[Show abstract][Hide abstract] ABSTRACT: In plants, epigenetic regulation is important in normal development and in modulating some agronomic traits. The potential contribution of DNA methylation mediated gene regulation to phenotypic diversity and development in cotton was investigated between cotton genotypes and various tissues. DNA methylation diversity, genetic diversity, and changes in methylation context were investigated using methylation-sensitive amplified polymorphism (MSAP) assays including a methylation insensitive enzyme (BsiSI), and the total DNA methylation level was measured by high-performance liquid chromatography (HPLC). DNA methylation diversity was greater than the genetic diversity in the selected cotton genotypes and significantly different levels of DNA methylation were identified between tissues, including fibre. The higher DNA methylation diversity (CHG methylation being more diverse than CG methylation) in cotton genotypes suggest epigenetic regulation may be important for cotton, and the change in DNA methylation between fibre and other tissues hints that some genes may be epigenetically regulated for fibre development. The novel approach using BsiSI allowed direct comparison between genetic and epigenetic diversity, and also measured CC methylation level that cannot be detected by conventional MSAP.
PLoS ONE 01/2014; 9(1):e86049. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The plant cell wall is a chemically complex structure composed mostly of polysaccharides. Detailed analyses of these cell wall polysaccharides are essential for our understanding of plant development and for our use of plant biomass (largely wall material) in the food, agriculture, fabric, timber, biofuel and biocomposite industries. We present analytical techniques not only to define the fine chemical structures of individual cell wall polysaccharides but also to estimate the overall polysaccharide composition of cell wall preparations. The procedure covers the preparation of cell walls, together with gas chromatography-mass spectrometry (GC-MS)-based methods, for both the analysis of monosaccharides as their volatile alditol acetate derivatives and for methylation analysis to determine linkage positions between monosaccharide residues as their volatile partially methylated alditol acetate derivatives. Analysis time will vary depending on both the method used and the tissue type, and ranges from 2 d for a simple neutral sugar composition to 2 weeks for a carboxyl reduction/methylation linkage analysis.
[Show abstract][Hide abstract] ABSTRACT: An exo-β-(1→3)-D-galactanase (SGalase1) that specifically cleaves the β-(1→3)-D-galactan backbone of arabinogalactan-proteins (AGPs) was isolated from culture filtrates of a soil Streptomyces sp. Internal peptide sequence information was used to clone and recombinantly express the gene in E. coli. The molecular mass of the isolated enzyme was ~45 kDa, similar to the 48.2 kDa mass predicted from the amino acid sequence. The pI, pH and temperature optima for the enzyme were ~7.45, 3.8 and 48 °C, respectively. The native and recombinant enzymes specifically hydrolysed β-(1→3)-D-galacto-oligo- or poly-saccharides from the upstream (non-reducing) end, typical of an exo-acting enzyme. A second homologous Streptomyces gene (SGalase2) was also cloned and expressed. SGalase2 was similar in size (47.9 kDa) and enzyme activity to SGalase1 but differed in its pH optimum (pH 5). Both SGalase1 and SGalase2 are predicted to belong to the CAZy glycosyl hydrolase family GH 43 based on activity, sequence homology and phylogenetic analysis. The K(m) and V(max) of the native exo-β-(1→3)-D-galactanase for de-arabinosylated gum arabic (dGA) were 19 mg/ml and 9.7 μmol D-Gal/min/mg protein, respectively. The activity of these enzymes is well suited for the study of type II galactan structures and provides an important tool for the investigation of the biological role of AGPs in plants. De-arabinosylated gum arabic (dGA) was used as a model to investigate the use of these enzymes in defining type II galactan structure. Exhaustive hydrolysis of dGA resulted in a limited number of oligosaccharide products with a trisaccharide of Gal(2)GlcA(1) predominating.
