Isolation and structural identification of diarabinosyl 8-O-4-dehydrodiferulate from maize bran insoluble fibre

The first saccharide ester of a dehydrodiferulic acid (DFA) other than 5-5-DFA has been isolated from maize bran insoluble fibre after acidic hydrolysis and fractionation by gel chromatography and semi-preparative RP-HPLC. HPLC-MS along with 1D, 2D and 3D NMR spectra provided the requisite structural evidence that it is the di-5-O-l-arabinosyl ester of 8-O-4-DFA. Although a range of DFAs have been well authenticated as components released from the cell walls of grasses, the only structural evidence for a DFA attached to polysaccharides had been from 5-5-DFA. The isolation of the 8-O-4-ester demonstrates that polysaccharides in maize cell walls, and presumably in all grasses, are cross-linked through dehydrodiferulates other than 5-5-dehydrodiferulate.     

White light promotes the formation of diferulic acid in maize coleoptile cell walls by enhancing PAL activity

To elucidate the mechanism by which white fluorescent light (5 W m^-2) stimulates the formation of diferulic acid (DFA) in cell walls, the effect of light on phenylalanine-and tyrosine-ammonia-lyase (PAL, EC 4.3.1.5 and TAL, EC 4.3.1.5) and peroxidase activities was studied using coleoptiles of maize ( Zea mays L. cv. Cross Bantam T51). Growth rate of dark-grown coleoptiles was highest at the basal zone and decreased towards the tip, while continuous irradiation caused an inhibition of growth, especially at the basal zone. Light decreased the cell wall extensibility in all zones of the coleoptile. The amounts of DFA, ferulic acid (FA) and p -coumaric acid ( p -CA) increased by severalfold in cell walls of light-grown maize coleoptiles as compared with those grown in the dark. Strong correlations were observed between the increase in the contents of either DFA, FA or p -CA and the decrease in cell wall extensibility. Light decreased the wall-bound peroxidase activity. No correlation was found between DFA content and peroxidase activity. The activities of PAL and TAL were enhanced upon white light irradiation. The increment in either DFA, FA or p -CA content was correlated with an increase in PAL activity, but not with that in TAL activity. White light may promote DFA formation in the cell walls of maize coleoptiles by enhancing PAL activity.     

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.     

Genetic and molecular basis of grass cell-wall degradability. I. Lignin-cell wall matrix interactions

Lignification limits grass cell-wall digestion by herbivores. Lignification is spatially and temporally regulated, and lignin characteristics differ between cell walls, plant tissues, and plant parts. Grass lignins are anchored within walls by ferulate and diferulate cross-links, p-coumarate cyclodimers, and possibly benzyl ester and ether cross-links. Cell-wall degradability is regulated by lignin concentration, cross-linking, and hydrophobicity but not directly by most variations in lignin composition or structure. Genetic manipulation of lignification can improve grass cell-wall degradability, but the degree of success will depend on genetic background, plant modification techniques employed, and analytical methods used to characterize cell walls.     

Matrix solubilization and cell wall weakening by β-expansin (group-1 allergen) from maize pollen

Beta-expansins accumulate to high levels in grass pollen, a feature apparently unique to grasses. These proteins, which are major human allergens, facilitate pollen tube penetration of the maize stigma and style (the silk). Here we report that treatment of maize silk cell walls with purified β-expansin from maize pollen led to solubilization of wall matrix polysaccharides, dominated by feruloyated highly substituted glucuronoarabinoxylan (60%) and homogalacturonan (35%). Such action was selective for cell walls of grasses, and indicated a target preferentially found in grass cell walls, probably the highly substituted glucuronoarabinoxylan. Several tests for lytic activities by β-expansin were negative and polysaccharide solubilization had weak temperature dependence, which indicated a non-enzymatic process. Concomitant with matrix solubilization, β-expansin treatment induced creep, reduced the breaking force and increased the plastic compliance of wall specimens. From comparisons of the pH dependencies of these processes, we conclude that matrix solubilization was linked closely to changes in wall plasticity and breaking force, but not so closely coupled to cell wall creep. Because matrix solubilization and increased wall plasticity have not been found with other expansins, we infer that these novel activities are linked to the specialized role of grass pollen β-expansins in promotion of penetration of the pollen tube through the stigma and style, most likely by weakening the middle lamella.     

Evaluation by electron microscopy and anaerobic culture of types of rumen bacteria associated with digestion of forage cell walls.

