Cell Wall Polysaccharide Composition Research Articles

STRUCTURE AND CHEMISTRY OF THE XYLEM OF ARBORESCENT SPECIES OF BLECHNUM FROM SOUTH AMERICA

The xylem in three arborescent species of Blechnum section Lomariocycas was studied in detail using SEM, TEM, FT-IR spectroscopy and sugar composition analysis. The overall structure of root and rhizome metaxylem tracheids was similar in the three species analyzed, and characterized by mostly scalariform pitting of these multifaceted cells. Pit membrane thickness and porosity varied according to the stage of tracheid maturation. Approximately rounded deposits resembling vestures were observed in the outer pit apertures of some tracheids. Under TEM, thickenings like one-sided tori appeared on the tracheid side of tracheid-to-parenchyma contact walls; some parenchyma cells showed, in addition, features of transfer cells. As the increase in stature creates new constraints in terms of biomechanical support and water transport in plants, the characteristics found in Blechnum xylem might be related to optimization of conductive efficiency and safety. Chemical analyses of roots and rhizomes of B. yungense revealed similar levels of G-type lignin deposited in the xylem cell walls. Such lignin is the most common in ferns, including other arborescent genera. Preliminary analysis of cell wall polysaccharide composition of both root and rhizome xylem, yielded cellulose, xyloglucans and xylans with low amounts of mannans and pectins. The xylem of rhizomes had higher amounts of cellulose than root xylem. Our results are discussed in the context of functional and evolutionary aspects of xylem ferns.

Investigating lignin key features in maize lignocelluloses using infrared spectroscopy.

Lignins and their cross-linking to hemicelluloses detrimentally affect the cellulose-to-ethanol conversion of grass lignocelluloses. Screening appropriate grass cell walls and their compositional changes during the various steps of the process calls for a high-throughput analytical technique. Such a performance can be fulfilled by Fourier transform mid-infrared (FT-MIR) spectroscopy. In the present paper, a set of maize cell walls from mature stems were selected, including brown midrib samples. Lignin fractions were isolated by mild acidolysis to obtain a set of purified maize lignin standards. The lignin content and the percentage of lignin-derived p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) thioacidolysis monomers were determined. In addition, the composition of cell wall polysaccharides, as well as the amount of ester-linked p-coumaric (CA) and ferulic (FA) acids, was measured by wet chemistry. Partial least square (PLS) analyses were applied to infrared and chemical data of cell walls. The resulting models showed a good predictive ability with regard to the lignin content, to the frequency of S (or G) thioacidolysis monomers, and to the level of ester-linked CA of maize cell walls. The loading plots and regression coefficients revealed relevant infrared absorption bands.

Cell-wall polysaccharide composition and glycanase activity of Silene vulgaris callus transformed with rolB and rolC genes.

The aim of this research is to investigate the effects of the Agrobacterium rhizogenes rol genes on the composition of cell-wall polysaccharides and glycanase activity in the campion callus. The expression of the rolC gene reduces the yield of campion pectin, while the expression of the rolB or rolC gene inhibits the volumetric production of both pectin and intracellular arabinogalactan. The rol genes are involved in regulating the activity of glycanases and esterases, thereby contributing to the modification of polysaccharide structures, their molecular weight (Mw) and the degree of pectin methyl esterification (DE). The increase in pectin arabinose residue appears to be connected to a decrease in intracellular and extracellular α-l-arabinofuranosidase activity in transgenic campion calluses. In transgenic calluses expressing the rolB and rolC genes, the increase in pectin galactose residue is likely due to a decrease in β-galactosidase activity. The decrease in the Mw of pectin and its d-galacturonic acid content appears to be connected to an increase in extracellular polygalacturonase activity. Finally, the increase in pectinesterase activity causes a decrease in the DE of pectin. Thus, the expression of rolB and rolC genes in campion callus has a considerable effect on pectin's sugar composition, DE and Mw, while it appears to have an insignificant influence on intracellular and extracellular arabinogalactans.