Carbohydrate research 05/2012; 352:70-81. · 2.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Immunolabeling, combined with chemical analyses and transcript profiling, have provided a comprehensive temporal and spatial picture of the deposition and modification of cell wall polysaccharides during barley (Hordeum vulgare) grain development, from endosperm cellularization at 3 d after pollination (DAP) through differentiation to the mature grain at 38 DAP. (1→3)-β-D-Glucan appears transiently during cellularization but reappears in patches in the subaleurone cell walls around 20 DAP. (1→3, 1→4)-β-Glucan, the most abundant polysaccharide of the mature barley grain, accumulates throughout development. Arabino-(1-4)-β-D-xylan is deposited significantly earlier than we previously reported. This was attributable to the initial deposition of the polysaccharide in a highly substituted form that was not recognized by antibodies commonly used to detect arabino-(1-4)-β-D-xylans in sections of plant material. The epitopes needed for antibody recognition were exposed by pretreatment of sections with α-L-arabinofuranosidase; this procedure showed that arabino-(1-4)-β-D-xylans were deposited as early as 5 DAP and highlighted their changing structures during endosperm development. By 28 DAP labeling of hetero-(1→4)-β-D-mannan is observed in the walls of the starchy endosperm but not in the aleurone walls. Although absent in mature endosperm cell walls we now show that xyloglucan is present transiently from 3 until about 6 DAP and disappears by 8 DAP. Quantitative reverse transcription-polymerase chain reaction of transcripts for GLUCAN SYNTHASE-LIKE, Cellulose Synthase, and CELLULOSE SYNTHASE-LIKE genes were consistent with the patterns of polysaccharide deposition. Transcript profiling of some members from the Carbohydrate-Active Enzymes database glycosyl transferase families GT61, GT47, and GT43, previously implicated in arabino-(1-4)-β-d-xylan biosynthesis, confirms their presence during grain development.
[Show abstract][Hide abstract] ABSTRACT: Recent studies indicate that mitochondrial functions impinge on cell wall integrity, drug tolerance, and virulence of human fungal pathogens. However, the mechanistic aspects of these processes are poorly understood. We focused on the mitochondrial outer membrane SAM (Sorting and Assembly Machinery) complex subunit Sam37 in Candida albicans. Inactivation of SAM37 in C. albicans leads to a large reduction in fitness, a phenotype not conserved with the model yeast Saccharomyces cerevisiae. Our data indicate that slow growth of the sam37ΔΔ mutant results from mitochondrial DNA loss, a new function for Sam37 in C. albicans, and from reduced activity of the essential SAM complex subunit Sam35. The sam37ΔΔ mutant was hypersensitive to drugs that target the cell wall and displayed altered cell wall structure, supporting a role for Sam37 in cell wall integrity in C. albicans. The sensitivity of the mutant to membrane-targeting antifungals was not significantly altered. The sam37ΔΔ mutant was avirulent in the mouse model, and bioinformatics showed that the fungal Sam37 proteins are distant from their animal counterparts and could thus represent potential drug targets. Our study provides the first direct evidence for a link between mitochondrial function and cell wall integrity in C. albicans and is further relevant for understanding mitochondrial function in fitness, antifungal drug tolerance, and virulence of this major pathogen. Beyond the relevance to fungal pathogenesis, this work also provides new insight into the mitochondrial and cellular roles of the SAM complex in fungi.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Numerous evolutionary innovations were required to enable freshwater green algae to colonize terrestrial habitats and thereby initiate the evolution of land plants (embryophytes). These adaptations probably included changes in cell-wall composition and architecture that were to become essential for embryophyte development and radiation. However, it is not known to what extent the polymers that are characteristic of embryophyte cell walls, including pectins, hemicelluloses, glycoproteins and lignin, evolved in response to the demands of the terrestrial environment or whether they pre-existed in their algal ancestors. Here we show that members of the advanced charophycean green algae (CGA), including the Charales, Coleochaetales and Zygnematales, but not basal CGA (Klebsormidiales and Chlorokybales), have cell walls that are comparable in several respects to the primary walls of embryophytes. Moreover, we provide both chemical and immunocytochemical evidence that selected Coleochaete species have cell walls that contain small amounts of lignin or lignin-like polymers derived from radical coupling of hydroxycinnamyl alcohols. Thus, the ability to synthesize many of the components that characterize extant embryophyte walls evolved during divergence within CGA. Our study provides new insight into the evolutionary window during which the structurally complex walls of embryophytes originated, and the significance of the advanced CGA during these events.