Different morphological types of rumen bacteria which degraded cell walls of forage grasses with various in vitro digestibilities were evaluated with electron microscopy. The majority of these bacteria (i.e., about 70% or more) consisted of two distinct types: (i) encapsulated cocci and (ii) irregularly shaped bacteria, resembling major fiber digesters found in the rumen. Each type was capable of degrading structurally intact cell walls. Differences (P less than or equal to 0.02) in the percent ratio of encapsulated cocci to irregularly shaped bacteria were observed between Bermuda grass and fescue; the ratio of encapsulated cocci to irregularly shaped bacteria between Bermuda grass and orchard grass was similar and variations were high. The proportion of irregularly shaped bacteria usually increased with increased time of digestion. Differences (P greater than 0.1) were not found in the percentage ratio of encapsulated cocci to irregularly shaped bacteria attached to specific tissue types in either Bermuda grass or fescue. However, encapsulated cocci tended to be more prevalent on sclerenchyma than other tissues in Bermuda grass, but less prevalent on sclerenchyma than other tissues in fescue. Transmission electron microscopy of tissue digestion of rapidly degraded orchard grass blades revealed that mesophyll, parenchyma bundle sheath, and parts of the epidermal cell wall apparently were degraded without direct attachment of bacteria although bacteria were near the cell walls undergoing digestion. Anaerobic growth studies showed that the total culturable bacteria developing on medium 10 and media containing carbohydrates similar to those in forage cell walls (i.e., pectin, xylan, and cellobiose) were 80% higher from rumen bacterial populations adapted in vitro to cell walls of orchard grass compared to those from Bermuda grass; the number of colonies from the orchard grass-adapted population was significantly (P less than or equal to 0.05) greater on the medium containing xylan. Filter paper tests showed that the cellulolytic activity of populations adapted to fescue was greater than that of orchard grass or Bermuda grass.     

Purification and characterization of four beta-expansins (Zea m 1 isoforms) from maize pollen

Four proteins with wall extension activity on grass cell walls were purified from maize (Zea mays) pollen by conventional column chromatography and high-performance liquid chromatography. Each is a basic glycoprotein (isoelectric point = 9.1-9.5) of approximately 28 kD and was identified by immunoblot analysis as an isoform of Zea m 1, the major group 1 allergen of maize pollen and member of the beta-expansin family. Four distinctive cDNAs for Zea m 1 were identified by cDNA library screening and by GenBank analysis. One pair (GenBank accession nos. AY104999 and AY104125) was much closer in sequence to well-characterized allergens such as Lol p 1 and Phl p 1 from ryegrass (Lolium perenne) and Phleum pretense, whereas a second pair was much more divergent. The N-terminal sequence and mass spectrometry fingerprint of the most abundant isoform (Zea m 1d) matched that predicted for AY197353, whereas N-terminal sequences of the other isoforms matched or nearly matched AY104999 and AY104125. Highly purified Zea m 1d induced extension of a variety of grass walls but not dicot walls. Wall extension activity of Zea m 1d was biphasic with respect to protein concentration, had a broad pH optimum between 5 and 6, required more than 50 micro g mL(-1) for high activity, and led to cell wall breakage after only approximately 10% extension. These characteristics differ from those of alpha-expansins. Some of the distinctive properties of Zea m 1 may not be typical of beta-expansins as a class but may relate to the specialized function of this beta-expansin in pollen function.     

Phylogenetic comparative methods complement discriminant function analysis in ecomorphology

In ecomorphology, Discriminant Function Analysis (DFA) has been used as evidence for the presence of functional links between morphometric variables and ecological categories. Here we conduct simulations of characters containing phylogenetic signal to explore the performance of DFA under a variety of conditions. Characters were simulated using a phylogeny of extant antelope species from known habitats. Characters were modeled with no biomechanical relationship to the habitat category; the only sources of variation were body mass, phylogenetic signal, or random “noise.” DFA on the discriminability of habitat categories was performed using subsets of the simulated characters, and Phylogenetic Generalized Least Squares (PGLS) was performed for each character. Analyses were repeated with randomized habitat assignments. When simulated characters lacked phylogenetic signal and/or habitat assignments were random, <5.6% of DFAs and <8.26% of PGLS analyses were significant. When characters contained phylogenetic signal and actual habitats were used, 33.27 to 45.07% of DFAs and <13.09% of PGLS analyses were significant. False Discovery Rate (FDR) corrections for multiple PGLS analyses reduced the rate of significance to <4.64%. In all cases using actual habitats and characters with phylogenetic signal, correct classification rates of DFAs exceeded random chance. In simulations involving phylogenetic signal in both predictor variables and predicted categories, PGLS with FDR was rarely significant, while DFA often was. In short, DFA offered no indication that differences between categories might be explained by phylogenetic signal, while PGLS did. As such, PGLS provides a valuable tool for testing the functional hypotheses at the heart of ecomorphology. Am J Phys Anthropol 153:663–674, 2014. © 2013 Wiley Periodicals, Inc.     

