AFM studies of water-soluble wheat arabinoxylans--effects of esterase treatment

The degradation products of water-soluble wheat arabinoxylans treated with Aspergillus niger ferulic acid esterase (FAEA-able to cleave 5,5'- and 8-O-4'-ferulic acid dimers) have been characterised by atomic force microscopy (AFM) and size exclusion chromatography. The AFM images of arabinoxylans confirmed that a small proportion ( approximately 15%) of the population of arabinoxylan molecules contain xylan-based branches attached to the xylan-based backbone. Treatment with FAEA reduced the contour length of the molecules suggesting that certain dimeric ferulic acid linkages may play a previously unconfirmed role in the elongation of arabinoxylans. Overnight treatment with FAEA led to a reduction in the density of branches suggesting that they may also be linked to the backbone through phenolic linkages.     

Dynamic changes in cell wall polysaccharides during wheat seedling development

Changes in arabinoxylan content and composition during development of wheat seedlings were investigated. The cell walls isolated from the seedlings showed an increasing content of arabinoxylan during development, which could be correlated to increased activity of xylan synthase and arabinoxylan arabinosyltransferase. Arabinoxylan changed from initially having a high degree of arabinose substitution to a much lower degree of substitution. beta-Glucan was present in the walls at the early stages of development, but was actively degraded after day 4. Increased deposition of arabinoxylan did not take place until beta-glucan had been fully degraded. Ferulic and p-coumaric acid esters were present at all points but increased significantly from day 3 to 6, where lignification began. Ferulic acid dimers did not appear in the cell wall until day three and the different ferulic acid dimers varied in the course of accumulation. The ratio of ferulic acid dimers to free ferulic acid was maximal at the time when the wall had been depleted for beta-glucan, which had not yet been fully replaced by arabinoxylan. This pattern suggests a role for ferulic acid dimers in stabilizing the wall during the transition from a flexible to a more rigid structure. To investigate if the same changes could be observed within a single seedling, 7 day old seedlings were divided into four sections and the walls were analyzed. Some of the changes observed during the seedling development could also be observed within a single seedling, when analyzing the segments from the elongation zone at the base to the top of the leaf. However, the expanding region of older seedlings was much richer in hydroxycinnamates than the expanding region of younger seedlings. Diferulic acids are stabilizing the wall in the transition phase from an expanding to a mature wall. This transition can take place in different manners depending on the cell and tissue type.     

Change in wall composition of transfer and aleurone cells during wheat grain development

In addition to the starchy endosperm, a specialized tissue accumulating storage material, the endosperm of wheat grain, comprises the aleurone layer and the transfer cells next to the crease. The transfer cells, located at the ventral region of the grain, are involved in nutrient transfer from the maternal tissues to the developing endosperm. Immunolabeling techniques, Raman spectroscopy, and synchrotron infrared micro-spectroscopy were used to study the chemistry of the transfer cell walls during wheat grain development. The kinetic depositions of the main cell wall polysaccharides of wheat grain endosperm, arabinoxylan, and (1–3)(1–4)-β-glucan in transfer cell walls were different from kinetics previously observed in the aleurone cell walls. While (1–3)(1–4)-β-glucan appeared first in the aleurone cell walls at 90°D, arabinoxylan predominated in the transfer cell walls from 90 to 445°D. Both aleurone and transfer cell walls were enriched in (1–3)(1–4)-β-glucan at the mature stage of wheat grain development. Arabinoxylan was more substituted in the transfer cell walls than in the aleurone walls. However, arabinoxylan was more feruloylated in the aleurone than in the transfer cell walls, whatever the stage of grain development. In the transfer cells, the ferulic acid was less abundant in the outer periclinal walls while para-coumarate was absent. Possible implications of such differences are discussed.     