The Plant Journal 06/2011; 68(2):201-11. · 6.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Changes in cell wall polysaccharides, transcript abundance, metabolite profiles, and hormone concentrations were monitored in the upper and lower regions of maize (Zea mays) pulvini in response to gravistimulation, during which maize plants placed in a horizontal position returned to the vertical orientation. Heteroxylan levels increased in the lower regions of the pulvini, together with lignin, but xyloglucans and heteromannan contents decreased. The degree of substitution of heteroxylan with arabinofuranosyl residues decreased in the lower pulvini, which exhibited increased mechanical strength as the plants returned to the vertical position. Few or no changes in noncellulosic wall polysaccharides could be detected on the upper side of the pulvinus, and crystalline cellulose content remained essentially constant in both the upper and lower pulvinus. Microarray analyses showed that spatial and temporal changes in transcript profiles were consistent with the changes in wall composition that were observed in the lower regions of the pulvinus. In addition, the microarray analyses indicated that metabolic pathways leading to the biosynthesis of phytohormones were differentially activated in the upper and lower regions of the pulvinus in response to gravistimulation. Metabolite profiles and measured hormone concentrations were consistent with the microarray data, insofar as auxin, physiologically active gibberellic acid, and metabolites potentially involved in lignin biosynthesis increased in the elongating cells of the lower pulvinus.
[Show abstract][Hide abstract] ABSTRACT: Exposure of the mature Arabidopsis (Arabidopsis thaliana) seed to water results in the rapid release of pectinaceous mucilage from the outer cells of the testa. Once released, mucilage completely envelops the seed in a gel-like capsule. The physical force required to rupture the outer cell wall of the testa comes from the swelling of the mucilage as it expands rapidly following hydration. In this study, we show that mutations in the transcriptional regulator LEUNIG_HOMOLOG (LUH) cause a mucilage extrusion defect due to altered mucilage swelling. Based on sugar linkage and immunomicroscopic analyses, we show that the structure of luh mucilage is altered, having both an increase in substituted rhamnogalacturonan I and in methyl-esterified homogalacturonan. Also correlated with the structural modification of luh mucilage is a significant decrease in MUCILAGE MODIFIED2 (MUM2; a β-galactosidase) expression in the luh seed coat, raising the possibility that reduced activity of this glycosidase is directly responsible for the luh mucilage defects. Consistent with this is the structural similarity between mum2 and luh mucilage as well as the observation that elevating MUM2 expression in luh mutants completely suppresses the mucilage extrusion defect. Suppression of the luh mutant phenotype was also observed when LEUNIG, a transcriptional corepressor closely related to LUH, was introduced in luh mutants under the control of the LUH promoter. Based on these data, we propose a new model for the regulation of pectin biosynthesis during plant growth and development.
[Show abstract][Hide abstract] ABSTRACT: The orange albedo alcohol insoluble solids (AIS) wall preparation, material prepared from the albedo tissue of mature orange fruit, contained 55mol% pectic polysaccharides, including homogalacturonan (HG), rhamnogalacturonan I (RG I) and rhamnogalacturonan II (RG II) together with Type I arabinogalactan (AG) and arabinan. It also contained cellulose (22mol%) and other non-cellulosic polysaccharides (14mol%), including xyloglucans (XGs) (10mol%), heteromannans (2mol%) and heteroxylans (2mol%). Sequential extraction of the AIS performed with NaOAc, CDTA, Na2CO3, 1M KOH and 4M KOH produced soluble fractions (Frs.) with different relative proportions of these polysaccharides. The NaOAc and CDTA Frs. were composed predominantly of pectic polysaccharides (89 and 97mol%, respectively) with only very low proportions of other non-cellulosic polysaccharides. Na2CO3 (50mM) released highly ramified pectic polysaccharides, whereas stronger alkali (1M and 4M KOH) liberated mostly non-cellulosic polysaccharide, comprised predominantly of XG. The NaOAc, CDTA and Na2CO3 Frs. contain low levels of 2-O-methyl-Fuc and 2-O-methyl-Xyl, suggesting the presence of RG II. To obtain the RG II for structural characterization, the NaOAc fraction was digested with a combination of endo-polygalacturonase (endo-PG; PG I and PG II) and exo-PG, and the degradation product was fractionated by anion exchange and size exclusion chromatography. The isolated product (approx. 0.5% of orange albedo AIS) contained all six diagnostic sugars and has a glycosyl linkage composition consistent with the structural model of RG II.