Difructose anhydrides: their mass-production and physiological functions

Difructose anhydrides (DFAs) are the smallest cyclic disaccharides consisting of two fructose residues, and are expected to have novel physiological functions from their unique structures and properties. For mass-production of alpha-D-fructofuranose-beta-D-fructofuranose-2',1:2,3'-dianhydride (DFA III) and beta-D-fructofuranose-beta-D-fructofuranose-2',6:2,6'-dianhydride (DFA IV), Arthrobacter sp. H65-7 and A. nicotinovorans GS-9 were selected as the best producers of inulase II, which produced DFA III from inulin and LFTase, which produced DFA IV from levan. The enzymes were purified and their genes were subsequently cloned and expressed in E. coli at higher levels than in the original bacteria. Thus, it became possible to provide a large amount of DFA III and DFA IV for evaluating their physiological properties. DFA III and DFA IV have half the sweetness of sucrose, but cannot be digested by the digestive system of rats. Their use by the intestinal microorganisms was observed in vivo even though their assimilation could not be detected in vitro. This implied that they were degraded by an unknown system in the intestine. It was also found that they affected calcium absorption mainly in the small intestine through mechanisms different from the known stimulants such as fructooligosaccharides and raffinose.     

Incorporation of 14C-l-arabinose into polysaccharides of maize root-tips

Summary [1-¹⁴C]-l-arabinose was supplied to maize roots over a range of concentrations extending from 0.1 M to 0.04 mM. In each case, only xylose and arabinose units in the cell wall polysaccharides became labelled. However, although uptake increased with concentration, the conversion of l-arabinose to these cell wall units was not greatly influenced by raising the external sugar concentration, and there was no marked accumulation of UDP-pentose under any of the experimental conditions tested. Furthermore, specific activity of the arabinose isolated from the cell wall hydrolysates was always higher than that of the xylose. Because the labelling was so specific, patterns of pentose deposition could be followed by preparing autoradiographs of sections from roots fed with ¹⁴C-l-arabinose. In the pith and cortex, which are typically parenchymatous tissues, the maximum rate of incorporation was observed in cell walls at around 2 mm from the cap-stele junction. These cells had just reached their full width and were about to undergo a phase of rapid elongation. Results are in essential aggrement with those obtained earlier with d-glucuronate in similar experiments.     

The influence of ageing on cell wall composition and degradability of three maize genotypes

Stem segments of the maize (Zea mavs L.) hybrids LGH, Eta Ipho (EI) and a brown midrib mutant. INRA 260 bm3 (bm3) were freeze-dried, ground and analysed for cell wall content, hemicellulose, cellulose, lignin and in vitro cell wall degradability with rumen fluid. Stem cross-sections, stained with acid phloroglucinol (AP) and chlorine sulphite (CS) showed an increased intensity in staining during maturation, but no considerable difference in staining intensity was observed between genotypes. The lignin content increased during maturation with evidently less lignin in bm3 than in EI and LGH. However, cell wall degradability did not differ between the older stem segments of EI and bm3, although the amount of lignin in LGH was twice that of bm3.

It can be concluded that an increase in lignin content occurs simultancously with a decrease in cell wall degradability within a genotype. However, between different genotypes the lignin content is not an indicator of degree of cell wall degradability.     

Modification of chemical properties of cell walls by silicon and its role in regulation of the cell wall extensibility in oat leaves

Effects of silicon on the mechanical and chemical properties of cell walls in the second leaf of oat (Avena sativa L.) seedlings were investigated. The cell wall extensibility in the basal region of the second leaf was considerably higher than that in the middle and subapical regions. Externally applied silicon increased the cell wall extensibility in the basal region, but it did not affect the extensibility in the middle and subapical regions. The amounts of cell wall polysaccharides and phenolic compounds, such as diferulic acid (DFA) and ferulic acid (FA), per unit length were lower in the basal region than in the middle and subapical regions of the leaf, and silicon altered these amounts in the basal region. In this region, silicon decreased the amounts of matrix polymers and cellulose per unit length and of DFA and FA, both per unit length and unit matrix polymer content. Silicon treatment also lowered the activity of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) in the basal region. In contrast, the amount of silicon in cell walls increased in response to silicon treatment in three regions. These results suggest that in the basal region, silicon reduces the net wall mass and the formation of phenolic acid-mediated cross-linkages between wall polysaccharides. Such modifications of wall architecture may be responsible for the silicon-induced increase in the cell wall extensibility in oat leaves.     

