Comprehensive Microarray Polymer Profiling Research Articles

Analyses of Aloe Polysaccharides Using Carbohydrate Microarray Profiling.

Background: As the popularity of Aloe vera extracts continues to rise, a desire to fully understand the individual polymer components of the leaf mesophyll, their relation to one another, and the effects they have on the human body are increasing. Polysaccharides present in the leaf mesophyll have been identified as the components responsible for the biological activities of A. vera, and they have been widely studied in the past decades. However, the commonly used methods do not provide the desired platform to conduct large comparative studies of polysaccharide compositions, as most of them require a complete or near-complete fractionation of the polymers. Objective: The objective for this study was to assess whether carbohydrate microarrays could be used for the high-throughput analysis of cell wall polysaccharides in aloe leaf mesophyll. Methods: The method we chose is known as comprehensive microarray polymer profiling (CoMPP) and combines the high-throughput capacity of microarray technology with the specificity of molecular probes. Results: Preliminary findings showed that CoMPP can successfully be used for high-throughput screening of aloe leaf mesophyll tissue. Seventeen species of Aloe and closely related genera were analyzed, and a clear difference in the polysaccharide compositions of the mesophyll tissues was seen. Conclusions: These preliminary data suggest that the polysaccharides vary between species and that true species of Aloe may differ from segregate genera.

Double blind microarray-based polysaccharide profiling enables parallel identification of uncharacterized polysaccharides and carbohydrate-binding proteins with unknown specificities

Marine algae are one of the largest sources of carbon on the planet. The microbial degradation of algal polysaccharides to their constitutive sugars is a cornerstone in the global carbon cycle in oceans. Marine polysaccharides are highly complex and heterogeneous, and poorly understood. This is also true for marine microbial proteins that specifically degrade these substrates and when characterized, they are frequently ascribed to new protein families. Marine (meta)genomic datasets contain large numbers of genes with functions putatively assigned to carbohydrate processing, but for which empirical biochemical activity is lacking. There is a paucity of knowledge on both sides of this protein/carbohydrate relationship. Addressing this ‘double blind’ problem requires high throughput strategies that allow large scale screening of protein activities, and polysaccharide occurrence. Glycan microarrays, in particular the Comprehensive Microarray Polymer Profiling (CoMPP) method, are powerful in screening large collections of glycans and we described the integration of this technology to a medium throughput protein expression system focused on marine genes. This methodology (Double Blind CoMPP or DB-CoMPP) enables us to characterize novel polysaccharide-binding proteins and to relate their ligands to algal clades. This data further indicate the potential of the DB-CoMPP technique to accommodate samples of all biological sources.

Surface properties correlate to the digestibility of hydrothermally pretreated lignocellulosic Poaceae biomass feedstocks

BackgroundUnderstanding factors that govern lignocellulosic biomass recalcitrance is a prerequisite for designing efficient 2nd generation biorefining processes. However, the reasons and mechanisms responsible for quantitative differences in enzymatic digestibility of various biomass feedstocks in response to hydrothermal pretreatment at different severities are still not sufficiently understood.ResultsPotentially important lignocellulosic feedstocks for biorefining, corn stover (Zea mays subsp. mays L.), stalks of Miscanthus × giganteus, and wheat straw (Triticum aestivum L.) were systematically hydrothermally pretreated; each at three different severities of 3.65, 3.83, and 3.97, respectively, and the enzymatic digestibility was assessed. Pretreated samples of Miscanthus × giganteus stalks were the least digestible among the biomass feedstocks producing ~24 to 66.6% lower glucose yields than the other feedstocks depending on pretreatment severity and enzyme dosage. Bulk biomass composition analyses, 2D nuclear magnetic resonance, and comprehensive microarray polymer profiling were not able to explain the observed differences in recalcitrance among the pretreated feedstocks. However, methods characterizing physical and chemical features of the biomass surfaces, specifically contact angle measurements (wettability) and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy (surface biopolymer composition) produced data correlating pretreatment severity and enzymatic digestibility, and they also revealed differences that correlated to enzymatic glucose yield responses among the three different biomass types.ConclusionThe study revealed that to a large extent, factors related to physico-chemical surface properties, namely surface wettability as assessed by contact angle measurements and surface content of hemicellulose, lignin, and wax as assessed by ATR-FTIR rather than bulk biomass chemical composition correlated to the recalcitrance of the tested biomass types. The data provide new insight into how hydrothermal pretreatment severity affects surface properties of key Poaceae lignocellulosic biomass and may help design new approaches to overcome biomass recalcitrance.

