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.

Commercial Yeast Strains Expressing Polygalacturonase and Glucanase Unravel the Cell Walls of Chardonnay Grape Pomace.

Industrial wine yeast strains expressing hydrolytic enzymes were fermented on Chardonnay pomace and were shown to unravel the cell walls of the berry tissues according to the enzyme activities. The yeasts produced a native endo-polygalacturonase (Saccharomyces cerevisiae × Saccharomyces paradoxus hybrid, named PR7) and/or a recombinant endo-glucanase (S. cerevisiae strains named VIN13 END1 and PR7 END1). The impact of the enzymes during the fermentations was evaluated by directly studying the cell wall changes in the berry tissues using a Comprehensive Microarray Polymer Profiling technique. By the end of the fermentation, the endo-glucanase did not substantially modify the berry tissue cell walls, whereas the endo-polygalacturonase removed some homogalacturonan. The recombinant yeast strain producing both enzymes (PR7 END1) unravelled the cell walls more fully, enabling polymers, such as rhamnogalacturonan-I, ß-1,4-D-galactan and α-1,5-L-arabinan, as well as cell wall proteins to be extracted in a pectin solvent. This enzyme synergism led to the enrichment of rhamnogalacturonan-type polymers in the subsequent NaOH fractions. This study illustrated the potential utilisation of a recombinant yeast in pomace valorisation processes and simulated consolidated bioprocessing. Furthermore, the cell wall profiling techniques were confirmed as valuable tools to evaluate and optimise enzyme producing yeasts for grape and plant cell wall degradation.

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.

Differences in berry skin and pulp cell wall polysaccharides from ripe and overripe Shiraz grapes evaluated using glycan profiling reveals extensin-rich flesh.

Shiraz is a widely planted cultivar in many of the world's top wine regions where it is used for the production of top-quality single varietal or blended red wines. Cell wall changes during grape ripening and over-ripening have been investigated, particularly in the context of understanding berry deconstruction thereby facilitating the release of favorable compounds during winemaking. However, no information is available on cell wall changes during berry shrinkage in Shiraz. Glycan microarray technology was used to directly profile Shiraz berries for cell wall polysaccharide and glycoprotein epitopes. Skins and pulp tissues were profiled separately and revealed that whereas the skin was rich in pectins and xyloglucans, the pulp tissues were mainly composed of extensin glycoproteins. Overripe (26-28°B) berries, particularly those from the warmer region site, revealed degradation of their pectin and extensin epitopes.

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.

Overexpression of VviPGIP1 and NtCAD14 in Tobacco Screened Using Glycan Microarrays Reveals Cell Wall Reorganisation in the Absence of Fungal Infection.

The expression of Vitis vinifera polygalacturonase inhibiting protein 1 (VviPGIP1) in Nicotiana tabacum has been linked to modifications at the cell wall level. Previous investigations have shown an upregulation of the lignin biosynthesis pathway and reorganisation of arabinoxyloglucan composition. This suggests cell wall tightening occurs, which may be linked to defence priming responses. The present study used a screening approach to test four VviPGIP1 and four NtCAD14 overexpressing transgenic lines for cell wall alterations. Overexpressing the tobacco-derived cinnamyl alcohol dehydrogenase (NtCAD14) gene is known to increase lignin biosynthesis and deposition. These lines, particularly PGIP1 expressing plants, have been shown to lead to a decrease in susceptibility towards grey rot fungus Botrytis cinerea. In this study the aim was to investigate the cell wall modulations that occurred prior to infection, which should highlight potential priming phenomena and phenotypes. Leaf lignin composition and relative concentration of constituent monolignols were evaluated using pyrolysis gas chromatography. Significant concentrations of lignin were deposited in the stems but not the leaves of NtCAD14 overexpressing plants. Furthermore, no significant changes in monolignol composition were found between transgenic and wild type plants. The polysaccharide modifications were quantified using gas chromatography (GC-MS) of constituent monosaccharides. The major leaf polysaccharide and cell wall protein components were evaluated using comprehensive microarray polymer profiling (CoMPP). The most significant changes appeared at the polysaccharide and protein level. The pectin fraction of the transgenic lines had subtle variations in patterning for methylesterification epitopes for both VviPGIP1 and NtCAD14 transgenic lines versus wild type. Pectin esterification levels have been linked to pathogen defence in the past. The most marked changes occurred in glycoprotein abundance for both the VviPGIP1 and NtCAD14 lines. Epitopes for arabinogalactan proteins (AGPs) and extensins were notably altered in transgenic NtCAD14 tobacco.

