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.

Importance of Water in Maintaining Softwood Secondary Cell Wall Nanostructure.

Water is one of the principal constituents by mass of living plant cell walls. However, its role and interactions with secondary cell wall polysaccharides and the impact of dehydration and subsequent rehydration on the molecular architecture are still to be elucidated. This work combines multidimensional solid-state 13C magic-angle-spinning (MAS) nuclear magnetic resonance (NMR) with molecular dynamics modeling to decipher the role of water in the molecular architecture of softwood secondary cell walls. The proximities between all main polymers, their molecular conformations, and interaction energies are compared in never-dried, oven-dried, and rehydrated states. Water is shown to play a critical role at the hemicellulose-cellulose interface. After significant molecular shrinkage caused by dehydration, the original molecular conformation is not fully recovered after rehydration. The changes include xylan becoming more closely and irreversibly associated with cellulose and some mannan becoming more mobile and changing conformation. These irreversible nanostructural changes provide a basis for explaining and improving the properties of wood-based materials.

Bio-Based and Robust Polydopamine Coated Nanocellulose/Amyloid Composite Aerogel for Fast and Wide-Spectrum Water Purification.

Water contamination resulting from human activities leads to the deterioration of aquatic ecosystems. This restrains the access to fresh water, which is the leading cause of mortality worldwide. In this work, we developed a bio-based and water-resistant composite aerogel from renewable nanofibrils for water remediation application. The composite aerogel consists of two types of cross-linked nanofibrils. Poly(dopamine)-coated cellulose nanofibrils and amyloid protein nanofibrils are forming a double networked crosslinked via periodate oxidation. The resulting aerogel exhibits good mechanical strength and high pollutants adsorption capability. Removal of dyes (rhodamine blue, acriflavine, crystal violet, malachite green, acid fuchsin and methyl orange), organic traces (atrazine, bisphenol A, and ibuprofen) and heavy metal ions (Pb(II) and Cu(II)) from water was successfully demonstrated with the composite aerogel. More specifically, the bio-based aerogel demonstrated good adsorption efficiencies for crystal violet (93.1% in 30 min), bisphenol A (91.7% in 5 min) and Pb(II) ions (94.7% in 5 min), respectively. Furthermore, the adsorption-desorption performance of aerogel for Pb(II) ions demonstrates that the aerogel has a high reusability as maintains satisfactory removal performances. The results suggest that this type of robust and bio-based composite aerogel is a promising adsorbent to decontaminate water from a wide range of pollutants in a sustainable and efficient way.

Nanoscale Wetting of Crystalline Cellulose.

Cellulose possesses considerable potential for a wide range of sustainable applications. Nanocellulose-based material properties are primarily dependent on the structural surface characteristics of its crystalline planes. Experimental measurements of the affinity of crystalline nanocellulose surfaces with water are scarce and challenging to obtain. Therefore, the relative hydrophilicity of different cellulose allomorphs crystalline planes is often inferred from qualitative assessments of their surface and the exposition of polar groups to the solvent. This work investigates the relative hydrophilicity of cellulose surfaces using molecular dynamics simulations. The behavior of a water droplet laid on different crystal planes was used to determine their relative hydrophilicity. The water molecules fully spread onto highly hydrophilic surfaces. However, a water droplet placed on less hydrophilic surfaces equilibrates as an oblate spheroidal cap allowing the measurement of a contact angle. The results indicate that the Iα (010), Iα (11̅0), Iß (010), and Iß (110) faces, as well as the faces of human-made celluloses II and III_I (100), (11̅0), (010), and (110) are all highly hydrophilic. They all have a contact angle value inferior to 11°. Not unexpectedly, the Iα (001) and Iß (100) surfaces are less hydrophilic with contact angles of 48 and 34°, respectively. However, the Iß (11̅0) plane, often referred to as a hydrophilic surface, forms a contact angle of about 32°. The results are rationalized in terms of structure, exposure of hydroxyl groups to the solvent, and degree of cellulose-cellulose versus cellulose-water hydrogen bonds on each face. The simulations also show that the surface oxidation degree tunes the surface hydrophilicity in a nonlinear manner due to cooperative effects involving water-cellulose interactions. Our study helps us to understand how the degree of hydrophilicity of cellulose emerges from specific structural features of each crystalline surface.

