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.

Structural differences of cell walls in earlywood and latewood of Pinus sylvestris and their contribution to biomass recalcitrance.

Scots pine (Pinus sylvestris L.) is an evergreen coniferous tree with wide distribution and good growth performance in a range of habitats. Therefore, wood from P. sylvestris is produced in many managed forests and is frequently used in industry. Despite the importance of pine wood, we still do not fully understand its molecular structure what limits improvements in its processing. One of the basic features leading to variation in wood properties is the presence of earlywood and latewood which form annual growth rings. Here, we characterise biochemical traits that differentiate cell walls of earlywood and latewood in Scots pine. We discover that latewood is less recalcitrant to enzymatic digestion, with galactoglucomannan showing particularly pronounced difference in accessibility. Interestingly, characterisation of lignin reveals a higher proportion of coniferaldehydes in pine latewood and suggests the presence of a different linkage landscape in this wood type. With complementary analysis of wood polysaccharides this enabled us to propose the first detailed molecular model of earlywood and latewood and to conclude that the variation in lignin structure is likely the main determinant of differences in recalcitrance observed between the two wood types in pine. Our discoveries lay the foundation for improvements in industrial processes that use pine wood since we show clear pathways for increasing the efficiency of enzymatic processing of this renewable material. Our work will help guide future breeding of pine trees with desired timber properties and can help link molecular structure of softwood cell walls to function of the different types of xylem in conifers.

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.

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.

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.

Upregulation of GLRs expression by light in Arabidopsis leaves.

BACKGROUND: Glutamate receptor-like (GLR) channels are plant homologs of iGluRs, animal ionotropic glutamate receptors which participate in neurotransmission. GLRs mediate plant adaptive processes and photomorphogenesis. Despite their contribution to light-dependent processes, signaling mechanisms that modulate GLR response to light remain unknown. Here we show that leaf expression of 7 out of 20 Arabidopsis GLRs is significantly up-regulated by monochromatic irradiation. RESULTS: Our data indicates that both red and blue light stimulate the expression of selected AtGLRs. Using a photosynthesis inhibitor and different irradiation regimes, we demonstrated that retrograde signaling from photosystem II is unlikely to be involved in light-dependent GLR up-regulation. Analysis of transcriptional patterns in mutants of key photoreceptors allowed us to observe that both phytochromes and cryptochromes are likely to be involved in the control of light-dependent up-regulation of AtGLR expression, with phytochromes playing a clearly dominating role in this process. CONCLUSIONS: In mature Arabidopsis leaves, phytochromes, assisted by cryptochromes, mediate light-driven transcriptional up-regulation of several genes encoding GLR proteins. Since GLRs are known to be involved in a wide range of plant developmental processes our results provide mechanistic insight into how light may influence plant growth and development.

Transformation of European Ash (Fraxinus excelsior L.) Callus as a Starting Point for Understanding the Molecular Basis of Ash Dieback.

The population of European ash (Fraxinus excelsior L.) is currently facing the risk of collapse, mainly due to ash dieback, a disease caused by a pathogenic fungus, Hymenoscyphus fraxineus. To facilitate studies into the molecular basis of ash dieback and design breeding strategies for a generation of resistant trees, it is necessary to develop tools enabling the study of gene function in F. excelsior. Despite this, a method for the genetic engineering of F. excelsior is still missing. Here, we report the first successful genetic transformation of F. excelsior callus and a selection process enabling the formation of stable transgenic callus lines. The protocol relies on the use of Agrobacterium tumefaciens to transform callus tissue derived from embryos of F. excelsior. In our experiments, we used the ß-glucuronidase (GUS) reporter system to demonstrate the transformation of callus cells and performed RT-PCR experiments to confirm the stable expression of the transgene. Since ash dieback threatens the long-term stability of many native F. excelsior populations, we hope that the transformation techniques described in this manuscript will facilitate rapid progress in uncovering the molecular basis of the disease and the validation of gene targets previously proposed to be linked to the resistance of trees to H. fraxineus pathogenicity.

Two conifer GUX clades are responsible for distinct glucuronic acid patterns on xylan.

Wood of coniferous trees (softwood), is a globally significant carbon sink and an important source of biomass. Despite that, little is known about the genetic basis of softwood cell wall biosynthesis. Branching of xylan, one of the main hemicelluloses in softwood secondary cell walls, with glucuronic acid (GlcA) is critical for biomass recalcitrance. Here, we investigate the decoration patterns of xylan by conifer GlucUronic acid substitution of Xylan (GUX) enzymes. Through molecular phylogenetics we identify two distinct conifer GUX clades. Using transcriptional profiling we show that the genes are preferentially expressed in secondary cell wall forming tissues. With in vitro and in planta assays we demonstrate that conifer GUX enzymes from both clades are active glucuronyltransferases. Conifer GUX enzymes from each clade have different specific activities. While members of clade one add evenly spaced GlcA branches, the members of clade two are also capable of glucuronidating two consecutive xyloses. Importantly, these types of xylan patterning are present in softwood. As xylan patterning might modulate xylan-cellulose and xylan-lignin interactions, our results further the understanding of softwood cell wall biosynthesis and provide breeding or genetic engineering targets that can be used to modify softwood properties.

