The legacy of terrestrial plant evolution on cell wall fine structure.

The evolution of land flora was an epochal event in the history of planet Earth. The success of plants, and especially flowering plants, in colonizing all but the most hostile environments required multiple mechanisms of adaptation. The mainly polysaccharide-based cell walls of flowering plants, which are indispensable for water transport and structural support, are one of the most important adaptations to life on land. Thus, development of vasculature is regarded as a seminal event in cell wall evolution, but the impact of further refinements and diversification of cell wall compositions and architectures on radiation of flowering plant families is less well understood. We approached this from a glyco-profiling perspective and, using carbohydrate microarrays and monoclonal antibodies, studied the cell walls of 287 plant species selected to represent important evolutionary dichotomies and adaptation to a variety of habitats. The results support the conclusion that radiation of flowering plant families was indeed accompanied by changes in cell wall fine structure and that these changes can obscure earlier evolutionary events. Convergent cell wall adaptations identified by our analyses do not appear to be associated with plants with similar lifestyles but that are taxonomically distantly related. We conclude that cell wall structure is linked to phylogeny more strongly than to habitat or lifestyle and propose that there are many approaches of adaptation to any given ecological niche.

Ancient origin of fucosylated xyloglucan in charophycean green algae.

The charophycean green algae (CGA or basal streptophytes) are of particular evolutionary significance because their ancestors gave rise to land plants. One outstanding feature of these algae is that their cell walls exhibit remarkable similarities to those of land plants. Xyloglucan (XyG) is a major structural component of the cell walls of most land plants and was originally thought to be absent in CGA. This study presents evidence that XyG evolved in the CGA. This is based on a) the identification of orthologs of the genetic machinery to produce XyG, b) the identification of XyG in a range of CGA and, c) the structural elucidation of XyG, including uronic acid-containing XyG, in selected CGA. Most notably, XyG fucosylation, a feature considered as a late evolutionary elaboration of the basic XyG structure and orthologs to the corresponding biosynthetic enzymes are shown to be present in Mesotaenium caldariorum.

Plant Protein O-Arabinosylation.

A wide range of proteins with diverse functions in development, defense, and stress responses are O-arabinosylated at hydroxyprolines (Hyps) within distinct amino acid motifs of continuous stretches of Hyps, as found in the structural cell wall extensins, or at non-continuous Hyps as, for example, found in small peptide hormones and a variety of plasma membrane proteins involved in signaling. Plant O-glycosylation relies on hydroxylation of Prolines to Hyps in the protein backbone, mediated by prolyl-4-hydroxylase (P4H) which is followed by O-glycosylation of the Hyp C4-OH group by either galactosyltransferases (GalTs) or arabinofuranosyltranferases (ArafTs) yielding either Hyp-galactosylation or Hyp-arabinosylation. A subset of the P4H enzymes with putative preference to hydroxylation of continuous prolines and presumably all ArafT enzymes needed for synthesis of the substituted arabinose chains of one to four arabinose units, have been identified and functionally characterized. Truncated root-hair phenotype is one common denominator of mutants of Hyp formation and Hyp-arabinosylation glycogenes, which act on diverse groups of O-glycosylated proteins, e.g., the small peptide hormones and cell wall extensins. Dissection of different substrate derived effects may not be regularly feasible and thus complicate translation from genotype to phenotype. Recently, lack of proper arabinosylation on arabinosylated proteins has been shown to influence their transport/fate in the secretory pathway, hinting to an additional layer of functionality of O-arabinosylation. Here, we provide an update on the prevalence and types of O-arabinosylated proteins and the enzymatic machinery responsible for their modifications.

Analytical implications of different methods for preparing plant cell wall material.

Almost all plant cells are surrounded by a wall constructed of co-extensive networks of polysaccharides and proteoglycans. The capability to analyse cell wall components is essential for both understanding their complex biology and to fully exploit their numerous practical applications. Several biochemical and immunological techniques are used to analyse cell walls and in almost all cases the first step is the preparation of an alcohol insoluble residue (AIR). There is significant variation in the protocols used for AIR preparation, which can have a notable impact on the downstream extractability and detection of cell wall components. To explore these effects, we have formally compared ten AIR preparation methods and analysed polysaccharides subsequently extracted using high-performance anion exchange chromatography (HPAEC-PAD) and Micro Array Polymer Profiling (MAPP). Our results reveal the impact that AIR preparation has on downstream detection of cell wall components and the need for optimisation and consistency when preparing AIR.