[Show abstract][Hide abstract] ABSTRACT: The cell wall is essential for viability of fungi and is an effective drug target in pathogens such as Candida albicans. The contribution of post-transcriptional gene regulators to cell wall integrity in C. albicans is unknown. We show that the C. albicans Ccr4-Pop2 mRNA deadenylase, a regulator of mRNA stability and translation, is required for cell wall integrity. The ccr4/pop2 mutants display reduced wall β-glucans and sensitivity to the echinocandin caspofungin. Moreover, the deadenylase mutants are compromised for filamentation and virulence. We demonstrate that defective cell walls in the ccr4/pop2 mutants are linked to dysfunctional mitochondria and phospholipid imbalance. To further understand mitochondrial function in cell wall integrity, we screened a Saccharomyces cerevisiae collection of mitochondrial mutants. We identify several mitochondrial proteins required for caspofungin tolerance and find a connection between mitochondrial phospholipid homeostasis and caspofungin sensitivity. We focus on the mitochondrial outer membrane SAM complex subunit Sam37, demonstrating that it is required for both trafficking of phospholipids between the ER and mitochondria and cell wall integrity. Moreover, in C. albicans also Sam37 is essential for caspofungin tolerance. Our study provides the basis for an integrative view of mitochondrial function in fungal cell wall biogenesis and resistance to echinocandin antifungal drugs.
[Show abstract][Hide abstract] ABSTRACT: Arabinogalactan-proteins (AGPs), found in the culture medium of suspension cells of Araucaria angustifolia grown in plant growth regulator-free and plant growth regulator-containing BM media, BM0 and BM2, respectively, were evaluated quantitatively and qualitatively. The concentrated extracellular fractions (CEFs), obtained from suspension cell cultures grown for 20 days in BM0 and BM2 media yielded two fractions, CEF-0 and CEF-2, respectively. CEF-0 and CEF-2 was submitted to selective precipitation using the beta-glucosyl Yariv reagent (beta-GlcY) to isolate AGPs for structural characterization; this yielded fractions designated CEF-0YPF and CEF-2YPF, respectively. The monosaccharide composition analysis established that samples were composed of Rha, Ara, Gal and uronic acid in a molar ratio 3:37:55:5 (CEF-0YPF) and 1:37:58:4 (CEF-2YPF), although trace amounts (<0.5 mol%) of Xyl were also found. Methylation analysis of CEF-YPF fractions showed similar results for both CEF-0YPF and CEF-2YPF, with non-reducing terminal units of Araf, Arap, Galp, Rhap and Xylp, as well as 3-O-substituted and 5-O-substituted Araf units and 3-O-substituted, 6-O-substituted and 3,6-di-O-substituted Galp units. The amino acid composition analysis established Ser, Ala, and Hyp as major amino acids in both samples. In conclusion, this investigation has shown that CEF-0YPF and CEF-2YPF contain macromolecules having typical AGP characteristics, including a Hyp/Ala/Ser-rich protein moiety, a (1-->3) and/or (1-->6) linked beta-d-galactopyranosyl main chain substituted by Gal, Ara, Rha and Xyl residues, and binding affinity for beta-GlcY and monoclonal anti-AGP antibodies.