Carbohydrate Microarray Technology Applied to High-Throughput Mapping of Plant Cell Wall Glycans Using Comprehensive Microarray Polymer Profiling (CoMPP).

Cell walls are an important feature of plant cells and a major component of the plant glycome. They have both structural and physiological functions and are critical for plant growth and development. The diversity and complexity of these structures demand advanced high-throughput techniques to answer questions about their structure, functions and roles in both fundamental and applied scientific fields. Microarray technology provides both the high-throughput and the feasibility aspects required to meet that demand. In this chapter, some of the most recent microarray-based techniques relating to plant cell walls are described together with an overview of related contemporary techniques applied to carbohydrate microarrays and their general potential in glycoscience. A detailed experimental procedure for high-throughput mapping of plant cell wall glycans using the comprehensive microarray polymer profiling (CoMPP) technique is included in the chapter and provides a good example of both the robust and high-throughput nature of microarrays as well as their applicability to plant glycomics.

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

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.

High-throughput microarray mapping of cell wall polymers in roots and tubers during the viscosity-reducing process.

Viscosity reduction has a great impact on the efficiency of ethanol production when using roots and tubers as feedstock. Plant cell wall-degrading enzymes have been successfully applied to overcome the challenges posed by high viscosity. However, the changes in cell wall polymers during the viscosity-reducing process are poorly characterized. Comprehensive microarray polymer profiling, which is a high-throughput microarray, was used for the first time to map changes in the cell wall polymers of sweet potato (Ipomoea batatas), cassava (Manihot esculenta), and Canna edulis Ker. over the entire viscosity-reducing process. The results indicated that the composition of cell wall polymers among these three roots and tubers was markedly different. The gel-like matrix and glycoprotein network in the C. edulis Ker. cell wall caused difficulty in viscosity reduction. The obvious viscosity reduction of the sweet potato and the cassava was attributed to the degradation of homogalacturonan and the released 1,4-β-d-galactan and 1,5-α-l-arabinan.

Dissecting the polysaccharide-rich grape cell wall changes during winemaking using combined high-throughput and fractionation methods

Limited information is available on grape wall-derived polymeric structure/composition and how this changes during fermentation. Commercial winemaking operations use enzymes that target the polysaccharide-rich polymers of the cell walls of grape tissues to clarify musts and extract pigments during the fermentations. In this study, we have assessed changes in polysaccharide composition/turnover throughout the winemaking process by applying recently developed cell wall profiling approaches for monosaccharide composition (GC–MS), infra-red (IR) spectroscopy and comprehensive microarray polymer profiling (CoMPP). CoMPP performed on the concentrated soluble wine polysaccharides showed a fraction rich in rhamnogalacturonan I (RGI), homogalacturonan (HG) and arabinogalactan proteins (AGPs). We also used chemical and enzymatic fractionation techniques in addition to CoMPP to understand the berry deconstruction process more in-depth. CoMPP and gravimetric analysis of the fractionated pomace used aqueous buffers and CDTA solutions to obtain a pectin-rich fraction (pulp tightly-bound to skins) containing HG, RGI and AGPs; and then alkali (sodium carbonate and potassium hydroxide), liberating a xyloglucan-rich fraction (mainly skins). Interestingly this fraction was found to include pectins consisting of tightly associated and highly methyl-esterified HG and RGI networks. This was supported by enzymatic fractionation targeting pectin and xyloglucan polymers. A unique aspect is datasets suggesting that enzyme-resistant pectin polymers ‘coat’ the inner xyloglucan-rich skin cells. This data has important implications for developing effective strategies for efficient release of favorable compounds (pigments, tannins, aromatics, etc.) from the berry tissues during winemaking. This study provides a framework to understand the complex interactions between the grape matrix and carbohydrate-active enzymes to produce wine of desired quality and consistency.

Resistant starch diet induces change in the swine microbiome and a predominance of beneficial bacterial populations.