Antitumor and immunomodulatory activity of arabinoxylans: a major constituent of wheat bran

We aimed to investigate the antitumor activity of wheat bran arabinoxylans, including the role of its immunostimulatory effect. In S180 tumor-bearing mice arabinoxylan administration significantly inhibited the growth of mouse transplantable tumors and remarkably promoted thymus and spleen indexes, splenocyte proliferation, natural killer cell and macrophage phagocytosis activity, interleukin 2 production, and delayed-type hypersensitivity reaction. In addition, it increased peripheral leukocyte count, and bone-marrow cellularity in tumor-bearing mice. As the antitumor activity of arabinoxylans may be mediated via the improvement in the immune response, they can be considered an antitumor agent with immunomodulatory activity.     

Studies on the oxidative cross-linking of feruloylated arabinoxylans from wheat flour and wheat bran

Feruloylated arabinoxylans isolated from wheat flour and wheat bran were compared in their cross-linking behaviour with respect to viscosity properties and cross-linking products formed when various oxidative agents were applied to dilute solutions. Optimal conditions for each oxidative agent were investigated. In case of hydrogen peroxide and peroxidase, similar conditions were found for both types of arabinoxylans but wheat bran arabinoxylans gave a larger viscosity increase upon cross-linking than those of wheat flour.

When glucose, glucoseoxidase and peroxidase or ammonium persulphate were used as oxidative agents, differences in the concentration of reagent needed to induce cross-linking and in viscosity increase were observed. The distribution of coupling products for both types of arabinoxylans and the different oxidative treatments was approximately 5 : 3 : 1 : 1 for 8-5, 8-O-4, 8-8 and 5-5, respectively. The low ferulate recovery after oxidative treatment was assumed to be caused by formation of unknown compounds, such as higher oligomers and lignin-linked products.

A 1 : 1 mixture of flour arabinoxylan and feruloylated pectin showed a maximum synergistic effect on viscosity upon oxidative treatment using hydrogen peroxide and peroxidase. Both polysaccharides were shown to participate in cross-linking.     

Hemicellulosic polymers from the cell walls of beeswing wheat bran: Part I,polymers solubilised by alkali at 2°

Cell-wall material from beeswing wheat bran was sequentially extracted with 0.05m NaOH at 2°, m KOH at 2° and 20°, and 4m KOH at 20° followed by delignification and further extraction with m and 4m KOH at 20°, to leave the α-cellulose residue which contained a significant amount of arabinoxylan. The hemicellulosic polymers solubilised by m KOH at 2°, which represented ∼20% of the dry weight of the cell walls, were fractionated by graded precipitation with alcohol prior to anion-exchange chromatography and then subjected to methylation analysis. The major polymers were closely related, highly branched arabinoxylans, slightly branched arabinoxylans, and arabinoxylans in close association with xylogucans (arabinoxylan-xyloglucan complexes); the arabinoxylans were acidic and were associated with various amounts of phenolics. The various polymers exhibit heterogeneity, and phenolic ester and phenolic ether cross-links play a major role in the architecture of the cell walls.     

Expression of a fungal ferulic acid esterase in suspension cultures of tall fescue (<Emphasis Type="Italic">Festuca arundinacea</Emphasis>) decreases cell wall feruloylation and increases rates of cell wall digestion

In the cell walls of grasses ferulic acid is esterified to arabinosyl residues in arabinoxylans that can then undergo oxidative coupling reactions to form ferulate dehydrodimers, trimers and oligomers which function to cross-link cell-wall polysaccharides, limiting cell wall degradability. Fungal ferulic acid esterase can release both esterified monomeric and dimeric ferulic acids from these cell wall arabinoxylans making the cell wall more susceptible to further enzymatic attack and increasing cell wall degradability. Non-embryogenic cell suspension cultures of Festuca arundinacea expressing a Aspergillus niger ferulic acid esterase (faeA) targeted to either the apoplast, or endoplasmic reticulum under the control of a constitutive actin promoter, or to the vacuole under the control of a soybean heat shock promoter, were established and FAE activity determined in the cells and medium during a growth cycle. Analysis of the ester-linked ferulates of the cell walls showed that all three transformed cell lines had both reduced ferulate levels and increased levels of xylanase mediated release of wall phenolics on autodigestion as well as increased rates of cell wall digestion in a simulated rumen environment, when compared to control non-transformed cells.     