[Show abstract][Hide abstract] ABSTRACT: The barley (Hordeum vulgare) brittle stem mutants, fs2, designated X054 and M245, have reduced levels of crystalline cellulose compared with their parental lines Ohichi and Shiroseto. A custom-designed microarray, based on long oligonucleotide technology and including genes involved in cell wall metabolism, revealed that transcript levels of very few genes were altered in the elongation zone of stem internodes, but these included a marked decrease in mRNA for the HvCesA4 cellulose synthase gene of both mutants. In contrast, the abundance of several hundred transcripts changed in the upper, maturation zones of stem internodes, which presumably reflected pleiotropic responses to a weakened cell wall that resulted from the primary genetic lesion. Sequencing of the HvCesA4 genes revealed the presence of a 964-bp solo long terminal repeat of a Copia-like retroelement in the first intron of the HvCesA4 genes of both mutant lines. The retroelement appears to interfere with transcription of the HvCesA4 gene or with processing of the mRNA, and this is likely to account for the lower crystalline cellulose content and lower stem strength of the mutants. The HvCesA4 gene maps to a position on chromosome 1H of barley that coincides with the previously reported position of fs2.
[Show abstract][Hide abstract] ABSTRACT: Cell walls in commercially important cereals and grasses are characterized by the presence of (1,3;1,4)-β-d-glucans. These polysaccharides are beneficial constituents of human diets, where they can reduce the risk of hypercholesterolemia, type II diabetes, obesity and colorectal cancer. The biosynthesis of cell wall (1,3;1,4)-β-d-glucans in the Poaceae is mediated, in part at least, by the cellulose synthase-like CslF family of genes. Over-expression of the barley CslF6 gene under the control of an endosperm-specific oat globulin promoter results in increases of more than 80% in (1,3;1,4)-β-d-glucan content in grain of transgenic barley. Analyses of (1,3;1,4)-β-d-glucan fine structure indicate that individual CslF enzymes might direct the synthesis of (1,3;1,4)-β-d-glucans with different structures. When expression of the CslF6 transgene is driven by the Pro35S promoter, the transgenic lines have up to sixfold higher levels of (1,3;1,4)-β-d-glucan in leaves, but similar levels as controls in the grain. Some transgenic lines of Pro35S:CslF4 also show increased levels of (1,3;1,4)-β-d-glucans in grain, but not in leaves. Thus, the effects of CslF genes on (1,3;1,4)-β-d-glucan levels are dependent not only on the promoter used, but also on the specific member of the CslF gene family that is inserted into the transgenic barley lines. Altering (1,3;1,4)-β-d-glucan levels in grain and vegetative tissues will have potential applications in human health, where (1,3;1,4)-β-d-glucans contribute to dietary fibre, and in tailoring the composition of biomass cell walls for the production of bioethanol from cereal crop residues and grasses.
[Show abstract][Hide abstract] ABSTRACT: Arabinogalactan-proteins (AGPs) are a family of highly glycosylated hydroxyproline-rich glycoproteins implicated in several
aspects of plant growth and development. (β-d-glucosyl)3 Yariv phenylglycoside (β-GlcY), commonly known as Yariv reagent, selectively binds AGPs. We treated cell suspension cultures
angustifolia, the Brazilian pine, with β-GlcY and observed inhibition of biomass increase in a culture medium with 50μM β-GlcY. However,
the growth was not inhibited by (α-d-galactosyl)3 Yariv phenylglycoside (α-GalY) which does not bind AGPs. Fluorescein diacetate staining of cells indicated that β-GlcY severely
affected cell viability. However, cell swelling, bursting and release of cellular contents, all characteristics of necrotic
cell death, were not observed in β-GlcY-treated cells. Instead, programmed cell death (PCD) structural changes such as cytoplasmic
shrinkage and condensation were observed in β-GlcY-treated cells. In addition, callose accumulation, which is another marker
of PCD, was also observed in β-GlcY-treated cells. The use of both, Ac-VEID-CHO, an inhibitor of caspase-like proteolytic
activity related to PCD, and phenyl methyl sulphonyl fluoride (PMSF), a protease inhibitor known to suppress PCD, in the culture