Amylose/cellulose nanofiber composites for all-natural, fully biodegradable and flexible bioplastics.

Thermoplastic, polysaccharide-based plastics are environmentally friendly. However, typical shortcomings include lack of water resistance and poor mechanical properties. Nanocomposite manufacturing using pure, highly linear, polysaccharides can overcome such limitations. Cast nanocomposites were fabricated with plant engineered pure amylose (AM), produced in bulk quantity in transgenic barley grain, and cellulose nanofibers (CNF), extracted from agrowaste sugar beet pulp. Morphology, crystallinity, chemical heterogeneity, mechanics, dynamic mechanical, gas and water permeability, and contact angle of the films were investigated. Blending CNF into the AM matrix significantly enhanced the crystallinity, mechanical properties and permeability, whereas glycerol increased elongation at break, mainly by plasticizing the AM. There was significant phase separation between AM and CNF. Dynamic plasticizing and anti-plasticizing effects of both CNF and glycerol were demonstrated by NMR demonstrating high molecular order, but also non-crystalline, and evenly distributed 20 nm-sized glycerol domains. This study demonstrates a new lead in functional polysaccharide-based bioplastic systems.

Golgi-localized exo-ß1,3-galactosidases involved in cell expansion and root growth in Arabidopsis.

Plant arabinogalactan proteins (AGPs) are a diverse group of cell surface- and wall-associated glycoproteins. Functionally important AGP glycans are synthesized in the Golgi apparatus, but the relationships among their glycosylation levels, processing, and functionalities are poorly understood. Here, we report the identification and functional characterization of two Golgi-localized exo-ß-1,3-galactosidases from the glycosyl hydrolase 43 (GH43) family in Arabidopsis thaliana GH43 loss-of-function mutants exhibited root cell expansion defects in sugar-containing growth media. This root phenotype was associated with an increase in the extent of AGP cell wall association, as demonstrated by Yariv phenylglycoside dye quantification and comprehensive microarray polymer profiling of sequentially extracted cell walls. Characterization of recombinant GH43 variants revealed that the exo-ß-1,3-galactosidase activity of GH43 enzymes is hindered by ß-1,6 branches on ß-1,3-galactans. In line with this steric hindrance, the recombinant GH43 variants did not release galactose from cell wall-extracted glycoproteins or AGP-rich gum arabic. These results indicate that the lack of exo-ß-1,3-galactosidase activity alters cell wall extensibility in roots, a phenotype that could be explained by the involvement of galactosidases in AGP glycan biosynthesis.

Phenolic cross-links: building and de-constructing the plant cell wall.

Covering: Up to 2019Phenolic cross-links and phenolic inter-unit linkages result from the oxidative coupling of two hydroxycinnamates or two molecules of tyrosine. Free dimers of hydroxycinnamates, lignans, play important roles in plant defence. Cross-linking of bound phenolics in the plant cell wall affects cell expansion, wall strength, digestibility, degradability, and pathogen resistance. Cross-links mediated by phenolic substituents are particularly important as they confer strength to the wall via the formation of new covalent bonds, and by excluding water from it. Four biopolymer classes are known to be involved in the formation of phenolic cross-links: lignins, extensins, glucuronoarabinoxylans, and side-chains of rhamnogalacturonan-I. Lignins and extensins are ubiquitous in streptophytes whereas aromatic substituents on xylan and pectic side-chains are commonly assumed to be particular features of Poales sensu lato and core Caryophyllales, respectively. Cross-linking of phenolic moieties proceeds via radical formation, is catalyzed by peroxidases and laccases, and involves monolignols, tyrosine in extensins, and ferulate esters on xylan and pectin. Ferulate substituents, on xylan in particular, are thought to be nucleation points for lignin polymerization and are, therefore, of paramount importance to wall architecture in grasses and for the development of technology for wall disassembly, e.g. for the use of grass biomass for production of 2nd generation biofuels. This review summarizes current knowledge on the intra- and extracellular acylation of polysaccharides, and inter- and intra-molecular cross-linking of different constituents. Enzyme mediated lignan in vitro synthesis for pharmaceutical uses are covered as are industrial exploitation of mutant and transgenic approaches to control cell wall cross-linking.