Remodelling of arabinoxylan in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>) endosperm cell walls during grain filling

Previous studies using spectroscopic imaging have allowed the spatial distribution of structural components in wheat endosperm cell walls to be determined. FT-IR microspectroscopy showed differing changes in arabinoxylan (AX) structure, during grain development under cool/wet and hot/dry growing conditions, for differing cultivars (Toole et al. in Planta 225:1393–1403, 2007). These studies have been extended using Raman microspectroscopy, providing more details of the impact of environment on the polysaccharide and phenolic components of the cell walls. NMR studies provide complementary information on the types and levels of AX branching both early in development and at maturity. Raman microspectroscopy has allowed the arabinose:xylose (A/X) ratio in the cell wall AX to be determined, and the addition of ferulic acid and related phenolic acids to be followed. The changes in the A/X ratio during grain development were affected by the environmental conditions, with the A/X ratio generally being slightly lower for samples grown under cool/wet conditions than for those from hot/dry conditions. The degree of esterification of the endosperm cell walls with ferulic acid was also affected by the environment, being lower under hot/dry conditions. The results support earlier suggestions that AX is either delivered to the cell wall in a highly substituted form and is remodelled through the action of arabinoxylan arabinofuranohydrolases or arabinofuranosidases, or that low level substituted AX are incorporated into the wall late in cell wall development, reducing the average degree of substitution, and that the rate of this remodelling is influenced by the environment. ¹H NMR provided a unique insight into the chemical structure of intact wheat endosperm cell walls, providing qualitative information on the proportions of mono- and disubstituted AX and the levels of branching of adjacent units. The A/X ratio did not change greatly with either the development stage or the growth conditions, but the ratio of mono- to disubstituted Xylp residues increased markedly (by about fourfold) in the more mature samples, confirming the changes in branching levels determined using FT-IR. To the best of our knowledge, this is the first time that intact endosperm cell walls have been studied by ¹H NMR.     

Enzymic Analysis of Feruloylated Arabinoxylans (Feraxan) Derived from Zea mays Cell Walls I : Purification of Novel Enzymes Capable of Dissociating Feraxan Fragments from Zea mays Coleoptile Cell Wall

Three novel β-xylan xylanohydrolases capable of dissociating ferulated arabinoxylan (Feraxan) from maize (Zea mays L. hybrid B73 × Mo17) coleoptile sections and two conventional β-xylan xylanohydrolases (xylanases) were purified from a Bacillus subtilis industrial enzyme preparation (Novo Ban L-120). The Feraxan-dissociating enzymes (designated as feraxanases) exhibit optimum activities between pH 6.5 and 7.0 and have common molecular weights of 45 kilodaltons as studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two xylanases exhibit their optimum activities between pH 4.5 and 6.0 and have common molecular weights of 27 kilodaltons. Feraxanases liberate oligomeric fragments, which accounted for the following percentages of walls of Zea mays coleoptile sections that had been pretreated by boiling in 80% ethanol: 76% of the ferulic acid, 96% of the arabinose, 71% of the xylose, 27% of the galactose, 50% of the uronic acid, and 4% of the glucose. Monomers, dimers, trimers, or tetramers were not found among enzyme digestion products. The enzymes hydrolyzed both Feraxan in intact cell wall and maize arabinoxylans extracted from walls by alkaline solutions but did not degrade other substrates including larch arabinoxylan and Rhodymenia xylan. Structural analyses of the fragments released by the enzymes from the maize cell wall indicated the presence of 2,4/3,4-linked-xylopyranosyl, terminal-arabinofuranosyl, 5-linked-arabinofuranosyl, 4-linked-xylopyranosyl, terminal-glucuronopyranosyl, and ferulic acid as major components. This result is consistent with the idea that most of the fragments were derived from Feraxan. Because of high enzyme specificity and substantial recovery of digestion products from maize cell walls, these new enzymes offer opportunities not only for enhanced structural analyses of cell walls but also for assistance in protoplast preparation from cereals.     