Suppression of cell wall stiffening along coleoptiles of wheat (Triticum aestivum L.) seedlings grown under osmotic stress conditions

Effects of polyethylene glycol (PEG)-induced osmotic stress on the mechanical properties of cell walls and the levels of their components were investigated along intact wheat (Triticum aestivum L.) coleoptiles. Stress-relaxation analysis showed that the cell walls of stressed coleoptiles were loosened as compared with those of unstressed ones not only in the apical but in the basal regions. The amounts of wall-bound ferulic acid (FA) and diferulic acid (DFA) of stressed coleoptiles were substantially lower than those of unstressed ones in all regions. The cellulose and hemicellulose contents increased toward the coleoptile base. Osmotic stress reduced the cellulose content in the basal region but it slightly affected the hemicellulose content. The molecular weight of hemicellulose in the apical region of stressed coleoptiles was higher than that of unstressed ones, while that in the basal region was almost the same in both coleoptiles. FA, DFA and cellulose contents correlated with the cell wall mechanical property. The amount and molecular weight of hemicellulose, however, did not correlate. These results suggest that the reduced levels of FA and DFA in all regions and cellulose in the basal region of wheat coleoptiles are involved in maintaining the cell wall extensibility under osmotic stress.     

Alterations in structure, chemistry, and biodegradability of grass lignocellulose treated with the white rot fungi Ceriporiopsis subvermispora and Cyathus stercoreus.

The white rot fungi Ceriporiopsis subvermispora FP-90031-sp and Cyathus stercoreus ATCC 36910 were evaluated for their ability to delignify Bermuda grass (Cynodon dactylon) stems and improve biodegradability. Compositional and structural alterations in plant cell walls effected by the fungi were determined by nuclear magnetic resonance spectroscopy, gas chromatography of alkali-treated residues, microspectrophotometry, and electron microscopy. Contaminating bacteria and fungi, which grew from unsterilized Bermuda grass stems, did not alter the improvement in grass biodegradability by either of the fungi from that of gas-sterilized stems. The biodegradation of stems by ruminal microorganisms, after treatment for 6 weeks with C. subvermispora or C. stercoreus, was improved by 29 to 32% and by 63 to 77%, respectively; dry weight losses caused by pretreatment with the fungi were about 20% over that in untreated, control stems. Both fungi preferentially removed aromatics to carbohydrates, and C. subvermispora removed proportionately more guaiacyl units than did C. stercoreus. Substantial amounts of ester-linked p-coumaric and ferulic acids were removed by both fungi, and about 23 and 41% of total aromatics (determined after 4 M NaOH direct treatment) were removed from the plant biomass after incubation with C. subvermispora and C. stercoreus, respectively. UV absorption microspectrophotometry indicated that ester-linked phenolic acids were totally removed from the parenchyma cell walls, and these cells were readily and completely degraded by both fungi. However, aromatic constituents were only partially removed from the more recalcitrant sclerenchyma cell walls, resulting in variation in electron density and random digestion pits after incubation with fiber-degrading bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron Microscopy of the Microbial Populations Present and Their Modes of Attack on Various Cellulosic Substrates Undergoing Digestion in the Sheep Rumen

Cotton fibers and various cell wall preparations from grass leaves and from the feces of sheep fed on dried grass were placed in the sheep rumen in bags made from 5-μm-mesh nylon cloth. After periods of from 3 to 48 h, bags were removed, and the contents were fixed, embedded, sectioned, and stained for electron microscopy. Some of the bacteria present were seen to be closely associated with the cell walls, either tunneling within them or making very close contact. Evidence was obtained for differential digestion of cell walls and of the layers within them. Distinct differences were noticed between bacterial populations attacking the more susceptible wall types and those attacking feces cell walls and cotton fibers. Among the latter, the dominant form was a long, thin rod with a typical gramnegative cell wall structure, different from that described for Bacteroides succinogenes S85 or for Butyrivibrio fibrisolvens.     