BackgroundDietary fibers contribute to health and physiology primarily via the fermentative actions of the host’s gut microbiome. Physicochemical properties such as solubility, fermentability, viscosity, and gel-forming ability differ among fiber types and are known to affect metabolism. However, few studies have focused on how they influence the gut microbiome and how these interactions influence host health. The aim of this study is to investigate how the gut microbiome of growing pigs responds to diets containing gel-forming alginate and fermentable resistant starch and to predict important interactions and functional changes within the microbiota.ResultsNine growing pigs (3-month-old), divided into three groups, were fed with either a control, alginate-, or resistant starch-containing diet (CON, ALG, or RS), and fecal samples were collected over a 12-week period. SSU (small subunit) rDNA amplicon sequencing data was annotated to assess the gut microbiome, whereas comprehensive microarray polymer profiling (CoMPP) of digested material was employed to evaluate feed degradation. Gut microbiome structure variation was greatest in pigs fed with resistant starch, where notable changes included the decrease in alpha diversity and increase in relative abundance of Lachnospiraceae- and Ruminococcus-affiliated phylotypes. Imputed function was predicted to vary significantly in pigs fed with resistant starch and to a much lesser extent with alginate; however, the key pathways involving degradation of starch and other plant polysaccharides were predicted to be unaffected. The change in relative abundance levels of basal dietary components (plant cell wall polysaccharides and proteins) over time was also consistent irrespective of diet; however, correlations between the dietary components and phylotypes varied considerably in the different diets.ConclusionsResistant starch-containing diet exhibited the strongest structural variation compared to the alginate-containing diet. This variation gave rise to a microbiome that contains phylotypes affiliated with metabolically reputable taxonomic lineages. Despite the significant microbiome structural shifts that occurred from resistant starch-containing diet, functional redundancy is seemingly apparent with respect to the microbiome’s capacity to degrade starch and other dietary polysaccharides, one of the key stages in digestion.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-015-0078-5) contains supplementary material, which is available to authorized users.

Non-cellulosic polysaccharides from cotton fibre are differently impacted by textile processing.

Cotton fibre is mainly composed of cellulose, although non-cellulosic polysaccharides play key roles during fibre development and are still present in the harvested fibre. This study aimed at determining the fate of non-cellulosic polysaccharides during cotton textile processing. We analyzed non-cellulosic cotton fibre polysaccharides during different steps of cotton textile processing using GC-MS, HPLC and comprehensive microarray polymer profiling to obtain monosaccharide and polysaccharide amounts and linkage compositions. Additionally, in situ detection was used to obtain information on polysaccharide localization and accessibility. We show that pectic and hemicellulosic polysaccharide levels decrease during cotton textile processing and that some processing steps have more impact than others. Pectins and arabinose-containing polysaccharides are strongly impacted by the chemical treatments, with most being removed during bleaching and scouring. However, some forms of pectin are more resistant than others. Xylan and xyloglucan are affected in later processing steps and to a lesser extent, whereas callose showed a strong resistance to the chemical processing steps. This study shows that non-cellulosic polysaccharides are differently impacted by the treatments used in cotton textile processing with some hemicelluloses and callose being resistant to these harsh treatments.

Understanding the relationship between cotton fiber properties and non-cellulosic cell wall polysaccharides.

A detailed knowledge of cell wall heterogeneity and complexity is crucial for understanding plant growth and development. One key challenge is to establish links between polysaccharide-rich cell walls and their phenotypic characteristics. It is of particular interest for some plant material, like cotton fibers, which are of both biological and industrial importance. To this end, we attempted to study cotton fiber characteristics together with glycan arrays using regression based approaches. Taking advantage of the comprehensive microarray polymer profiling technique (CoMPP), 32 cotton lines from different cotton species were studied. The glycan array was generated by sequential extraction of cell wall polysaccharides from mature cotton fibers and screening samples against eleven extensively characterized cell wall probes. Also, phenotypic characteristics of cotton fibers such as length, strength, elongation and micronaire were measured. The relationship between the two datasets was established in an integrative manner using linear regression methods. In the conducted analysis, we demonstrated the usefulness of regression based approaches in establishing a relationship between glycan measurements and phenotypic traits. In addition, the analysis also identified specific polysaccharides which may play a major role during fiber development for the final fiber characteristics. Three different regression methods identified a negative correlation between micronaire and the xyloglucan and homogalacturonan probes. Moreover, homogalacturonan and callose were shown to be significant predictors for fiber length. The role of these polysaccharides was already pointed out in previous cell wall elongation studies. Additional relationships were predicted for fiber strength and elongation which will need further experimental validation.

A polysaccharide utilization locus from an uncultured bacteroidetes phylotype suggests ecological adaptation and substrate versatility.