Unusual linear polysaccharides: (1→5)-α-l-Arabinan, (1→3)-(1→4)-α-d-glucan and (1→4)-β-d-xylan from pulp of buriti (Mauritia flexuosa), an edible palm fruit from the Amazon region

A structural characterisation of polysaccharides extracted from ripe pulp of edible buriti, an exotic tropical fruit from Mauritia flexuosa palm was carried out. After fractionation by freeze–thaw treatment of an aqueous extract (BW), a fraction containing a linear (1→5)-α-l-arabinan and a linear (1→3)-(1→4)-α-d-glucan was obtained (0.2% yield). The proportion of (1→3)- and (1→4)-linked Glcp units was 1:4. After fractionation by freeze–thaw and Fehling treatments of an alkaline extract (BK), a linear (1→4)-β-d-xylan was purified (0.6% yield). Their chemical structures were performed by sugar composition, methylation and NMR spectroscopy analysis. In addition, these results together with monosaccharide analysis of SBW and SBK are discussed in relation to literature data on polysaccharide compositions of primary cell walls of palms (commelinid monocotyledons).

A combined chemical and enzymatic method to determine quantitatively the polysaccharide components in the cell wall of yeasts.

A reliable method to determine cell wall polysaccharides composition in yeast is presented, which combines acid and enzymatic hydrolysis. Sulphuric acid treatment is used to determine mannans, whereas specific hydrolytic enzymes are employed in a two sequential steps to quantify chitin and the proportion of β-(1,3) and β-(1,6)-glucan in the total β-glucan of the cell wall. In the first step, chitin and β-(1,3)-glucan were hydrolysed into their corresponding monomers N-acetylglucosamine and glucose, respectively, by the combined action of a chitinase from Streptomyces griseus and a pure preparation of endo/exo-β-(1,3)-glucanase from Trichoderma species. This step was followed by addition of recombinant endo-β-(1,6)-glucanase from Trichoderma harzianum with β-glucosidase from Aspergillus niger to hydrolyse the remaining β-glucan. This latter component corresponded to a highly branched β-(1,6)-glucan that contained about 75-80% of linear β-(1,6)-glucose linked units as deduced from periodate oxidation. We validated this novel method by showing that the content of β-(1,3), β-(1,6)-glucan or chitin was dramatically decreased in yeast mutants defective in the biosynthesis of these cell wall components. Moreover, we found that heat shock at 42°C in Saccharomyces cerevisiae and treatment of this yeast species and Candida albicans with the antifungal drug caspofungin resulted in 2- to 3-fold increase of chitin and in a reduction of β-(1,3)-glucan accompanied by an increase of β-(1,6)-glucan, whereas ethanol stress had apparently no effect on yeast cell wall composition.

Effect of soluble and insoluble fibers within the in vitro fermentation of chicory root pulp by human gut bacteria.

The aim of this research was to study the in vitro fermentation of chicory root pulp (CRP) and ensiled CRP (ECRP) using human fecal inoculum. Analysis of carbohydrate levels in fermentation digests showed that 51% of all CRP carbohydrates were utilized after 24 h of fermentation. For ECRP, having the same cell wall polysaccharide composition as CRP, but with solubilization of 4 times more of CRP pectin due to ensiling, the fermentation was quicker than with CRP as 11% more carbohydrates were utilized within the first 12 h. The level of fiber utilization for ECRP after 24 h was increased by 8% compared to CRP. This effect on fiber utilization from ECRP seemed to arise from (i) increased levels of soluble pectin fibers (arabinan, homogalacturonan, and galactan) and (ii) ahypothesized more open structure of the remaining cell walls in ECRP, which was more accessible to degradation than the CRP cell wall network.

Serotype specific polymerase chain reaction identifies a higher prevalence of streptococcus mutans serotype k and e in a random group of children with dental caries from the Southern region of India.

Background:The development of dental caries has been associated with the oral prevalence of Streptococcus mutans. Four serotypes of S. mutans have been reported, namely serotype c, e, f, and k that are classified based on the composition and linkages of cell wall polysaccharides, response to physiological reactions, sero-specificity and 16s rRNA homology. Although the oral prevalence of S. mutans serotype c in Indian subjects with or without caries is known, the prevalence of the other three serotypes, e, f, and k are not known. Hence in this study, we have investigated the occurrence of the e, f, and k serotypes in children with or without caries within the age group of 6-12 years.Materials and Methods:Genomic DNA isolated from whole saliva of caries active (CA) and caries free (CF) groups were first screened for the presence of S. mutans by strain specific polymerase chain reaction (PCR). Those samples that tested positive for the presence of S. mutans were further analyzed by serotype specific PCR to identify the prevalence of the serotypes.Results:Strain specific PCR indicated a higher prevalence of S. mutans in CA group (80%) relative to CF group (43%). Further analysis of the S. mutans positive samples in both groups indicated a higher prevalence of serotype k and e, followed by serotype f in CA group.Conclusion:The present data clearly establishes a novel S. mutans serotype prevalence hierarchy in children from this region, compared with those that have been reported elsewhere. Besides, the data are also clinically significant as the occurrence of serotype k has been associated with infective endocarditis.