Sustainable production of cellulose nanofiber gels and paper from sugar beet waste using enzymatic pre-treatment.

Removal of non-cellulosic polymers from vegetable pulp to obtain cellulose nanofibers (CNF) is normally achieved by chemical pre-treatments which requires several washing steps. In the present study, it is demonstrated how incubation of sugar beet pulp at pH 9, followed by treatment with polysaccharide-degrading enzymes and subsequent bleaching can be done in a one-pot procedure to make CNF. The new method consumes 67% less water and removes non-cellulosic polysaccharides with similar efficiency as a chemical method. In addition, CNF produced by the new method contained slightly more pectin and formed gels with 2.7 times higher storage modulus. Nanopapers cast from chemically- and enzymatically produced CNF showed similar mechanical properties. However, without the pH 9 incubation step, the enzymes accessibility to cell-wall polymers was limited resulting in lower gel and paper strengths. In conclusion, the new method offers a sustainable route for producing high quality CNF from sugar beet waste.

Metabolism of polysaccharides in dynamic middle lamellae during cotton fibre development.

MAIN CONCLUSION: Evidence is presented that cotton fibre adhesion and middle lamella formation are preceded by cutin dilution and accompanied by rhamnogalacturonan-I metabolism. Cotton fibres are single cell structures that early in development adhere to one another via the cotton fibre middle lamella (CFML) to form a tissue-like structure. The CFML is disassembled around the time of initial secondary wall deposition, leading to fibre detachment. Observations of CFML in the light microscope have suggested that the development of the middle lamella is accompanied by substantial cell-wall metabolism, but it has remained an open question as to which processes mediate adherence and which lead to detachment. The mechanism of adherence and detachment were investigated here using glyco-microarrays probed with monoclonal antibodies, transcript profiling, and observations of fibre auto-digestion. The results suggest that adherence is brought about by cutin dilution, while the presence of relevant enzyme activities and the dynamics of rhamnogalacturonan-I side-chain accumulation and disappearance suggest that both attachment and detachment are accompanied by rhamnogalacturonan-I metabolism.

Extensin arabinoside chain length is modulated in elongating cotton fibre.

Cotton fibre provides a unicellular model system for studying cell expansion and secondary cell wall deposition. Mature cotton fibres are mainly composed of cellulose while the walls of developing fibre cells contain a variety of polysaccharides and proteoglycans required for cell expansion. This includes hydroxyproline-rich glycoproteins (HRGPs) comprising the subgroup, extensins. In this study, extensin occurrence in cotton fibres was assessed using carbohydrate immunomicroarrays, mass spectrometry and monosaccharide profiling. Extensin amounts in three species appeared to correlate with fibre quality. Fibre cell expression profiling of the four cotton cultivars, combined with extensin arabinoside chain length measurements during fibre development, demonstrated that arabinoside side-chain length is modulated during development. Implications and mechanisms of extensin side-chain length dynamics during development are discussed.

Nanofibers Produced from Agro-Industrial Plant Waste Using Entirely Enzymatic Pretreatments.