Recent metagenomic analyses have identified uncultured bacteria that are abundant in the rumen of herbivores and that possess putative biomass-converting enzyme systems. Here we investigate the saccharolytic capabilities of a polysaccharide utilization locus (PUL) that has been reconstructed from an uncultured Bacteroidetes phylotype (SRM-1) that dominates the rumen microbiome of Arctic reindeer. Characterization of the three PUL-encoded outer membrane glycoside hydrolases was performed using chromogenic substrates for initial screening, followed by detailed analyses of products generated from selected substrates, using high-pressure anion-exchange chromatography with electrochemical detection. Two glycoside hydrolase family 5 (GH5) endoglucanases (GH5_g and GH5_h) demonstrated activity against β-glucans, xylans, and xyloglucan, whereas GH5_h and the third enzyme, GH26_i, were active on several mannan substrates. Synergy experiments examining different combinations of the three enzymes demonstrated limited activity enhancement on individual substrates. Binding analysis of a SusE-positioned lipoprotein revealed an affinity toward β-glucans and, to a lesser extent, mannan, but unlike the two SusD-like lipoproteins previously characterized from the same PUL, binding to cellulose was not observed. Overall, these activities and binding specificities correlated well with the glycan content of the reindeer rumen, which was determined using comprehensive microarray polymer profiling and showed an abundance of various hemicellulose glycans. The substrate versatility of this single PUL putatively expands our perceptions regarding PUL machineries, which so far have demonstrated gene organization that suggests one cognate PUL for each substrate type. The presence of a PUL that possesses saccharolytic activity against a mixture of abundantly available polysaccharides supports the dominance of SRM-1 in the Svalbard reindeer rumen microbiome.

Pectic-β(1,4)-galactan, extensin and arabinogalactan-protein epitopes differentiate ripening stages in wine and table grape cell walls.

Cell wall changes in ripening grapes (Vitis vinifera) have been shown to involve re-modelling of pectin, xyloglucan and cellulose networks. Newer experimental techniques, such as molecular probes specific for cell wall epitopes, have yet to be extensively used in grape studies. Limited general information is available on the cell wall properties that contribute to texture differences between wine and table grapes. This study evaluates whether profiling tools can detect cell wall changes in ripening grapes from commercial vineyards. Standard sugar analysis and infra-red spectroscopy were used to examine the ripening stages (green, véraison and ripe) in grapes collected from Cabernet Sauvignon and Crimson Seedless vineyards. Comprehensive microarray polymer profiling (CoMPP) analysis was performed on cyclohexanediaminetetraacetic acid (CDTA) and NaOH extracts of alcohol-insoluble residue sourced from each stage using sets of cell wall probes (mAbs and CBMs), and the datasets were analysed using multivariate software. The datasets obtained confirmed previous studies on cell wall changes known to occur during grape ripening. Probes for homogalacturonan (e.g. LM19) were enriched in the CDTA fractions of Crimson Seedless relative to Cabernet Sauvignon grapes. Probes for pectic-β-(1,4)-galactan (mAb LM5), extensin (mAb LM1) and arabinogalactan proteins (AGPs, mAb LM2) were strongly correlated with ripening. From green stage to véraison, a progressive reduction in pectic-β-(1,4)-galactan epitopes, present in both pectin-rich (CDTA) and hemicellulose-rich (NaOH) polymers, was observed. Ripening changes in AGP and extensin epitope abundance also were found during and after véraison. Combinations of cell wall probes are able to define distinct ripening phases in grapes. Pectic-β-(1,4)-galactan epitopes decreased in abundance from green stage to véraison berries. From véraison there was an increase in abundance of significant extensin and AGP epitopes, which correlates with cell expansion events. This study provides new ripening biomarkers and changes that can be placed in the context of grape berry development.

Extractability and digestibility of plant cell wall polysaccharides during hydrothermal and enzymatic degradation of wheat straw (Triticum aestivum L.)