Development and application of a high throughput carbohydrate profiling technique for analyzing plant cell wall polysaccharides and carbohydrate active enzymes

BackgroundPlant cell wall polysaccharide composition varies substantially between species, organs and genotypes. Knowledge of the structure and composition of these polysaccharides, accompanied by a suite of well characterised glycosyl hydrolases will be important for the success of lignocellulosic biofuels. Current methods used to characterise enzymatically released plant oligosaccharides are relatively slow.ResultsA method and software was developed allowing the use of a DNA sequencer to profile oligosaccharides derived from plant cell wall polysaccharides (DNA sequencer-Assisted Saccharide analysis in High throughput, DASH). An ABI 3730xl, which can analyse 96 samples simultaneously by capillary electrophoresis, was used to separate fluorophore derivatised reducing mono- and oligo-saccharides from plant cell walls. Using electrophoresis mobility markers, oligosaccharide mobilities were standardised between experiments to enable reproducible oligosaccharide identification. These mobility markers can be flexibly designed to span the mobilities of oligosaccharides under investigation, and they have a fluorescence emission that is distinct from that of the saccharide labelling. Methods for relative and absolute quantitation of oligosaccharides are described. Analysis of a large number of samples is facilitated by the DASHboard software which was developed in parallel. Use of this method was exemplified by comparing xylan structure and content in Arabidopsis thaliana mutants affected in xylan synthesis. The product profiles of specific xylanases were also compared in order to identify enzymes with unusual oligosaccharide products.ConclusionsThe DASH method and DASHboard software can be used to carry out large-scale analyses of the compositional variation of plant cell walls and biomass, to compare plants with mutations in plant cell wall synthesis pathways, and to characterise novel carbohydrate active enzymes.

Plant cell wall profiling by fast maximum likelihood reconstruction (FMLR) and region-of-interest (ROI) segmentation of solution-state 2D 1H–13C NMR spectra

BackgroundInterest in the detailed lignin and polysaccharide composition of plant cell walls has surged within the past decade partly as a result of biotechnology research aimed at converting biomass to biofuels. High-resolution, solution-state 2D 1H–13C HSQC NMR spectroscopy has proven to be an effective tool for rapid and reproducible fingerprinting of the numerous polysaccharides and lignin components in unfractionated plant cell wall materials, and is therefore a powerful tool for cell wall profiling based on our ability to simultaneously identify and comparatively quantify numerous components within spectra generated in a relatively short time. However, assigning peaks in new spectra, integrating them to provide relative component distributions, and producing color-assigned spectra, are all current bottlenecks to the routine use of such NMR profiling methods.ResultsWe have assembled a high-throughput software platform for plant cell wall profiling that uses spectral deconvolution by Fast Maximum Likelihood Reconstruction (FMLR) to construct a mathematical model of the signals present in a set of related NMR spectra. Combined with a simple region of interest (ROI) table that maps spectral regions to NMR chemical shift assignments of chemical entities, the reconstructions can provide rapid and reproducible fingerprinting of numerous polysaccharide and lignin components in unfractionated cell wall material, including derivation of lignin monomer unit (S:G:H) ratios or the so-called SGH profile. Evidence is presented that ROI-based amplitudes derived from FMLR provide a robust feature set for subsequent multivariate analysis. The utility of this approach is demonstrated on a large transgenic study of Arabidopsis requiring concerted analysis of 91 ROIs (including both assigned and unassigned regions) in the lignin and polysaccharide regions of almost 100 related 2D 1H–13C HSQC spectra.ConclusionsWe show that when a suitable number of replicates are obtained per sample group, the correlated patterns of enriched and depleted cell wall components can be reliably and objectively detected even prior to multivariate analysis. The analysis methodology has been implemented in a publicly-available, cross-platform (Windows/Mac/Linux), web-enabled software application that enables researchers to view and publish detailed annotated spectra in addition to summary reports in simple spreadsheet data formats. The analysis methodology is not limited to studies of plant cell walls but is amenable to any NMR study where ROI segmentation techniques generate meaningful results.Please see Research Article: http://www.biotechnologyforbiofuels.com/content/6/1/46/.