Structural characteristics of the cold-water-soluble arabinoxylans from the white flour of the soft wheat variety Kadet

Cold-water-soluble arabinoxylan polymers from the soft wheat variety Kadet were isolated by a stepwise (NH₄)₂SO₄ precipitation and freed from co-precipitating proteins by pronase digestion. The purified arabinoxylans, representing 0·5% of the dry weight of the white flour, were fractionated by graded precipitation with ethanol. Monosaccharide analysis revealed the major carbohydrate fractions to contain arabinoxylans only. The arabinoxylans are heterogeneous in molecular size. Determination of the carbohydrate composition in conjunction with methylation analysis and ¹³C-NMR spectroscopy, indicated that they can be divided into specific groups, based on characteristic differences with respect to -xylopyranose to -arabinofuranose ratio and in 2,3,4-tri-:3,4-di-:4-mono-substituted -xylopyranose ratio.     

Enzymic Analysis of Feruloylated Arabinoxylans (Feraxan) Derived from Zea mays Cell Walls : III. Structural Changes in the Feraxan during Coleoptile Elongation

Changes in structural features of feraxan (feruloylated arabinoxylans) in cell walls during development of maize (Zea mays L.) coleoptiles were investigated by analysis of fragments released by feraxanase, a specific enzyme purified from Bacillus subtilis. The following patterns were identified: (a) The total quantity of carbohydrate dissociated from a given dry weight of cell wall by feraxanase remained rather constant throughout the initial 10 days of coleoptile development. However, during the same period the proportion of ferulic acid in the fraction increased 12-fold. The absolute amount of ferulic acid per coleoptile also increased rapidly during this developmental phase. (b) Fragments dissociated by the enzyme were resolved into feruloylated and nonferuloylated components by reversed phase chromatography. While the quantity of feruloylated fractions represented a small portion of the total arabinoxylan during the phase of maximum coleoptile elongation (days 2-4) this component increased in abundance to reach a plateau (after 8-10 days). In contrast, nonferuloylated fractions were most abundant during the stage of maximum elongation but declined to a constant level by day 6. (c) Glycosidic linkage analysis of carbohydrate reveals that substitution of the xylan backbone of feraxan by arabinosyl residues decreased during coleoptile growth. We conclude that significant incorporation of ferulic acid occurs and/or more feruloyated domains are added to the arabinoxylan during development. This augmentation in phenolic acids is accompanied by a concerted displacement of arabinosyl residues and/or a reduction in the incorporation of regions enriched in arabinosyl sidechains.     

Effects of soluble corn bran arabinoxylans on cecal digestion, lipid metabolism, and mineral balance (Ca, Mg) in rats

The effects of soluble corn bran arabinoxylans on cecal digestion, lipid metabolism, and mineral utilization [calcium (Ca) and magnesium (Mg)] were investigated in rats adapted to semipurified diets. The diets provided either 710 g/kg wheat starch alone (control) or 610 g/kg wheat starch plus 100 g/kg corn soluble fiber (arabinoxylans) and either 0 or 2 g/kg cholesterol (control + cholesterol and arabinoxylans + cholesterol, respectively). Compared with rats fed the control diets, rats fed the arabinoxylan diets had significant cecal hypertrophy (+50% after 3 days of the fiber adaptation) and an accumulation of short-chain fatty acids, especially propionic acid (up to 45% in molar percentage). Arabinoxylans enhanced the cecal absorption of Ca and Mg (from 0.07 to 0.19 micromol/min for Ca and from 0.05 to 0.23 micromol/min for Mg). Mg balance was enhanced by arabinoxylans (+25%). The arabinoxylan diet markedly reduced the cholesterol absorption from 50% of ingested cholesterol in controls up to approximately 15% in rats adapted to the arabinoxylans diet. Arabinoxylans were effective in lowering plasma cholesterol (approximately -20%). There was practically no effect of the diets on cholesterol in d > 1.040 lipoproteins (high density lipoproteins) whereas arabinoxylans were very effective in depressing cholesterol in d < 1.040 lipoproteins (especially in triglyceride-rich lipoproteins). Corn fermentable fiber decreased the accumulation of cholesterol in the liver. In parallel, the arabinoxylan diet counteracted the downregulation of 3-hydroxy-3-methylglutaryl-CoA by cholesterol. These data suggest that arabinoxylans may have a great impact on intestinal fermentation, mineral utilization, and cholesterol metabolism.     