Analysis of Plant Cell Walls Using High-Throughput Profiling Techniques with Multivariate Methods.

Plant cell walls are composed of a number of coextensive polysaccharide-rich networks (i.e., pectin, hemicellulose, protein). Polysaccharide-rich cell walls are important in a number of biological processes including fruit ripening, plant-pathogen interactions (e.g., pathogenic fungi), fermentations (e.g., winemaking), and tissue differentiation (e.g., secondary cell walls). Applying appropriate methods is necessary to assess biological roles as for example in putative plant gene functional characterization (e.g., experimental evaluation of transgenic plants). Obtaining datasets is relatively easy, using for example gas chromatography-mass spectrometry (GC-MS) methods for monosaccharide composition, Fourier transform infrared spectroscopy (FT-IR) and comprehensive microarray polymer profiling (CoMPP); however, analyzing the data requires implementing statistical tools for large-scale datasets. We have validated and implemented a range of multivariate data analysis methods on datasets from tobacco, grapevine, and wine polysaccharide studies. Here we present the workflow from processing samples to acquiring data to performing data analysis (particularly principal component analysis (PCA) and orthogonal projection to latent structure (OPLS) methods).

Tracking polysaccharides during white winemaking using glycan microarrays reveals glycoprotein-rich sediments.

Winemaking results in a significant amount of sediments that are formed in the tanks, the vats and in the bottles before and after fermentation. Little is known about the biochemical composition of these sediments apart from the fact that they are assumed to be derived in large part from the grape matrix. Glycan microarray technology offers a relatively rapid means to track the polysaccharides from their origin in the grape material and throughout the various steps in the winemaking process. In this study Comprehensive Microarray Polymer Profiling (CoMPP) was used to investigate the glycan-rich composition of particularly white grapes during winemaking and then investigate the effects of recombinant and commercial enzyme formulations on wine sediment compositions. The gross lees or sediments produced in the absence of enzymes were found to be composed of an abundance of homogalacturonans, rhamnogalacturonans, arabinans and galactans in addition to an abundance of extensins and arabinogalactan proteins. The addition of enzymes was shown to strip off the homogalacturonan and much of the rhamnogalacturonan with its side chains revealing a sediment layer composed almost exclusively of extensins and arabinogalactan proteins. The effect of winemaking techniques was shown to have an effect on the glycan-rich wine sediment compositions and holds implications for the management of gross lees in a winery environment.

Understanding Changes in Tomato Cell Walls in Roots and Fruits: The Contribution of Arbuscular Mycorrhizal Colonization.

Modifications in cell wall composition, which can be accompanied by changes in its structure, were already reported during plant interactions with other organisms, such as the mycorrhizal fungi. Arbuscular mycorrhizal (AM) fungi are among the most widespread soil organisms that colonize the roots of land plants, where they facilitate mineral nutrient uptake from the soil in exchange for plant-assimilated carbon. In AM symbiosis, the host plasma membrane invaginates and proliferates around all the developing intracellular fungal structures, and cell wall material is laid down between this membrane and the fungal cell surface. In addition, to improve host nutrition and tolerance/resistance to environmental stresses, AM symbiosis was shown to modulate fruit features. In this study, Comprehensive Microarray Polymer Profiling (CoMMP) technique was used to verify the impact of the AM symbiosis on the tomato cell wall composition both at local (root) and systemic level (fruit). Multivariate data analyses were performed on the obtained datasets looking for the effects of fertilization, inoculation with AM fungi, and the fruit ripening stage. Results allowed for the discernment of cell wall component modifications that were correlated with mycorrhizal colonization, showing a different tomato response to AM colonization and high fertilization, both at the root and the systemic level.