Composition and Structure of Sugarcane Cell Wall Polysaccharides: Implications for Second-Generation Bioethanol Production

The structure and fine structure of leaf and culm cell walls of sugarcane plants were analyzed using a combination of microscopic, chemical, biochemical, and immunological approaches. Fluorescence microscopy revealed that leaves and culm display autofluorescence and lignin distributed differently through different cell types, the former resulting from phenylpropanoids associated with vascular bundles and the latter distributed throughout all cell walls in the tissue sections. Polysaccharides in leaf and culm walls are quite similar, but differ in the proportions of xyloglucan and arabinoxylan in some fractions. In both cases, xyloglucan (XG) and arabinoxylan (AX) are closely associated with cellulose, whereas pectins, mixed-linkage-β-glucan (BG), and less branched xylans are strongly bound to cellulose. Accessibility to hydrolases of cell wall fraction increased after fractionation, suggesting that acetyl and phenolic linkages, as well as polysaccharide–polysaccharide interactions, prevented enzyme action when cell walls are assembled in its native architecture. Differently from other hemicelluloses, BG was shown to be readily accessible to lichenase when in intact walls. These results indicate that wall architecture has important implications for the development of more efficient industrial processes for second-generation bioethanol production. Considering that pretreatments such as steam explosion and alkali may lead to loss of more soluble fractions of the cell walls (BG and pectins), second-generation bioethanol, as currently proposed for sugarcane feedstock, might lead to loss of a substantial proportion of the cell wall polysaccharides, therefore decreasing the potential of sugarcane for bioethanol production in the future.

Determining the polysaccharide composition of plant cell walls

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.

Plant Cell Biology: The ABC of Monolignol Transport

Lignins are complex aromatic heteropolymers that reinforce the cell walls of terrestrial plants. A new study identifies an ATP-binding cassette ABC transporter that pumps a monolignol lignin precursor across the plasma membrane.

Assessment of Genetic Variability of Cell Wall Degradability for the Selection of Alfalfa with Improved Saccharification Efficiency

Alfalfa (Medicago sativa L.) has a high potential for sustainable bioethanol production, particularly because of its low reliance on N fertilizer. We assessed near-infrared reflectance spectroscopy (NIRS) as a high-throughput technique to measure cell wall (CW) degradability in a large number of lignified alfalfa stem samples. We also used a powerful immunological approach, glycome profiling, and chemical analyses to increase our knowledge of the composition of CW polysaccharides of alfalfa stems with various levels of degradability. NIRS accurately predicted CW degradability in four different alfalfa cultivars, as assessed by glucose released following enzymatic saccharification (R2 = 0.94). There was a large genetic diversity for enzyme-released glucose. The 10 genotypes with the highest (D+) and 10 genotypes with the lowest (D−) amounts of enzyme-released glucose of a biomass-type (Orca) and a winterhardy-type (54V54) cultivar were further characterized. Glycome profiling showed that there were very few differences in CW polysaccharide composition between the two groups, although the D+ genotypes were at least 35 % more degradable than the D− genotypes. Determination of CW composition by chemical analyses showed that a higher lignin content of the D− genotypes was closely related to their lower enzyme-released glucose (R = −0.83). In each cultivar tested, 20 D+ genotypes and 20 D− genotypes were intercrossed to generate D+ and D− populations. Assessment of CW enzyme-released glucose in the progenies showed that this trait is genetically inherited. The large genetic diversity for enzyme-released glucose and its potential for selection support the huge potential of alfalfa for the sustainable production of bio-ethanol.

Acetylesterase-mediated deacetylation of pectin impairs cell elongation, pollen germination, and plant reproduction.