Modification of esterified cell wall phenolics increases vulnerability of tall fescue to herbivory by the fall armyworm

Feruloylation of arabinoxylan in grass cell walls leads to cross-linked xylans. Such cross-linking appears to play a role in plant resistance to pathogens and insect herbivores. In this study, we investigated the effect of ferulate cross-linking on resistance to herbivory by fall armyworm (Spodoptera frugiperda) making use of genetically modified tall fescue [Schedonorus arundinaceus (Festuca arundinacea)] expressing a ferulic acid esterase gene. Mature leaves of these plants have significant reduced levels of cell wall ferulates and diferulates but no change in acid detergent lignin. These reduced levels of esterified cell wall ferulates in transgenic plants had a positive effect on all measures of armyworm larval performance examined. More larvae survived (89 vs. 57?%) and grew faster (pupated 2.1?days sooner) when fed transgenic leaves with reduced levels of cell wall ferulates, than when fed control tall fescue leaves where levels of cell wall ferulates were not altered. Overall, mortality, growth and food utilization were negatively associated with level of esterified cell wall ferulates and diferulates in leaves they were fed. This study is the first to use transgenic plants with modified level of cell wall esterified ferulates to test the role of feruloylation in plant resistance to insects. It is concluded that the accumulation of ferulates and the cross-linking of arabinoxylans via diferulate esters in the leaves of tall fescue underlies the physical barrier to insect herbivory. Reducing ferulate cross-linking in grass cell walls could increase susceptibility of these plants to insect folivores.     

Intracellular feruloylation of arabinoxylan in wheat: evidence for feruloyl-glucose as precursor

Incorporation of [(3)H]arabinose and [(14)C]ferulic acid into soluble and polymeric fractions from suspension-cultured wheat (Triticum aestivum L.) cells and the corresponding extracellular medium was studied. The major part of these products was identified as arabinoxylan and two proteins of 40 and 100 kDa. The time course suggests an intracellular synthesis of feruloylated arabinoxylan with feruloyl-glucose as substrate. In contrast, synthesis of feruloylated proteins appears to occur with feruloyl-CoA as precursor. Intracellular formation of ferulic acid dimers is limited to 8,5'-diferulic acid, while other dimers appear to be formed extracellularly. [(3)H]Arabinose was incorporated into polymeric material in both the cellular and in the medium fraction while [(14)C]ferulic was only found in polymers from the cellular fraction, indicating synthesis of both feruloylated and non-feruloylated arabinoxylan by the cells.     

Feruloyl oligosaccharides stimulate the growth of Bifidobacterium bifidum

Insoluble dietary fiber from wheat bran contains some feruloyl groups linked to the arabinose residues in the cell wall arabinoxylan. Treatment of wheat bran insoluble dietary fiber with xylanase from Bacillus subtilis yielded feruloyl oligosacchairdes, which were purified with Amberlite XAD-2. Saponification of the feruloyl oligosaccharides released ferulic acid and arabinoxylan oligosaccharides which consist of arabinose and xylose. The effect of the feruloyl oligosacchairdes on the growth of Bifidobacterium bifidum F-35 was investigated in vitro. The B. bifidum produced acid when cultivated anaerobically in TPY broth with 0.5% feruloyl oligosacchairdes as the carbohydrate source. The biomass yield of the B. bifidum increased with increasing the concentration of feruloyl oligosaccharides in TPY broth. The maximum cell growth was increased by 50% in TPY broth supplemented with 0.1% feruloyl oligosaccharides compared to TPY broth. These results indicated that the growth of B. bifidum F-35 was promoted by the feruloyl oligosaccharides from wheat bran insoluble dietary fiber, and not suppressed by the ferulic acid moiety of them.     