medium did not reverse the growth inhibition caused by β-GlcY. These data indicate that the β-GlcY-induced inhibition of Araucaria cell’s growth is related to AGP perturbation, and also that this growth inhibition is due to increased cell death not driven
-Caspase-6-inhibitor-Gymnospermae-Phenyl methyl sulphonyl fluoride-Programmed cell death-Yariv phenylglycoside
Trees 01/2010; 24(2):391-398. · 1.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A chromogenic assay using RB5-dGA, Reactive Black 5 (RB5) dye covalently coupled to de-arabinosylated gum arabic (dGA), was developed for rapid screening of beta-galactanases. dGA was prepared by partial acid hydrolysis (0.25M trifluoroacetic acid for 2h at 90-95 degrees C) of gum Arabic (GA) from Acacia senegal. The dGA exhibited a median molecular mass of approximately 10kDa, corresponding to a degree of polymerisation (DP) approximately 60. It was devoid of Ara residues, and contained mostly Galp (68mol%) together with GlcpA (30mol%). The Galp residues were (1,6)- (34mol%), (1,3)- (3mol%) and (1,3,6)- (26mol%) linked, and the GlcAp residues were primarily terminal (28mol%) together with a small amount of (1,4)-linked (2mol%), as expected for a type II (3,6)-galactan. The new chromogenic assay is simple, cost effective, relatively sensitive, and is specific for either beta-(1-->3)- and/or beta-(1-->6)-d-galactanases. It will enable routine large-scale screening of beta-galactanases from crude enzyme preparations and microorganism cultures, and is suitable for profiling activity during purification processes.
Carbohydrate research 08/2009; 344(15):1941-6. · 2.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Maintaining cell wall integrity is essential for fungal growth and development. We describe two mutants with altered expression of a gene, LmIFRD, from the ascomycete Leptosphaeria maculans. Truncation of the LmIFRD transcript in a T-DNA insertional mutant led to slower germination, less sporulation and loss-of-pathogenicity towards Brassica napus, whereas silencing of the LmIFRD transcript led to increased germination, sporulation and earlier infection. The increased tolerance to cell wall lysing enzymes and cell wall-disrupting compounds of the T-DNA mutant contrasts with decreased tolerance of the silenced mutant and suggests altered cell wall integrity and accessibility to 1,3-linked glucan and chitin. Lectin binding experiments and monosaccharide analysis revealed altered polysaccharide content and structure within the cell wall of the LmIFRD mutants, notably increased 1,3-linked galactose and chitin within the cell wall of the T-DNA mutant. This is the first analysis of monosaccharide linkage composition of cell walls of spores and mycelia for any dothideomycete.
Fungal Genetics and Biology 07/2009; 46(9):695-706. · 3.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A procedure has been developed for the isolation of cell walls from the hyphae of the causal agent for barley leaf scald, Rhynchosporium secalis (Oudem) J.J. Davis. Based primarily on monosaccharide linkage analysis, but also on the limited use of linkage-specific glucan hydrolases and solvent fractionation, the walls consist predominantly of (1,3/1,6)-beta-D-glucans, (1,3;1,4)-beta-D-glucans, galactomannans of (1,2;1,6)-Manp residues and (1,5)-galactofuranosyl [(1,5)-Galf] side chains, rhamnomannans of (1,6)-Manp residues and rhamnopyranosyl [(1,2)-Rhap] side chains, and chitin; the walls also contain approximately 23% (w/w) protein. Electron microscopy shows the presence of distinct inner and outer wall layers. Treatment of wall preparations with guanidine hydrochloride dissolves the outer layer and enables separate analysis of the inner and outer walls. The insoluble, inner wall layer is composed of (1,3/1,6)-beta-D-glucans, galacto- and rhamnomannans, (1,3;1,4)-beta-D-glucans and chitin, whereas the soluble outer wall material contains a high proportion of rhamnomannan, and smaller proportions of galactomannan, (1,3;1,4)-beta-D-glucan and (1,3/1,6)-beta-D-glucan with only trace levels of chitin. It was confirmed by immunochemical and enzymatic analysis that at least a portion of the (1,3;1,4)-beta-D-glucan component of the inner wall exists as a (1,3;1,4)-beta-D-glucan. The analyses not only provide information that is important for a complete understanding of the interactions between R. secalis and barley, but they also identify potential targets for the development of fungicides or resistant transgenic barley varieties.