Pectin is a major component of the primary cell wall of higher plants. Some galacturonyl residues in the backbone of pectinaceous polysaccharides are often O-acetylated at the C-2 or C-3 position, and the resulting acetylesters change dynamically during the growth and development of plants. The processes involve both enzymatic acetylation and deacetylation. Through genomic sequence analysis, we identified a pectin acetylesterase (PAE1) from black cottonwood (Populus trichocarpa). Recombinant Pt PAE1 exhibited preferential activity in releasing the acetate moiety from sugar beet (Beta vulgaris) and potato (Solanum tuberosum) pectin in vitro. Overexpressing Pt PAE1 in tobacco (Nicotiana tabacum) decreased the level of acetyl esters of pectin but not of xylan. Deacetylation engendered differential changes in the composition and/or structure of cell wall polysaccharides that subsequently impaired the cellular elongation of floral styles and filaments, the germination of pollen grains, and the growth of pollen tubes. Consequently, plants overexpressing PAE1 exhibited severe male sterility. Furthermore, in contrast to the conventional view, PAE1-mediated deacetylation substantially lowered the digestibility of pectin. Our data suggest that pectin acetylesterase functions as an important structural regulator in planta by modulating the precise status of pectin acetylation to affect the remodeling and physiochemical properties of the cell wall's polysaccharides, thereby affecting cell extensibility.

Variability of cell wall polysaccharides composition and hemicellulose enzymatic profile in an apple progeny

The genetic variability of apple cell walls polysaccharides chemical composition and structure was assessed in a progeny of 141 individuals harvested over 2 years. The variability of the hemicelluloses oligosaccharides released by glucanase was analyzed by MALDI-TOF MS. The genetic contribution was distinguished from harvest year as well as from parental crossing patterns and scab resistance selection. Results showed that harvest year had a major impact on cell wall polysaccharide composition and structure. Within each harvest, genetic effect impact more significantly cell wall polysaccharide chemistry than does reciprocal crossing or early scab selection. Uronic acids, glucose, galactose and xylose contents as well as some glucomannan and xyloglucan structures have a high heritability. This first cell wall chemotyping of an apple progeny opens the way for future searches of genetic markers for the chemical variability of cell wall polysaccharides.

QUASIMODO 3 (QUA3) is a putative homogalacturonan methyltransferase regulating cell wall biosynthesis in Arabidopsis suspension-cultured cells.

Pectins are complex polysaccharides that are essential components of the plant cell wall. In this study, a novel putative Arabidopsis S-adenosyl-L-methionine (SAM)-dependent methyltransferase, termed QUASIMODO 3 (QUA3, At4g00740), has been characterized and it was demonstrated that it is a Golgi-localized, type II integral membrane protein that functions in methylesterification of the pectin homogalacturonan (HG). Although transgenic Arabidopsis seedlings with overexpression, or knock-down, of QUA3 do not show altered phenotypes or changes in pectin methylation, this enzyme is highly expressed and abundant in Arabidopsis suspension-cultured cells. In contrast, in cells subjected to QUA3 RNA interference (RNAi) knock-down there is less pectin methylation as well as altered composition and assembly of cell wall polysaccharides. Taken together, these observations point to a Golgi-localized QUA3 playing an essential role in controlling pectin methylation and cell wall biosynthesis in Arabidopsis suspension cell cultures.

Polysaccharide composition of mycelium and cell walls of the fungus Penicillium roqueforti

Preliminary data on the polysaccharide composition of mycelium and cell walls of the submergedly grown fungus Penicillium roqueforti were obtained. Mild acid hydrolysis of mycelium and cell walls led to formation of glucose, mannose and galactose, whereas acid treatment under drastic conditions afforded glucosamine as the hydrolysis product of chitin, which content in the cell walls was estimated as 19%. Sequential treatment of the mycelium with hot water and 1 M NaOH at room temperature gave rise to several polysaccharide fractions, which were characterized by their monosaccharide composition. The main fraction obtained by the action of alkali, according to NMR spectroscopy, mass spectrometry and chemical methods of structural analysis data, is a linear alpha-D-glucopyranan containing blocks of (1 --> 3)-linked glucose residues interconnected by (1 --> 4)-linkages. Water-soluble polysaccharides contained linear blocks of (1 --> 5)-linked beta-galactofuranose residues, probably connected with a mannan core. The data obtained may be important for chemotaxonomy of the genus Penicillium.