Polyploidy Affects Plant Growth and Alters Cell Wall Composition.

Polyploidization has played a key role in plant breeding and crop improvement. Although its potential to increase biomass yield is well described, the effect of polyploidization on biomass composition has largely remained unexplored. Here, we generated a series of Arabidopsis (Arabidopsis thaliana) plants with different somatic ploidy levels (2n, 4n, 6n, and 8n) and performed rigorous phenotypic characterization. Kinematic analysis showed that polyploids developed slower compared to diploids; however, tetra- and hexaploids, but not octaploids, generated larger rosettes due to delayed flowering. In addition, morphometric analysis of leaves showed that polyploidy affected epidermal pavement cells, with increased cell size and reduced cell number per leaf blade with incrementing ploidy. However, the inflorescence stem dry weight was highest in tetraploids. Cell wall characterization revealed that the basic somatic ploidy level negatively correlated with lignin and cellulose content, and positively correlated with matrix polysaccharide content (i.e. hemicellulose and pectin) in the stem tissue. In addition, higher ploidy plants displayed altered sugar composition. Such effects were linked to the delayed development of polyploids. Moreover, the changes in polyploid cell wall composition promoted saccharification yield. The results of this study indicate that induction of polyploidy is a promising breeding strategy to further tailor crops for biomass production.

The impact of silicon on cell wall composition and enzymatic saccharification of Brachypodium distachyon.

BACKGROUND: Plants and in particular grasses benefit from a high uptake of silicon (Si) which improves their growth and productivity by alleviating adverse effects of biotic and abiotic stress. However, the silicon present in plant tissues may have a negative impact on the processing and degradation of lignocellulosic biomass. Solutions to reduce the silicon content either by biomass engineering or development of downstream separation methods are therefore targeted. Different cell wall components have been proposed to interact with the silica pool in plant shoots, but the understanding of the underlying processes is still limited. RESULTS: In the present study, we have characterized silicon deposition and cell wall composition in Brachypodium distachyon wild-type and low-silicon 1 (Bdlsi1-1) mutant plants. Our analyses included different organs and plant developmental stages. In the mutant defective in silicon uptake, low silicon availability favoured deposition of this element in the amorphous form or bound to cell wall polymers rather than as silicified structures. Several alterations in non-cellulosic polysaccharides and lignin were recorded in the mutant plants, indicating differences in the types of linkages and in the three-dimensional organization of the cell wall network. Enzymatic saccharification assays showed that straw from mutant plants was marginally more degradable following a 190 °C hydrothermal pretreatment, while there were no differences without or after a 120 °C hydrothermal pretreatment. CONCLUSIONS: We conclude that silicon affects the composition of plant cell walls, mostly by altering linkages of non-cellulosic polymers and lignin. The modifications of the cell wall network and the reduced silicon concentration appear to have little or no implications on biomass recalcitrance to enzymatic saccharification.

Tracking polysaccharides through the brewing process.

Brewing is a highly complex stepwise process that starts with a mashing step during which starch is gelatinized and converted into oligo- and/or monosaccharides by enzymes and heat. The starch is mostly degraded and utilised during the fermentation process, but grains and hops both contain additional soluble and insoluble complex polysaccharides within their cell walls that persist and can have beneficial or detrimental effects on the brewing process. Previous studies have mostly been restricted to analysing the grain and/or malt prior to entering the brewing process, but here we track the fates of polysaccharides during the entire brewing process. To do this, we utilised a novel approach based on carbohydrate microarray technology. We demonstrate the successful application of this technology to brewing science and show how it can be utilised to obtain an unprecedented level of knowledge about the underlying molecular mechanisms at work.

Exploring the Glycans of Euglena gracilis.