Expression of a fungal ferulic acid esterase increases cell wall digestibility of tall fescue (Festuca arundinacea)

In the cell walls of forage grasses, ferulic acid is esterified to arabinoxylans and participates with lignin monomers in oxidative coupling pathways to generate ferulate–polysaccharide–lignin complexes that cross-link the cell wall. Such cross-links hinder cell wall degradation by ruminant microbes, reducing plant digestibility. In this study, genetically modified Festuca arundinacea plants were produced expressing an Aspergillus niger ferulic acid esterase (FAEA) targeted to the vacuole. The rice actin promoter proved to be effective for FAEA expression, as did the cauliflower mosaic virus (CaMV) 35S and maize ubiquitin promoters. Higher levels of expression were, however, found with inducible heat-shock and senescence promoters. Following cell death and subsequent incubation, vacuole-targeted FAEA resulted in the release of both monomeric and dimeric ferulic acids from the cell walls, and this was enhanced several fold by the addition of exogenous endo-1,4-β-xylanase. Most of the FAEA-expressing plants showed increased digestibility and reduced levels of cell wall esterified phenolics relative to non-transformed plants. It is concluded that targeted FAEA expression is an effective strategy for improving wall digestibility in Festuca and, potentially, other grass species used for fodder or cellulosic ethanol production.     

Phenolic acids and their carbohydrate esters in rice endosperm cell walls

Ferulic acid, p-coumaric acid and diferulic acid were detected in the alkaline extract of rice endosperm cell walls. The amount of each component was estimated as 9.1, 2.5 and 0.56 mg/g cell wall, respectively. Several phenolic-carbohydrate esters were isolated from the enzymatic digest of this cell wall, which included a series of ferulic acid esters of arabinoxylan fragments and also some fractions containing a high proportion of diferulic acid.     

A wheat biorefining strategy based on solid-state fermentation for fermentative production of succinic acid

In this study, a novel generic feedstock production strategy based on solid-state fermentation (SSF) has been developed and applied to the fermentative production of succinic acid. Wheat was fractionated into bran, gluten and gluten-free flour by milling and gluten extraction processes. The bran, which would normally be a waste product of the wheat milling industry, was used to produce glucoamylase and protease enzymes via SSF using Aspergillus awamori and Aspergillus oryzae, respectively. The resulting solutions were separately utilised for the hydrolysis of gluten-free flour and gluten to generate a glucose-rich stream of over 140gl(-1) glucose and a nitrogen-rich stream of more than 3.5gl(-1) free amino nitrogen. A microbial feedstock consisting of these two streams contained all the essential nutrients required for succinic acid fermentations using Actinobacillus succinogenes. In a fermentation using only the combined hydrolysate streams, around 22gl(-1) succinic acid was produced. The addition of MgCO(3) into the wheat-derived medium improved the succinic acid production further to more than 64gl(-1). These results demonstrate the SSF-based strategy is a successful approach for the production of a generic feedstock from wheat, and that this feedstock can be efficiently utilised for succinic acid production.     

Hemicellulosic complexes from the cell walls of runner bean (Phaseolus coccineus).

The 1 M-KOH extract from the depectinated cell walls of parenchymatous tissues of mature runner bean (Phaseolus coccineus) on neutralization, dialysis and concentration gave insoluble (hemicellulose A) and soluble (hemicellulose B) carbohydrate complexes in the weight ratio 2:1. Both fractions contained polysaccharide, protein and polyphenolic material. The structural features of the carbohydrates were examined by methylation analysis. Hemicellulose A contained mainly pectic arabinogalactan, with lesser amounts of arabinoxylan and glucan. Sequential fractionation of hemicellulose B by anion-exchange and hydroxyapatite chromatography gave a range of polysaccharide-protein-polyphenolic complexes. The main polysaccharides in these complexes were (acidic) arabinoxylans, galactans, arabinogalactans 1 and 2 and xyloglucans. The proteins contained small amounts of hydroxyproline, but were rich in aspartic acid and glutamic acid. Attempts to determine the nature of the polyphenolic material were unsuccessful. The structural features of the polysaccharide-protein-polyphenolic complexes are discussed in relation to the structure of the cell walls of parenchymatous tissues.