Fuels and chemicals derived through biochemical conversion of agricultural by-products such as wheat straw (Triticum aestivum L.) is an area currently under intense research. In this study, separate leaves and stems were hydrothermally pretreated and enzymatically hydrolysed and analysed chemically and by comprehensive microarray polymer profiling (CoMPP). This way, the effects of each degradation step to the intermolecular organisation of specific polysaccharides in the cell walls were elucidated. After pretreatment, the degree of polymerisation (DP) of released xylo-oligosaccharides in both samples was up to about 20, but mostly around 3–8, and notably more acetylated in stems. Arabinoxylan (AX) and mixed-linkage glucan (MLG) became water-extractable while xylan, xyloglucan (XG), mannan and glucan remained only alkali-extractable. All polysaccharides became partly digestible after pretreatment however, regardless their extractability in water or only alkali. Based on the results, AX and MLG appear to be loosely bound in the cell wall matrix while the other polysaccharides are bound more tightly and shielded from enzymatic attack by AX and MLG until pretreatment. The gradual solubilisation and digestion of the polysaccharides during pretreatment and hydrolysis correlate well with previous models of the polysaccharides’ structural organisation in the cell wall.

Selection and phenotypic characterization of a core collection of Brachypodium distachyon inbred lines

BackgroundThe model grass Brachypodium distachyon is increasingly used to study various aspects of grass biology. A large and genotypically diverse collection of B. distachyon germplasm has been assembled by the research community. The natural variation in this collection can serve as a powerful experimental tool for many areas of inquiry, including investigating biomass traits.ResultsWe surveyed the phenotypic diversity in a large collection of inbred lines and then selected a core collection of lines for more detailed analysis with an emphasis on traits relevant to the use of grasses as biofuel and grain crops. Phenotypic characters examined included plant height, growth habit, stem density, flowering time, and seed weight. We also surveyed differences in cell wall composition using near infrared spectroscopy (NIR) and comprehensive microarray polymer profiling (CoMPP). In all cases, we observed extensive natural variation including a two-fold variation in stem density, four-fold variation in ferulic acid bound to hemicellulose, and 1.7-fold variation in seed mass.ConclusionThese characterizations can provide the criteria for selecting diverse lines for future investigations of the genetic basis of the observed phenotypic variation.

Assessment of leaf/stem ratio in wheat straw feedstock and impact on enzymatic conversion

AbstractThe composition of wheat straw leaf and stem fractions were characterized using traditional strong acid hydrolysis, and monoclonal antibodies using comprehensive microarray polymer profiling (CoMPP). These results are then related to high throughput lignocellulose pretreatment and saccharification screening data. Pure leaf fraction of wheat straw was the least recalcitrant compared to pure stem and easily digested by commercial cellulases after moderate hydrothermal pretreatment; 63% and 31% (w/w) of glucan, 88% and 61% of xylan were released from the leaf and stem fractions, respectively. By preparing samples of various leaf‐to‐stem (L/S) ratios, we found shifting conversion behavior as processing parameters were modified. Increasing the enzyme dosage, pretreatment temperature and pretreatment time all significantly improved conversion rates in samples with more than 50% leaf content, whereas less impact was observed on samples with less than 50% leaf content. Enzyme affinity, desorption and readsorption with leaf and stem fractions may affect the sugar yield in wheat straw saccharification. The data suggest that the L/S ratio is an important parameter when adjusting or optimizing conversion processes and additionally in feedstock breeding. Furthermore, this highlights the need for rapid techniques for determining L/S ratio in wheat straw harvests. The CoMPP data on specific carbohydrates and leaf pectin highlight carbohydrate epitopes that may be useful as markers in the development of novel screening techniques; especially pectin or arabinogalactan proteins related epitopes are promising.

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)(1,2). However, understanding the biosynthesis and function of these components remains challenging. Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall(3). Furthermore, their composition is also modified during development(1), or in response to environmental cues(4). In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis(5). However, relatively few of the biosynthetic genes have been functionally characterized (4,5). Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types(6). Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained(7). Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification. Monoclonal antibodies (mAbs)(8,9) have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously(9,10,11). Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 & 2)(10,11) that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems(12) and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

Arabinose-rich polymers as an evolutionary strategy to plasticize resurrection plant cell walls against desiccation