Euglena gracilis is an alga of great biotechnological interest and extensive metabolic capacity, able to make high levels of bioactive compounds, such as polyunsaturated fatty acids, vitamins and ß-glucan. Previous work has shown that Euglena expresses a wide range of carbohydrate-active enzymes, suggesting an unexpectedly high capacity for the synthesis of complex carbohydrates for a single-celled organism. Here, we present an analysis of some of the carbohydrates synthesised by Euglena gracilis. Analysis of the sugar nucleotide pool showed that there are the substrates necessary for synthesis of complex polysaccharides, including the unusual sugar galactofuranose. Lectin- and antibody-based profiling of whole cells and extracted carbohydrates revealed a complex galactan, xylan and aminosugar based surface. Protein N-glycan profiling, however, indicated that just simple high mannose-type glycans are present and that they are partially modified with putative aminoethylphosphonate moieties. Together, these data indicate that Euglena possesses a complex glycan surface, unrelated to plant cell walls, while its protein glycosylation is simple. Taken together, these findings suggest that Euglena gracilis may lend itself to the production of pharmaceutical glycoproteins.

Development of novel monoclonal antibodies against starch and ulvan - implications for antibody production against polysaccharides with limited immunogenicity.

Monoclonal antibodies (mAbs) are widely used and powerful research tools, but the generation of mAbs against glycan epitopes is generally more problematic than against proteins. This is especially significant for research on polysaccharide-rich land plants and algae (Viridiplantae). Most antibody production is based on using single antigens, however, there are significant gaps in the current repertoire of mAbs against some glycan targets with low immunogenicity. We approached mAb production in a different way and immunised with a complex mixture of polysaccharides. The multiplexed screening capability of carbohydrate microarrays was then exploited to deconvolute the specificities of individual mAbs. Using this strategy, we generated a set of novel mAbs, including one against starch (INCh1) and one against ulvan (INCh2). These polysaccharides are important storage and structural polymers respectively, but both are generally considered as having limited immunogenicity. INCh1 and INCh2 therefore represent important new molecular probes for Viridiplantae research. Moreover, since the α-(1-4)-glucan epitope recognised by INCh1 is also a component of glycogen, this mAb can also be used in mammalian systems. We describe the detailed characterisation of INCh1 and INCh2, and discuss the potential of a non-directed mass-screening approach for mAb production against some glycan targets.

Combining hydrothermal pretreatment with enzymes de-pectinates and exposes the innermost xyloglucan-rich hemicellulose layers of wine grape pomace.

Chardonnay grape pomace was treated with pressurized heat followed by enzymatic hydrolysis, with commercial or pure enzymes, in buffered conditions. The pomace was unfermented as commonly found for white winemaking wastes and treatments aimed to simulate biovalorization processing. Cell wall profiling techniques showed that the pretreatment led to depectination of the outer layers thereby exposing xylan polymers and increasing the extractability of arabinans, galactans, arabinogalactan proteins and mannans. This higher extractability is believed to be linked with partial degradation and opening-up of cell wall networks. Pectinase-rich enzyme preparations were presumably able to access the inner rhamnogalacturonan I dominant coating layers due to the hydrothermal pretreatment. Patterns of epitope abundance and the sequential release of cell wall polymers with specific combinations of enzymes led to a working model of the hitherto, poorly understood innermost xyloglucan-rich hemicellulose layers of unfermented grape pomace.

Genome-wide association mapping in winter barley for grain yield and culm cell wall polymer content using the high-throughput CoMPP technique.

A collection of 112 winter barley varieties (Hordeum vulgare L.) was grown in the field for two years (2008/09 and 2009/10) in northern Italy and grain and straw yields recorded. In the first year of the trial, a severe attack of barley yellow mosaic virus (BaYMV) strongly influenced final performances with an average reduction of ~ 50% for grain and straw harvested in comparison to the second year. The genetic determination (GD) for grain yield was 0.49 and 0.70, for the two years respectively, and for straw yield GD was low in 2009 (0.09) and higher in 2010 (0.29). Cell wall polymers in culms were quantified by means of the monoclonal antibodies LM6, LM11, JIM13 and BS-400-3 and the carbohydrate-binding module CBM3a using the high-throughput CoMPP technique. Of these, LM6, which detects arabinan components, showed a relatively high GD in both years and a significantly negative correlation with grain yield (GYLD). Overall, heritability (H2) was calculated for GYLD, LM6 and JIM and resulted to be 0.42, 0.32 and 0.20, respectively. A total of 4,976 SNPs from the 9K iSelect array were used in the study for the analysis of population structure, linkage disequilibrium (LD) and genome-wide association study (GWAS). Marker-trait associations (MTA) were analyzed for grain yield and cell wall determination by LM6 and JIM13 as these were the traits showing significant correlations between the years. A single QTL for GYLD containing three MTAs was found on chromosome 3H located close to the Hv-eIF4E gene, which is known to regulate resistance to BaYMV. Subsequently the QTL was shown to be tightly linked to rym4, a locus for resistance to the virus. GWAs on arabinans quantified by LM6 resulted in the identification of major QTLs closely located on 3H and hypotheses regarding putative candidate genes were formulated through the study of gene expression levels based on bioinformatics tools.