Genome editing with CRISPR/Cas9 in Pinus radiata (D. Don).

BACKGROUND: To meet increasing demand for forest-based products and protect natural forests from further deforestation requires increased productivity from planted forests. Genetic improvement of conifers by traditional breeding is time consuming due to the long juvenile phase and genome complexity. Genetic modification (GM) offers the opportunity to make transformational changes in shorter time frames but is challenged by current genetically modified organism (GMO) regulations. Genome editing, which can be used to generate site-specific mutations, offers the opportunity to rapidly implement targeted improvements and is globally regulated in a less restrictive way than GM technologies. RESULTS: We have demonstrated CRISPR/Cas9 genome editing in P. radiata targeting a single-copy cell wall gene GUX1 in somatic embryogenic tissue and produced plantlets from the edited tissue. We generated biallelic INDELs with an efficiency of 15 % using a single gRNA. 12 % of the transgenic embryogenic tissue was edited when two gRNAs were used and deletions of up to 1.3 kb were identified. However, the regenerated plants did not contain large deletions but had single nucleotide insertions at one of the target sites. We assessed the use of CRISPR/Cas9 ribonucleoproteins (RNPs) for their ability to accomplish DNA-free genome editing in P. radiata. We chose a hybrid approach, with RNPs co-delivered with a plasmid-based selectable marker. A two-gRNA strategy was used which produced an editing efficiency of 33 %, and generated INDELs, including large deletions. Using the RNP approach, deletions found in embryogenic tissue were also present in the plantlets. But, all plants produced using the RNP strategy were monoallelic. CONCLUSIONS: We have demonstrated the generation of biallelic and monoallelic INDELs in the coniferous tree P. radiata with the CRISPR/Cas9 system using plasmid expressed Cas9 gRNA and RNPs respectively. This opens the opportunity to apply genome editing in conifers to rapidly modify key traits of interest.

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.

Luminescent and Hydrophobic Wood Films as Optical Lighting Materials.

Most materials used for optical lighting applications need to produce a uniform illumination and require high mechanical and hydrophobic properties. However, they are rarely eco-friendly. Herein, a bio-based, polymer matrix-free, luminescent, and hydrophobic film with excellent mechanical properties for optical lighting purposes is demonstrated. A template is prepared by turning a wood veneer into porous scaffold from which most of the lignin and half of the hemicelluloses are removed. The infiltration of quantum dots (CdSe/ZnS) into the porous template prior to densification resulted in almost uniform luminescence (isotropic light scattering) and could be extended to various quantum dot particles, generating different light colors. In a subsequent step, the luminescent wood film is coated with hexadecyltrimethoxysilane (HDTMS) via chemical vapor deposition. The presence of the quantum dots coupled with the HDTMS coating renders the film hydrophobic (water contact angle ≈ 140°). This top-down process strongly eliminates lumen cavities and preserves the orientation of the original cellulose fibrils to create luminescent and polymer matrix-free films with high modulus and strength in the direction of fibers. The proposed optical lighting material could be attractive for interior designs (e.g., lamps and laminated cover panels), photonics, and laser devices.

Ligno-Cellulosic Fibre Sized with Nucleating Agents Promoting Transcrystallinity in Isotactic Polypropylene Composites.

The mechanical performance of composites made from isotactic polypropylene reinforced with natural fibres depends on the interface between fibre and matrix, as well as matrix crystallinity. Sizing the fibre surface with nucleating agents to promote transcrystallinity is a potential route to improve the mechanical properties. The sizing of thermo-mechanical pulp and regenerated cellulose (Tencel™) fibres with α- and ß-nucleating agents, to improve tensile strength and impact strength respectively, was assessed in this study. Polarised microscopy, electron microscopy and differential scanning calorimetry (DSC) showed that transcrystallinity was achieved and that the bulk crystallinity of the matrix was affected during processing (compounding and injection moulding). However, despite substantial changes in crystal structure in the final composite, the sizing method used did not lead to significant changes regarding the overall composite mechanical performance.

Wood-Based Flexible Electronics.