Tissue-specific biomass recalcitrance in corn stover pretreated with liquid hot-water: SEM imaging (part 2)

In the first part of our work, we combined compositional analysis, pretreatment and enzyme hydrolysis for fractionated pith, rind, and leaf tissues from a hybrid stay-green corn, in order to identify the role of structural characteristics on enzyme hydrolysis of cell walls. Hydrolysis experiments coupled with chemical analysis of the different fractions of corn stover showed significant differences in cell wall structure before and after liquid hot water pretreatment. The extent of enzyme hydrolysis followed the sequence rind < leaves < pith with 90% conversion of cellulose to glucose in 24 h in the best cases. Since similar lignin contents remained after liquid hot water pretreatment of leaves, rind, and pith, our results indicated that the amount of lignin alone is not sufficient to explain the different enzymatic hydrolysis characteristics of the fractions. While the role of structural characteristics on enzyme hydrolysis of cell walls is measured as described in part I, the SEM images presented in this part II of our work show that sugar yields from enzymatic hydrolysis of corn fractions correlate with changes in plant cell wall structure both before and after liquid hot water pretreatment.     

Chromosomal and cell size analysis of cold tolerant maize

Summary C-band number, guard cell length, and chloroplast number per guard cell were determined for eight maize populations. These populations consisted of maize selected for cold tolerance at the University of Nebraska as well as the original unselected populations. The genome size of these populations had previously been determined. C-band number fluctuated concertedly with the changes in genome size indicating that deletions and additions of constitutive heterochromatin occurred during selection, resulting in altered genome sizes. Guard cell size of all the cold tolerant populations was greater than the cell size of the respective nonselected populations. Chloroplast number per guard cell was also higher in all the cold tolerant populations than in their parental populations, but the increases were not statistically significant. The results indicate that changes in genome size that occurred during selection for cold tolerance are the result of changes in amounts of C-band heterochromatin and that the selection process results in an increase in cell size in the cold tolerant populations.     

Chemical composition,cell wall features and degradability of stem,leaf blade and sheath in untreated and alkali-treated rice straw

Three dominant morphological fractions (i.e. leaf blade (LB), leaf sheath (LS) and stem) were analysed for chemical composition and ruminal degradability in three rice straw varieties. In one variety treated with alkali, cell wall features were also characterized using Fourier Transform Infrared Spectroscopy (FTIR) and Scanning Electron Microscopy. The highest concentrations of cell wall carbohydrates (hemicellulose and cellulose) were observed in LS, whereas the highest concentrations of non-fibre (silica, phenolic compounds and CP) and lignin were recorded for LB. The stem had the lowest silica and hemicellulose contents but intermediate levels of other components. In terms of ruminal degradability, stem ranked higher than LB, which was followed by LS. Hemicellulose was found to be less degradable than either dry matter or cellulose in all the three fractions investigated. FTIR results indicated that the highest levels of hydrogen bonding, esterification and crystallinity within the cell wall components belonged to LS. In the alkaline treatment, these indices decreased to a larger extent for leaf fractions and a greater improvement was achieved in the degradability of LB and LS compared with that of stem. In the 24-h ruminal incubation, the silicified layer of epidermis and the underlying cell walls showed a rigid structure in the control fractions, whereas the treatment with NaOH resulted in crimping of the silicified cuticle layer and the loss of integrity in cell structure. Despite the highest silica and lignin contents observed in LB, LS showed the lowest degradability, which might be due to its high level of hydrogen bonding, crystallinity and esterification within its cell wall components as well as its high hemicellulose content.     

Cell wall oxalate oxidase modifies the ferulate metabolism in cell walls of wheat shoots

Oxalate oxidase (OXO) utilizes oxalate to generate hydrogen peroxide, and thereby acts as a source of hydrogen peroxide. The present study was carried out to investigate whether apoplastic OXO modifies the metabolism of cell wall-bound ferulates in wheat seedlings. Histochemical staining of OXO showed that cell walls were strongly stained, indicating the presence of OXO activity in shoot walls. When native cell walls prepared from shoots were incubated with oxalate or hydrogen peroxide, the levels of ester-linked diferulic acid (DFA) isomers were significantly increased. On the other hand, the level of ester-linked ferulic acid (FA) was substantially decreased. The decrease in FA level was accounted neither by the increases in DFA levels nor by the release of FA from cell walls during the incubation. After the extraction of ester-linked ferulates, considerable ultraviolet absorption remained in the hemicellulosic and cellulose fractions, which was increased by the treatment with oxalate or hydrogen peroxide. Therefore, a part of FA esters may form tight linkages within cell wall architecture. These results suggest that cell wall OXO is capable of modifying the metabolism of ester-linked ferulates in cell walls of wheat shoots by promoting the peroxidase action via supply of hydrogen peroxide.