest an alternative function of XAX1 in the transfer of hydroxycinnamic acid-modified arabinosyl substitutions to xylan, rather than xylosyl transfer to arabinosyl substitutions. Ultimately, XAX1 plays a fundamental role in cross-linking, providing a potential target for the improvement of use of grass biomass.

ß-1,4-Xylan backbone synthesis in higher plants: How complex can it be?

Xylan is a hemicellulose present in the cell walls of all land plants. Glycosyltransferases of the GT43 (IRX9/IRX9L and IRX14/IRX14L) and GT47 (IRX10/IRX10L) families are involved in the biosynthesis of its ß-1,4-linked xylose backbone, which can be further modified by acetylation and sugar side chains. However, it remains unclear how the different enzymes work together to synthesize the xylan backbone. A xylan synthesis complex (XSC) has been described in the monocots wheat and asparagus, and co-expression of asparagus AoIRX9, AoIRX10 and AoIRX14A is required to form a catalytically active complex for secondary cell wall xylan biosynthesis. Here, we argue that an equivalent XSC exists for the synthesis of the primary cell wall of the eudicot Arabidopsis thaliana, consisting of IRX9L, IRX10L and IRX14. This would suggest the existence of distinct XSCs for primary and secondary cell wall xylan synthesis, reminiscent of the distinct cellulose synthesis complexes (CSCs) of the primary and secondary cell wall. In contrast to the CSC, in which each CESA protein has catalytic activity, the XSC seems to contain proteins with non-catalytic function with each component bearing potentially unique but crucial roles. Moreover, the core XSC formed by a combination of IRX9/IRX9L, IRX10/IRX10L and IRX14/IRX14L might not be stable in its composition during transit from the endoplasmic reticulum to the Golgi apparatus. Instead, potential dynamic changes of the XSC might be a means of regulating xylan biosynthesis to facilitate coordinated deposition of tailored polysaccharides in the plant cell wall.

Characterisation of the enzyme transport path between shipworms and their bacterial symbionts.

BACKGROUND: Shipworms are marine xylophagus bivalve molluscs, which can live on a diet solely of wood due to their ability to produce plant cell wall-degrading enzymes. Bacterial carbohydrate-active enzymes (CAZymes), synthesised by endosymbionts living in specialised shipworm cells called bacteriocytes and located in the animal's gills, play an important role in wood digestion in shipworms. However, the main site of lignocellulose digestion within these wood-boring molluscs, which contains both endogenous lignocellulolytic enzymes and prokaryotic enzymes, is the caecum, and the mechanism by which bacterial enzymes reach the distant caecum lumen has remained so far mysterious. Here, we provide a characterisation of the path through which bacterial CAZymes produced in the gills of the shipworm Lyrodus pedicellatus reach the distant caecum to contribute to the digestion of wood. RESULTS: Through a combination of transcriptomics, proteomics, X-ray microtomography, electron microscopy studies and in vitro biochemical characterisation, we show that wood-digesting enzymes produced by symbiotic bacteria are localised not only in the gills, but also in the lumen of the food groove, a stream of mucus secreted by gill cells that carries food particles trapped by filter feeding to the mouth. Bacterial CAZymes are also present in the crystalline style and in the caecum of their shipworm host, suggesting a unique pathway by which enzymes involved in a symbiotic interaction are transported to their site of action. Finally, we characterise in vitro four new bacterial glycosyl hydrolases and a lytic polysaccharide monooxygenase identified in our transcriptomic and proteomic analyses as some of the major bacterial enzymes involved in this unusual biological system. CONCLUSION: Based on our data, we propose that bacteria and their enzymes are transported from the gills along the food groove to the shipworm's mouth and digestive tract, where they aid in wood digestion.