Cellulose fibers can be freed from the cell-wall skeleton via high-shear homogenization, to produce cellulose nanofibers (CNF) that can be used, for example, as the reinforcing phase in composite materials. Nanofiber production from agro-industrial byproducts normally involves harsh chemical-pretreatments and high temperatures to remove noncellulosic polysaccharides (20-70% of dry weight). However, this is expensive for large-scale processing and environmentally damaging. An enzyme-only pretreatment to obtain CNF from agro-industrial byproducts (potato and sugar beet) was developed with targeted commercial enzyme mixtures. It is hypothesized that cellulose can be isolated from the biomass, using enzymes only, due to the low lignin content, facilitating greater liberation of CNF via high-shear homogenization. Comprehensive Microarray Polymer Profiling (CoMPP) measured remaining extractable polysaccharides, showing that the enzyme-pretreatment was more successful at removing noncellulosic polysaccharides than alkaline- or acid-hydrolysis alone. While effective alone, the effect of the enzyme-pretreatment was bolstered via combination with a mild high-pH pretreatment. Dynamic rheology was used to estimate the proportion of CNF in resultant suspensions. Enzyme-pretreated suspensions showed 4-fold and 10-fold increases in the storage modulus for potato and sugar beet, respectively, compared to untreated samples. A greener yet facile method for producing CNF from vegetable waste is presented here.

The Chara Genome: Secondary Complexity and Implications for Plant Terrestrialization.

Land plants evolved from charophytic algae, among which Charophyceae possess the most complex body plans. We present the genome of Chara braunii; comparison of the genome to those of land plants identified evolutionary novelties for plant terrestrialization and land plant heritage genes. C. braunii employs unique xylan synthases for cell wall biosynthesis, a phragmoplast (cell separation) mechanism similar to that of land plants, and many phytohormones. C. braunii plastids are controlled via land-plant-like retrograde signaling, and transcriptional regulation is more elaborate than in other algae. The morphological complexity of this organism may result from expanded gene families, with three cases of particular note: genes effecting tolerance to reactive oxygen species (ROS), LysM receptor-like kinases, and transcription factors (TFs). Transcriptomic analysis of sexual reproductive structures reveals intricate control by TFs, activity of the ROS gene network, and the ancestral use of plant-like storage and stress protection proteins in the zygote.

Identification of an algal xylan synthase indicates that there is functional orthology between algal and plant cell wall biosynthesis.

Insights into the evolution of plant cell walls have important implications for comprehending these diverse and abundant biological structures. In order to understand the evolving structure-function relationships of the plant cell wall, it is imperative to trace the origin of its different components. The present study is focused on plant 1,4-ß-xylan, tracing its evolutionary origin by genome and transcriptome mining followed by phylogenetic analysis, utilizing a large selection of plants and algae. It substantiates the findings by heterologous expression and biochemical characterization of a charophyte alga xylan synthase. Of the 12 known gene classes involved in 1,4-ß-xylan formation, XYS1/IRX10 in plants, IRX7, IRX8, IRX9, IRX14 and GUX occurred for the first time in charophyte algae. An XYS1/IRX10 ortholog from Klebsormidium flaccidum, designated K. flaccidumXYLAN SYNTHASE-1 (KfXYS1), possesses 1,4-ß-xylan synthase activity, and 1,4-ß-xylan occurs in the K. flaccidum cell wall. These data suggest that plant 1,4-ß-xylan originated in charophytes and shed light on the origin of one of the key cell wall innovations to occur in charophyte algae, facilitating terrestrialization and emergence of polysaccharide-based plant cell walls.

Corrigendum: Identification and evolution of a plant cell wall specific glycoprotein glycosyl transferase, ExAD.

This corrects the article DOI: 10.1038/srep45341.

Pea Border Cell Maturation and Release Involve Complex Cell Wall Structural Dynamics.

The adhesion of plant cells is vital for support and protection of the plant body and is maintained by a variety of molecular associations between cell wall components. In some specialized cases, though, plant cells are programmed to detach, and root cap-derived border cells are examples of this. Border cells (in some species known as border-like cells) provide an expendable barrier between roots and the environment. Their maturation and release is an important but poorly characterized cell separation event. To gain a deeper insight into the complex cellular dynamics underlying this process, we undertook a systematic, detailed analysis of pea (Pisum sativum) root tip cell walls. Our study included immunocarbohydrate microarray profiling, monosaccharide composition determination, Fourier-transformed infrared microspectroscopy, quantitative reverse transcription-PCR of cell wall biosynthetic genes, analysis of hydrolytic activities, transmission electron microscopy, and immunolocalization of cell wall components. Using this integrated glycobiology approach, we identified multiple novel modes of cell wall structural and compositional rearrangement during root cap growth and the release of border cells. Our findings provide a new level of detail about border cell maturation and enable us to develop a model of the separation process. We propose that loss of adhesion by the dissolution of homogalacturonan in the middle lamellae is augmented by an active biophysical process of cell curvature driven by the polarized distribution of xyloglucan and extensin epitopes.