Next-generation electronics (e.g., substrate and conductor) need to be high performance, multifunctional, and environmentally friendly. Here, we report the creation of a fully wood-based flexible electronics circuit meeting these requirements, where the substrate, a strong, flexible and transparent wood film, is printed with a lignin-derived carbon nanofibers conductive ink. The wood film fabrication involves extensive removal of lignin and hemicellulose to tailor the nanostructure of the material followed by collapsing of the cell walls. This process preserves the original alignment of the cellulose nanofibers and promotes their binding. The film is flexible, yet strong in fiber direction with a Young's modulus and a tensile strength of 49.9 GPa and 469.9 MPa, respectively. Furthermore, a sustainable and bio-based conductive ink is formulated with lignin-derived carbon nanofibers. The bio-based ink is printed on transparent wood film, and a strain sensor application of the printed circuit is demonstrated. Combining the transparent wood film with the conductive ink produces environmental friendly and sustainable wood-based electronics for potential applications such as flexible circuits and sensors. Moreover, we envision the potential for a scalable and continuous fabrication process as well as end-of-life recyclability.

Two members of the DUF579 family are responsible for arabinogalactan methylation in Arabidopsis.

All members of the DUF579 family characterized so far have been described to affect the integrity of the hemicellulosic cell wall component xylan: GXMs are glucuronoxylan methyltransferases catalyzing 4-O-methylation of glucuronic acid on xylan; IRX15 and IRX15L, although their enzymatic activity is unknown, are required for xylan biosynthesis and/or xylan deposition. Here we show that the DUF579 family members, AGM1 and AGM2, are required for 4-O-methylation of glucuronic acid of a different plant cell wall component, the highly glycosylated arabinogalactan proteins (AGPs).

Development of an oligosaccharide library to characterise the structural variation in glucuronoarabinoxylan in the cell walls of vegetative tissues in grasses.

BACKGROUND: Grass glucuronoarabinoxylan (GAX) substitutions can inhibit enzymatic degradation and are involved in the interaction of xylan with cell wall cellulose and lignin, factors which contribute to the recalcitrance of biomass to saccharification. Therefore, identification of xylan characteristics central to biomass biorefining improvement is essential. However, the task of assessing biomass quality is complicated and is often hindered by the lack of a reference for a given crop. RESULTS: In this study, we created a reference library, expressed in glucose units, of Miscanthus sinensis GAX stem and leaf oligosaccharides, using DNA sequencer-Assisted Saccharide analysis in high throughput (DASH), supported by liquid chromatography (LC), nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). Our analysis of a number of grass species highlighted variations in substitution type and frequency of stem and leaf GAX. In miscanthus, for example, the ß-Xylp-(1 → 2)-α-Araf-(1 → 3) side chain is more abundant in leaf than stem. CONCLUSIONS: The reference library allows fast identification and comparison of GAX structures from different plants and tissues. Ultimately, this reference library can be used in directing biomass selection and improving biorefining.

Golgi-localized STELLO proteins regulate the assembly and trafficking of cellulose synthase complexes in Arabidopsis.

As the most abundant biopolymer on Earth, cellulose is a key structural component of the plant cell wall. Cellulose is produced at the plasma membrane by cellulose synthase (CesA) complexes (CSCs), which are assembled in the endomembrane system and trafficked to the plasma membrane. While several proteins that affect CesA activity have been identified, components that regulate CSC assembly and trafficking remain unknown. Here we show that STELLO1 and 2 are Golgi-localized proteins that can interact with CesAs and control cellulose quantity. In the absence of STELLO function, the spatial distribution within the Golgi, secretion and activity of the CSCs are impaired indicating a central role of the STELLO proteins in CSC assembly. Point mutations in the predicted catalytic domains of the STELLO proteins indicate that they are glycosyltransferases facing the Golgi lumen. Hence, we have uncovered proteins that regulate CSC assembly in the plant Golgi apparatus.

Plant Fibre: Molecular Structure and Biomechanical Properties, of a Complex Living Material, Influencing Its Deconstruction towards a Biobased Composite.