A variety of Southern African resurrection plants were surveyed using high-throughput cell wall profiling tools. Species evaluated were the dicotyledons, Myrothamnus flabellifolia and Craterostigma plantagineum; the monocotyledons, Xerophyta viscosa, Xerophyta schlecterii, Xerophyta humilis and the resurrection grass Eragrostis nindensis, as well as a pteridophyte, the resurrection fern, Mohria caffrorum. Comparisons were made between hydrated and desiccated leaf and frond material, with respect to cell wall composition and polymer abundance, using monosaccharide composition analysis, FT-IR spectroscopy and comprehensive microarray polymer profiling in combination with multivariate data analysis. The data obtained suggest that three main functional strategies appear to have evolved to prepare plant cell walls for desiccation. Arabinan-rich pectin and arabinogalactan proteins are found in the resurrection fern M. caffrorum and the basal angiosperm M. flabellifolia where they appear to act as 'pectic plasticizers'. Dicotyledons with pectin-rich walls, such as C. plantagineum, seem to use inducible mechanisms which consist of up-regulating wall proteins and osmoprotectants. The hemicellulose-rich walls of the grass-like Xerophyta spp. and the resurrection grass E. nindensis were found to contain highly arabinosylated xylans and arabinogalactan proteins. These data support a general mechanism of 'plasticising' the cell walls of resurrection plants to desiccation and implicate arabinose-rich polymers (pectin-arabinans, arabinogalactan proteins and arabinoxylans) as the major contributors in ensuring flexibility is maintained and rehydration is facilitated in these plants.

The Cell Wall Polymers of the Charophycean Green Alga Chara corallina: Immunobinding and Biochemical Screening

Immunocytochemistry, comprehensive microarray polymer profiling (CoMPP), and biochemical analysis were used to elucidate the distribution of polymers in the cell walls of seven distinct parts of the shoot region of the thallus of the charophycean green alga Chara corallina. The cell walls contain a diverse set of polymers, many of which are commonly found in the cell walls of land plants, including cellulose, pectic polymers, arabinogalactan proteins, and several polymers that are classified as cross‐linking polysaccharides in embryophytes, including heteroxylans and heteromannans. Evidence of the presence of xyloglucan was also found. Cellulose and homogalacturonan were found in virtually all parts of the vegetative and reproductive shoot thallus, whereas the occurrence of other polymers in the cell walls within the different cell types was more varied. These results, which demonstrate the shared biochemical characteristics of the cell walls of Chara and of embryophytes, support previous molecular, ultra...

High‐throughput microarray profiling of cell wall polymers during hydrothermal pre‐treatment of wheat straw

Lignocellulosic plant material is potentially a sustainable source of fermentable sugars for bioethanol production. However, a barrier to this is the high resistance or recalcitrance of plant cell walls to be hydrolyzed. Therefore, a detailed knowledge of the structural features of plant cell walls that contribute to recalcitrance is important for improving the efficiency of bioethanol production. In this work we have used a technique known as Comprehensive Microarray Polymer Profiling (CoMPP) to analyze wheat straw before and after being subjected to hydrothermal pre-treatments at four different temperatures. The CoMPP technique combines the specificity of monoclonal antibodies with the high-throughput capacity of microarrays. Changes in the relative abundance of cell wall polysaccharides could be tracked during processing, and a reduction in xylan, arabinoxylans, xyloglucan, and mixed-linked glucan epitopes was detected at the two highest temperatures of pre-treatment used. This work demonstrates the potential of CoMPP as a complementally technique to conventional methods for analyzing biomass composition.

A specialized outer layer of the primary cell wall joins elongating cotton fibers into tissue-like bundles.

Cotton (Gossypium hirsutum) provides the world's dominant renewable textile fiber, and cotton fiber is valued as a research model because of its extensive elongation and secondary wall thickening. Previously, it was assumed that fibers elongated as individual cells. In contrast, observation by cryo-field emission-scanning electron microscopy of cotton fibers developing in situ within the boll demonstrated that fibers elongate within tissue-like bundles. These bundles were entrained by twisting fiber tips and consolidated by adhesion of a cotton fiber middle lamella (CFML). The fiber bundles consolidated via the CFML ultimately formed a packet of fiber around each seed, which helps explain how thousands of cotton fibers achieve their great length within a confined space. The cell wall nature of the CFML was characterized using transmission electron microscopy, including polymer epitope labeling. Toward the end of elongation, up-regulation occurred in gene expression and enzyme activities related to cell wall hydrolysis, and targeted breakdown of the CFML restored fiber individuality. At the same time, losses occurred in certain cell wall polymer epitopes (as revealed by comprehensive microarray polymer profiling) and sugars within noncellulosic matrix components (as revealed by gas chromatography-mass spectrometry analysis of derivatized neutral and acidic glycosyl residues). Broadly, these data show that adhesion modulated by an outer layer of the primary wall can coordinate the extensive growth of a large group of cells and illustrate dynamic changes in primary wall structure and composition occurring during the differentiation of one cell type that spends only part of its life as a tissue.