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.

Structural Imaging of Native Cryo-Preserved Secondary Cell Walls Reveals the Presence of Macrofibrils and Their Formation Requires Normal Cellulose, Lignin and Xylan Biosynthesis.

The woody secondary cell walls of plants are the largest repository of renewable carbon biopolymers on the planet. These walls are made principally from cellulose and hemicelluloses and are impregnated with lignin. Despite their importance as the main load bearing structure for plant growth, as well as their industrial importance as both a material and energy source, the precise arrangement of these constituents within the cell wall is not yet fully understood. We have adapted low temperature scanning electron microscopy (cryo-SEM) for imaging the nanoscale architecture of angiosperm and gymnosperm cell walls in their native hydrated state. Our work confirms that cell wall macrofibrils, cylindrical structures with a diameter exceeding 10 nm, are a common feature of the native hardwood and softwood samples. We have observed these same structures in Arabidopsis thaliana secondary cell walls, enabling macrofibrils to be compared between mutant lines that are perturbed in cellulose, hemicellulose, and lignin formation. Our analysis indicates that the macrofibrils in Arabidopsis cell walls are dependent upon the proper biosynthesis, or composed, of cellulose, xylan, and lignin. This study establishes that cryo-SEM is a useful additional approach for investigating the native nanoscale architecture and composition of hardwood and softwood secondary cell walls and demonstrates the applicability of Arabidopsis genetic resources to relate fibril structure with wall composition and biosynthesis.

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.

An engineered GH1 ß-glucosidase displays enhanced glucose tolerance and increased sugar release from lignocellulosic materials.

ß-glucosidases play a critical role among the enzymes in enzymatic cocktails designed for plant biomass deconstruction. By catalysing the breakdown of ß-1, 4-glycosidic linkages, ß-glucosidases produce free fermentable glucose and alleviate the inhibition of other cellulases by cellobiose during saccharification. Despite this benefit, most characterised fungal ß-glucosidases show weak activity at high glucose concentrations, limiting enzymatic hydrolysis of plant biomass in industrial settings. In this study, structural analyses combined with site-directed mutagenesis efficiently improved the functional properties of a GH1 ß-glucosidase highly expressed by Trichoderma harzianum (ThBgl) under biomass degradation conditions. The tailored enzyme displayed high glucose tolerance levels, confirming that glucose tolerance can be achieved by the substitution of two amino acids that act as gatekeepers, changing active-site accessibility and preventing product inhibition. Furthermore, the enhanced efficiency of the engineered enzyme in terms of the amount of glucose released and ethanol yield was confirmed by saccharification and simultaneous saccharification and fermentation experiments using a wide range of plant biomass feedstocks. Our results not only experimentally confirm the structural basis of glucose tolerance in GH1 ß-glucosidases but also demonstrate a strategy to improve technologies for bioethanol production based on enzymatic hydrolysis.

The Patterned Structure of Galactoglucomannan Suggests It May Bind to Cellulose in Seed Mucilage.

The interaction between mannan polysaccharides and cellulose microfibrils contributes to cell wall properties in some vascular plants, but the molecular arrangement of mannan in the cell wall and the nature of the molecular bonding between mannan and cellulose remain unknown. Previous studies have shown that mannan is important in maintaining Arabidopsis (Arabidopsis thaliana) seed mucilage architecture, and that Cellulose Synthase-Like A2 (CSLA2) synthesizes a glucomannan backbone, which Mannan α-Galactosyl Transferase1 (MAGT1/GlycosylTransferase-Like6/Mucilage Related10) might decorate with single α-Gal branches. Here, we investigated the ratio and sequence of Man and Glc and the arrangement of Gal residues in Arabidopsis mucilage mannan using enzyme sequential digestion, carbohydrate gel electrophoresis, and mass spectrometry. We found that seed mucilage galactoglucomannan has a backbone consisting of the repeating disaccharide [4)-ß-Glc-(1,4)-ß-Man-(1,], and most of the Man residues in the backbone are substituted by single α-1,6-Gal. CSLA2 is responsible for the synthesis of this patterned glucomannan backbone and MAGT1 catalyses the addition of α-Gal. In vitro activity assays revealed that MAGT1 transferred α-Gal from UDP-Gal only to Man residues within the CSLA2 patterned glucomannan backbone acceptor. These results indicate that CSLAs and galactosyltransferases are able to make precisely defined galactoglucomannan structures. Molecular dynamics simulations suggested this patterned galactoglucomannan is able to bind stably to some hydrophilic faces and to hydrophobic faces of cellulose microfibrils. A specialization of the biosynthetic machinery to make galactoglucomannan with a patterned structure may therefore regulate the mode of binding of this hemicellulose to cellulose fibrils.