Plant cell walls form an organic complex composite material that fulfils various functions. The hierarchical structure of this material is generated from the integration of its elementary components. This review provides an overview of wood as a composite material followed by its deconstruction into fibres that can then be incorporated into biobased composites. Firstly, the fibres are defined, and their various origins are discussed. Then, the organisation of cell walls and their components are described. The emphasis is on the molecular interactions of the cellulose microfibrils, lignin and hemicelluloses in planta. Hemicelluloses of diverse species and cell walls are described. Details of their organisation in the primary cell wall are provided, as understanding of the role of hemicellulose has recently evolved and is likely to affect our perception and future study of their secondary cell wall homologs. The importance of the presence of water on wood mechanical properties is also discussed. These sections provide the basis for understanding the molecular arrangements and interactions of the components and how they influence changes in fibre properties once isolated. A range of pulping processes can be used to individualise wood fibres, but these can cause damage to the fibres. Therefore, issues relating to fibre production are discussed along with the dispersion of wood fibres during extrusion. The final section explores various ways to improve fibres obtained from wood.

An unusual xylan in Arabidopsis primary cell walls is synthesised by GUX3, IRX9L, IRX10L and IRX14.

Xylan is a crucial component of many plant primary and secondary cell walls. However, the structure and function of xylan in the dicotyledon primary cell wall is not well understood. Here, we characterized a xylan that is specific to tissues enriched in Arabidopsis primary cell walls. Unlike previously described xylans, this xylan carries a pentose linked 1-2 to the α-1,2-d-glucuronic acid (GlcA) side chains on the ß-1,4-Xyl backbone. The frequent and precisely regular spacing of GlcA substitutions every six xylosyl residues along the backbone is also unlike that previously observed in secondary cell wall xylan. Molecular genetics, in vitro assays, and expression data suggest that IRX9L, IRX10L and IRX14 are required for xylan backbone synthesis in primary cell wall synthesising tissues. IRX9 and IRX10 are not involved in the primary cell wall xylan synthesis but are functionally exchangeable with IRX9L and IRX10L. GUX3 is the only glucuronyltransferase required for the addition of the GlcA decorations on the xylan. The differences in xylan structure in primary versus secondary cell walls might reflect the different roles in cross-linking and interaction with other cell wall components.

Fluorescence recovery after photobleaching reveals high cycling dynamics of plasma membrane aquaporins in Arabidopsis roots under salt stress.

The constitutive cycling of plant plasma membrane (PM) proteins is an essential component of their function and regulation under resting or stress conditions. Transgenic Arabidopsis plants that express GFP fusions with AtPIP1;2 and AtPIP2;1, two prototypic PM aquaporins, were used to develop a fluorescence recovery after photobleaching (FRAP) approach. This technique was used to discriminate between PM and endosomal pools of the aquaporin constructs, and to estimate their cycling between intracellular compartments and the cell surface. The membrane trafficking inhibitors tyrphostin A23, naphthalene-1-acetic acid and brefeldin A blocked the latter process. By contrast, a salt treatment (100 mm NaCl for 30 min) markedly enhanced the cycling of the aquaporin constructs and modified their pharmacological inhibition profile. Two distinct models for PM aquaporin cycling in resting or salt-stressed root cells are discussed.

An Exo2 derivative affects ER and Golgi morphology and vacuolar sorting in a tissue-specific manner in arabidopsis.

We screened a panel of compounds derived from Exo2 - a drug that perturbs post-Golgi compartments and trafficking in mammalian cells - for their effect on the secretory pathway in Arabidopsis root epidermal cells. While Exo2 and most related compounds had no significant effect, one Exo2 derivative, named LG8, induced severe morphological alterations in both the Golgi (at high concentrations) and the endoplasmic reticulum (ER). LG8 causes the ER to form foci of interconnecting tubules, which at the ultrastructural level appear similar to those previously reported in Arabidopsis roots after treatment with the herbicide oryzalin. In cotyledonary leaves, LG8 causes redistribution of a trans Golgi network (TGN) marker to the vacuole. LG8 affects the anterograde secretory pathway by inducing secretion of vacuolar cargo and preventing the brassinosteroid receptor BRI1 from reaching the plasma membrane. Uptake and arrival at the TGN of the endocytic marker FM4-64 is not affected. Unlike the ADP ribosylation factor-GTP exchange factor (ARF-GEF) inhibitor brefeldin A (BFA), LG8 affects these post-Golgi events without causing the formation of BFA bodies. Up to concentrations of 50 µm, the effects of LG8 are reversible.