Identification and evolution of a plant cell wall specific glycoprotein glycosyl transferase, ExAD.

Extensins are plant cell wall glycoproteins that act as scaffolds for the deposition of the main wall carbohydrate polymers, which are interlocked into the supramolecular wall structure through intra- and inter-molecular iso-di-tyrosine crosslinks within the extensin backbone. In the conserved canonical extensin repeat, Ser-Hyp4, serine and the consecutive C4-hydroxyprolines (Hyps) are substituted with an α-galactose and 1-5 ß- or α-linked arabinofuranoses (Arafs), respectively. These modifications are required for correct extended structure and function of the extensin network. Here, we identified a single Arabidopsis thaliana gene, At3g57630, in clade E of the inverting Glycosyltransferase family GT47 as a candidate for the transfer of Araf to Hyp-arabinofuranotriose (Hyp-ß1,4Araf-ß1,2Araf-ß1,2Araf) side chains in an α-linkage, to yield Hyp-Araf4 which is exclusively found in extensins. T-DNA knock-out mutants of At3g57630 showed a truncated root hair phenotype, as seen for mutants of all hitherto characterized extensin glycosylation enzymes; both root hair and glycan phenotypes were restored upon reintroduction of At3g57630. At3g57630 was named Extensin Arabinose Deficient transferase, ExAD, accordingly. The occurrence of ExAD orthologs within the Viridiplantae along with its' product, Hyp-Araf4, point to ExAD being an evolutionary hallmark of terrestrial plants and charophyte green algae.

Degradation of lignin ß-aryl ether units in Arabidopsis thaliana expressing LigD, LigF and LigG from Sphingomonas paucimobilis SYK-6.

Lignin is a major polymer in the secondary plant cell wall and composed of hydrophobic interlinked hydroxyphenylpropanoid units. The presence of lignin hampers conversion of plant biomass into biofuels; plants with modified lignin are therefore being investigated for increased digestibility. The bacterium Sphingomonas paucimobilis produces lignin-degrading enzymes including LigD, LigF and LigG involved in cleaving the most abundant lignin interunit linkage, the ß-aryl ether bond. In this study, we expressed the LigD, LigF and LigG (LigDFG) genes in Arabidopsis thaliana to introduce postlignification modifications into the lignin structure. The three enzymes were targeted to the secretory pathway. Phenolic metabolite profiling and 2D HSQC NMR of the transgenic lines showed an increase in oxidized guaiacyl and syringyl units without concomitant increase in oxidized ß-aryl ether units, showing lignin bond cleavage. Saccharification yield increased significantly in transgenic lines expressing LigDFG, showing the applicability of our approach. Additional new information on substrate specificity of the LigDFG enzymes is also provided.

Rhamnogalacturonan-I Based Microcapsules for Targeted Drug Release.

Drug targeting to the colon via the oral administration route for local treatment of e.g. inflammatory bowel disease and colonic cancer has several advantages such as needle-free administration and low infection risk. A new source for delivery is plant-polysaccharide based delivery platforms such as Rhamnogalacturonan-I (RG-I). In the gastro-intestinal tract the RG-I is only degraded by the action of the colonic microflora. For assessment of potential drug delivery properties, RG-I based microcapsules (~1 µm in diameter) were prepared by an interfacial poly-addition reaction. The cross-linked capsules were loaded with a fluorescent dye (model drug). The capsules showed negligible and very little in vitro release when subjected to media simulating gastric and intestinal fluids, respectively. However, upon exposure to a cocktail of commercial RG-I cleaving enzymes, ~ 9 times higher release was observed, demonstrating that the capsules can be opened by enzymatic degradation. The combined results suggest a potential platform for targeted drug delivery in the terminal gastro-intestinal tract.