Dissecting the polysaccharide-rich grape cell wall matrix using recombinant pectinases during winemaking.

The effectiveness of enzyme-mediated-maceration in red winemaking relies on the use of an optimum combination of specific enzymes. A lack of information on the relevant enzyme activities and the corresponding polysaccharide-rich berry cell wall structure is a major limitation. This study used different combinations of purified recombinant pectinases with cell wall profiling tools to follow the deconstruction process during winemaking. Multivariate data analysis of the glycan microarray (CoMPP) and gas chromatography (GC) results revealed that pectin lyase performed almost as effectively in de-pectination as certain commercial enzyme mixtures. Surprisingly the combination of endo-polygalacturonase and pectin-methyl-esterase only unraveled the cell walls without de-pectination. Datasets from the various combinations used confirmed pectin-rich and xyloglucan-rich layers within the grape pomace. These data support a proposed grape cell wall model which can serve as a foundation to evaluate testable hypotheses in future studies aimed at developing tailor-made enzymes for winemaking scenarios.

Understanding plant cell-wall remodelling during the symbiotic interaction between Tuber melanosporum and Corylus avellana using a carbohydrate microarray.

MAIN CONCLUSION: A combined approach, using a carbohydrate microarray as a support for genomic data, has revealed subtle plant cell-wall remodelling during Tuber melanosporum and Corylus avellana interaction. Cell walls are involved, to a great extent, in mediating plant-microbe interactions. An important feature of these interactions concerns changes in the cell-wall composition during interaction with other organisms. In ectomycorrhizae, plant and fungal cell walls come into direct contact, and represent the interface between the two partners. However, very little information is available on the re-arrangement that could occur within the plant and fungal cell walls during ectomycorrhizal symbiosis. Taking advantage of the Comprehensive Microarray Polymer Profiling (CoMPP) technology, the current study has had the aim of monitoring the changes that take place in the plant cell wall in Corylus avellana roots during colonization by the ascomycetous ectomycorrhizal fungus T. melanosporum. Additionally, genes encoding putative plant cell-wall degrading enzymes (PCWDEs) have been identified in the T. melanosporum genome, and RT-qPCRs have been performed to verify the expression of selected genes in fully developed C. avellana/T. melanosporum ectomycorrhizae. A localized degradation of pectin seems to occur during fungal colonization, in agreement with the growth of the ectomycorrhizal fungus through the middle lamella and with the fungal gene expression of genes acting on these polysaccharides.

Effect of Commercial Enzymes on Berry Cell Wall Deconstruction in the Context of Intravineyard Ripeness Variation under Winemaking Conditions.

Significant intravineyard variation in grape berry ripening occurs within vines and between vines. However, no cell wall data are available on such variation. Here we used a checkerboard panel design to investigate ripening variation in pooled grape bunches for enzyme-assisted winemaking. The vineyard was dissected into defined panels, which were selected for winemaking with or without enzyme addition. Cell wall material was prepared and subjected to high-throughput profiling combined with multivariate data analysis. The study showed that significant ripening-related variation was present at the berry cell wall polymer level and occurred within the experimental vineyard block. Furthemore, all enzyme treatments reduced cell wall variation via depectination. Interestingly, cell wall esterification levels were unaffected by enzyme treatments. This study provides clear evidence that enzymes can positively influence the consistency of winemaking and provides a foundation for further research into the relationship between grape berry cell wall architecture and enzyme formulations.