An even pattern of xylan substitution is critical for interaction with cellulose in plant cell walls.

Xylan and cellulose are abundant polysaccharides in vascular plants and essential for secondary cell wall strength. Acetate or glucuronic acid decorations are exclusively found on even-numbered residues in most of the glucuronoxylan polymer. It has been proposed that this even-specific positioning of the decorations might permit docking of xylan onto the hydrophilic face of a cellulose microfibril 1-3 . Consequently, xylan adopts a flattened ribbon-like twofold screw conformation when bound to cellulose in the cell wall 4 . Here we show that ESKIMO1/XOAT1/TBL29, a xylan-specific O-acetyltransferase, is necessary for generation of the even pattern of acetyl esters on xylan in Arabidopsis. The reduced acetylation in the esk1 mutant deregulates the position-specific activity of the xylan glucuronosyltransferase GUX1, and so the even pattern of glucuronic acid on the xylan is lost. Solid-state NMR of intact cell walls shows that, without the even-patterned xylan decorations, xylan does not interact normally with cellulose fibrils. We conclude that the even pattern of xylan substitutions seen across vascular plants enables the interaction of xylan with hydrophilic faces of cellulose fibrils, and is essential for development of normal plant secondary cell walls.

Removal of glucuronic acid from xylan is a strategy to improve the conversion of plant biomass to sugars for bioenergy.

BACKGROUND: Plant lignocellulosic biomass can be a source of fermentable sugars for the production of second generation biofuels and biochemicals. The recalcitrance of this plant material is one of the major obstacles in its conversion into sugars. Biomass is primarily composed of secondary cell walls, which is made of cellulose, hemicelluloses and lignin. Xylan, a hemicellulose, binds to the cellulose microfibril and is hypothesised to form an interface between lignin and cellulose. Both softwood and hardwood xylan carry glucuronic acid side branches. As xylan branching may be important for biomass recalcitrance and softwood is an abundant, non-food competing, source of biomass it is important to investigate how conifer xylan is synthesised. RESULTS: Here, we show using Arabidopsis gux mutant biomass that removal of glucuronosyl substitutions of xylan can allow 30% more glucose and over 700% more xylose to be released during saccharification. Ethanol yields obtained through enzymatic saccharification and fermentation of gux biomass were double those obtained for non-mutant material. Our analysis of additional xylan branching mutants demonstrates that absence of GlcA is unique in conferring the reduced recalcitrance phenotype. As in hardwoods, conifer xylan is branched with GlcA. We use transcriptomic analysis to identify conifer enzymes that might be responsible for addition of GlcA branches onto xylan in industrially important softwood. Using a combination of in vitro and in vivo activity assays, we demonstrate that a white spruce (Picea glauca) gene, PgGUX, encodes an active glucuronosyl transferase. Glucuronic acid introduced by PgGUX reduces the sugar release of Arabidopsis gux mutant biomass to wild-type levels indicating that it can fulfil the same biological function as native glucuronosylation. CONCLUSION: Removal of glucuronic acid from xylan results in the largest increase in release of fermentable sugars from Arabidopsis plants that grow to the wild-type size. Additionally, plant material used in this work did not undergo any chemical pretreatment, and thus increased monosaccharide release from gux biomass can be achieved without the use of environmentally hazardous chemical pretreatment procedures. Therefore, the identification of a gymnosperm enzyme, likely to be responsible for softwood xylan glucuronosylation, provides a mutagenesis target for genetically improved forestry trees.

Label-Free Analysis and Sorting of Microalgae and Cyanobacteria in Microdroplets by Intrinsic Chlorophyll Fluorescence for the Identification of Fast Growing Strains.

Microalgae and cyanobacteria are promising organisms for sustainable biofuel production, but several challenges remain to make this economically viable, including identification of optimized strains with high biomass productivity. Here we report on a novel methodology for the label-free screening and sorting of cyanobacteria and microalgae in a microdroplet platform. We show for the first time that chlorophyll fluorescence can be used to measure differences in biomass between populations of picoliter microdroplets containing different species of cyanobacteria, Synechocystis PCC 6803 and Synechococcus PCC 7002, which exhibit different growth dynamics in bulk culture. The potential and robustness of this label-free screening approach is further demonstrated by the screening and sorting of cells of the green alga Chlamydomonas reinhardtii encapsulated in droplets.

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.

Fusion of pyruvate decarboxylase and alcohol dehydrogenase increases ethanol production in Escherichia coli.

Ethanol is an important biofuel. Heterologous expression of Zymomonas mobilis pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (AdhB) increases ethanol production in Escherichia coli. A fusion of PDC and ADH was generated and expressed in E. coli. The fusion enzyme was demonstrated to possess both activities. AdhB activity was significantly lower when fused to PDC than when the two enzymes were expressed separately. However, cells expressing the fusion protein generated ethanol more rapidly and to higher levels than cells coexpressing Pdc and AdhB, suggesting a specific rate enhancement due to the fusion of the two enzymes.