Mechanisms and effects of retention of over-expressed aquaporin AtPIP2;1 in the endoplasmic reticulum.

Plasma membrane intrinsic proteins (PIPs) are aquaporins that mediate water transport across the plant plasma membrane (PM). The present work addresses, using Arabidopsis AtPIP2;1 as a model, the mechanisms and significance of trafficking of newly synthesized PIPs from the endoplasmic reticulum (ER) to the Golgi apparatus. A functional diacidic export motif (Asp4-Val5-Glu6) was identified in the N-terminal tail of AtPIP2;1, using expression in transgenic Arabidopsis of site-directed mutants tagged with the green fluorescent protein (GFP). Confocal fluorescence imaging and a novel fluorescence recovery after photobleaching application based on the distinct diffusion of PM and intracellular AtPIP2;1-GFP forms revealed a retention in the ER of diacidic mutated forms, but with quantitative differences. Thus, the individual role of the two acidic Asp4 and Glu6 residues was established. In addition, expression in transgenic Arabidopsis of ER-retained AtPIP2;1-GFP constructs reduced the root hydraulic conductivity. Co-expression of AtPIP2;1-GFP and AtPIP1;4-mCherry constructs suggested that ER-retained AtPIP2;1-GFP may interact with other PIPs to hamper their trafficking to the PM, thereby contributing to inhibition of root cell hydraulic conductivity.

Mapping of tonoplast intrinsic proteins in maturing and germinating Arabidopsis seeds reveals dual localization of embryonic TIPs to the tonoplast and plasma membrane.

We have mapped the expression of the tonoplast intrinsic protein (TIP) gene family members in Arabidopsis seeds by fluorescent protein tagging of their genomic sequences and confocal microscopy. Three isoforms (TIP1;1, TIP2;1, and TIP2;2) have distinct patterns of expression in maternal tissues (outer integument and placento-chalazal region). Two isoforms, TIP3;1 and the previously uncharacterized TIP3;2, are the only detectable TIPs in embryos during seed maturation and the early stages of seed germination. Throughout these developmental stages, both isoforms co-locate to the tonoplast of the protein storage vacuoles, but also appear to label the plasma membrane. Plasma membrane labeling is specific to TIP3;1 and TIP3;2, is independent of the position of the fluorescent protein tag, and appears to be specific to early seed maturation and early germination stages. We discuss these results in the context of the predicted distribution of aquaporins in Arabidopsis seeds.

Tonoplast intrinsic proteins and vacuolar identity.

TIPs (tonoplast intrinsic proteins) have been traditionally used as markers for vacuolar identity in a variety of plant species and tissues. In the present article, we review recent attempts to compile a detailed map of TIP expression in Arabidopsis, in order to understand vacuolar identity and distribution in this model species. We discuss the general applicability of these findings. We also review the issue of the intracellular targeting of TIPs and propose key emerging questions relative to the cell biology of this protein family.

In vivo imaging of the tonoplast intrinsic protein family in Arabidopsis roots.

BACKGROUND: Tonoplast intrinsic proteins (TIPs) are widely used as markers for vacuolar compartments in higher plants. Ten TIP isoforms are encoded by the Arabidopsis genome. For several isoforms, the tissue and cell specific pattern of expression are not known. RESULTS: We generated fluorescent protein fusions to the genomic sequences of all members of the Arabidopsis TIP family whose expression is predicted to occur in root tissues (TIP1;1 and 1;2; TIP2;1, 2;2 and 2;3; TIP4;1) and expressed these fusions, both individually and in selected pairwise combinations, in transgenic Arabidopsis. Analysis by confocal microscopy revealed that TIP distribution varied between different cell layers within the root axis, with extensive co-expression of some TIPs and more restricted expression patterns for other isoforms. TIP isoforms whose expression overlapped appeared to localise to the tonoplast of the central vacuole, vacuolar bulbs and smaller, uncharacterised structures. CONCLUSION: We have produced a comprehensive atlas of TIP expression in Arabidopsis roots, which reveals novel expression patterns for not previously studied TIPs.