Overexpression of PhEXPA1 increases cell size, modifies cell wall polymer composition and affects the timing of axillary meristem development in Petunia hybrida

• Expansins are cell wall proteins required for cell enlargement and cell wall loosening during many developmental processes. The involvement of the Petunia hybrida expansin A1 (PhEXPA1) gene in cell expansion, the control of organ size and cell wall polysaccharide composition was investigated by overexpressing PhEXPA1 in petunia plants. • PhEXPA1 promoter activity was evaluated using a promoter-GUS assay and the protein's subcellular localization was established by expressing a PhEXPA1-GFP fusion protein. PhEXPA1 was overexpressed in transgenic plants using the cauliflower mosaic virus (CaMV) 35S promoter. Fourier transform infrared (FTIR) and chemical analysis were used for the quantitative analysis of cell wall polymers. • The GUS and GFP assays demonstrated that PhEXPA1 is present in the cell walls of expanding tissues. The constitutive overexpression of PhEXPA1 significantly affected expansin activity and organ size, leading to changes in the architecture of petunia plants by initiating premature axillary meristem outgrowth. Moreover, a significant change in cell wall polymer composition in the petal limbs of transgenic plants was observed. • These results support a role for expansins in the determination of organ shape, in lateral branching, and in the variation of cell wall polymer composition, probably reflecting a complex role in cell wall metabolism.

Cell wall composition as a maize defense mechanism against corn borers

European and Mediterranean corn borers are two of the most economically important insect pests of maize (Zea mays L.) in North America and southern Europe, respectively. Cell wall structure and composition were evaluated in pith and rind tissues of resistant and susceptible inbred lines as possible corn borer resistance traits. Composition of cell wall polysaccharides, lignin concentration and composition, and cell wall bound forms of hydroxycinnamic acids were measured. As expected, most of the cell wall components were found at higher concentrations in the rind than in the pith tissues, with the exception of galactose and total diferulate esters. Pith of resistant inbred lines had significantly higher concentrations of total cell wall material than susceptible inbred lines, indicating that the thickness of cell walls could be the initial barrier against corn borer larvae attack. Higher concentrations of cell wall xylose and 8-O-4-coupled diferulate were found in resistant inbreds. Stem tunneling by corn borers was negatively correlated with concentrations of total diferulates, 8-5-diferulate and p-coumarate esters. Higher total cell wall, xylose, and 8-coupled diferulates concentrations appear to be possible mechanisms of corn borer resistance.

Composition of cell wall phenolics and polysaccharides of the potential bioenergy crop -Miscanthus

The composition and concentrations of cell wall polysaccharides and phenolic compounds were analyzed in mature stems of several Miscanthus genotypes, in comparison with switchgrass and reed (Arundo donax), and biomass characteristics were correlated with cell wall saccharification efficiency. The highest cellulose content was found in cell walls of M. sinensis‘Grosse Fontaine’ (55%) and in A. donax (47%) and lowest (about 32%) in M. sinensis‘Adagio’. There was little variation in lignin contents across M. sinensis samples (all about 22–24% of cell wall), however, Miscanthus×giganteus (M × g) cell walls contained about 28% lignin, reed – 23% and switchgrass – 26%. The highest ratios of cellulose/lignin and cellulose/xylan were in M. sinensis‘Grosse Fontaine’ across all samples tested. About the same total content of ester-bound phenolics was found in different Miscanthus genotypes (23–27 μg/mg cell wall), while reed cell walls contained 17 μg/mg cell wall and switchgrass contained a lower amount of ester-bound phenolics, about 15 μg/mg cell wall. Coumaric acid was a major phenolic compound ester-bound to cell walls in plants analyzed and the ratio of coumaric acid/ferulic acid varied from 2.1 to 4.3, with the highest ratio being in M × g samples. Concentration of ether-bound hydroxycinnamic acids varied greatly (about two-three-fold) within Miscanthus genotypes and was also the highest in M × g cell walls, but at a concentration lower than ester-bound hydroxycinnamic acids. We identified four different forms of diferulic acid esters bound to Miscanthus cell walls and their concentration and proportion varied in genotypes analyzed with the 5-5-coupled dimer being the predominant type of diferulate in most samples tested. The contents of lignin and ether-bound phenolics in the cell wall were the major determinants of the biomass degradation caused by enzymatic hydrolysis.