Correlation between cell wall extensibility and the content of diferulic and ferulic acids in cell walls of Oryza sativa coleoptiles grown under water and in air

Rice ( Oryza sativa L. cv. Sasanishiki) coleoptiles grown under water achieved greater length than those grown either in air or under water with constant air bubbling. The extensibility of cell walls in coleoptiles grown under water was larger than that in the other treatments. Per unit length of the coleoptile, the content of ferulic and diferulic acids ester-linked to hemicelluloses was higher in air and bubbling type coleoptiles than in water type ones. The extensibility of the coleoptile cell walls correlated with the content of diferulic acids per unit length and per hemicellulose, suggesting that the enhancement of the formation of diferulic acid bridges in hemicelluloses in air or under water with air bubbling makes the cell walls mechanically rigid; thereby inhibiting cell elongation in rice coleoptiles. In addition, the ratio of diferulic acid to ferulic acid was almost constant irrespective of coleoptile age, zone and growth conditions, suggesting that the feruloylation of hemicelluloses is rate-limiting in the formation of diferulic acid bridges in the cell walls of rice coleoptiles.     

Diferulic and ferulic acid in the cell wall of Avena coleoptiles—Their relationships to mechanical properties of the cell wall

Alkaline hydrolysis liberated ferulic and diferulic acid from polysaccharides of the Avena coleoptile ( Avena sativa L. cv. Victory I) cell walls. The amount of the two phenolic acids bound to cell walls increased substantially at day 4–5 after sowing, when the growth rate of the coleoptile started to decrease. The level of these acids was almost constant from the tip to base in 3-day-old coleoptiles, but increased toward the basal zone in 4- and 5-day-old ones. The ratio of diferulic acid to ferulic acid was almost constant irrespective of coleoptile age and zone. An increase in the amount of ferulic and diferulic acids bound to cell wall polysaccharides correlated with a decrease in extensibility and with an increase in minimum stress-relaxation time and relaxation rate of the cell wall. The level of lignin in the cellulose fraction increased as coleoptiles aged, but this increase did not correlate with changes in mechanical properties of the cell walls. These results suggest that ferulic acid, ester-linked to cell wall polysaccharides, is oxidized to give diferulic acid, which makes the cell wall mechanically rigid by cross-linking matrix polysaccharides and results in limited cell extension growth. In addition, it is probable that the step of feruloylation of cell wall polysaccharides is rate-limiting in the formation of in-termolecular bridges by diferulic acid in Avena coleoptile cell walls.     

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.     

Involvement of Cell Wall-Bound Diferulic Acid in Light-Induced Decrease in Growth Rate and Cell Wall Extensibility of Oryza Coleoptiles

Irradiation of white fluorescent light (5 W m^–2) inhibitedthe growth of Oryza coleoptiles. Light irradiation increasedstress-relaxation parameters of coleoptile cell walls, minimumstressrelaxationtime and relaxation rate, and decreased cellwall extensibility (strain/load). Under light conditions, thecontents of ferulic and diferulic acids ester-linked to thehemicellulosic arabinose residue in cell walls increased andcorrelated with the modification of the cell wall mechanicalproperties. These results suggest that light irradiation enhancesthe formation of diferulic acid bridges in hemicelluloses, makingcell walls mechanically rigid and thus inhibits cell elongationin rice coleoptiles. Also, irrespective of coleoptile age orthe presence of light, the ratio of diferulic acid to ferulicacid was almost constant, suggesting that the rate limitingstep in the formation of diferulic acid bridges in Oryza cellwalls is in the step of feruloylation. (Received September 24, 1991; Accepted December 3, 1991)     

Involvement of cell wall-bound phenolic acids in decrease in cell wall susceptibility to expansins during the cessation of rapid growth in internodes of floating rice

The cell walls in the elongating zone of submerged floating rice internodes show high susceptibility to expansins. When internode sections corresponding to such an elongation zone were incubated for 24 h under osmotic stress conditions produced by treatment with 100 mM polyethylene glycol 4000 (PEG), the cell wall susceptibility to expansins remained at its initial level, while the susceptibility of internode sections incubated under unstressed conditions decreased considerably during the same period. The contents of polysaccharides and phenolic acids as ferulic, diferulic and p-coumaric acids in the cell walls of internode sections increased substantially under unstressed conditions, but the increases were almost completely prevented by osmotic stress. Ferulic acid applied to internode sections under osmotic stress reduced the susceptibility of the cell walls to expansins and increased the levels of ferulic and diferulic acids in the cell walls, with little effect on the accumulation of polysaccharides. In contrast, applied p-coumaric acid increased the level of p-coumaric acid in the cell walls without a change in the levels of ferulic and diferulic acids but did not reduce the susceptibility to expansins. These results suggest that the deposition of ferulic and diferulic acids is a primary determinant in regulating the reduction of the susceptibility of cell walls to expansins in floating rice internodes.     

Superoxide scavenging by polyphenols: effect of conjugation and dimerization

The superoxide anion scavenging capacity of two flavonols (quercetin and kaempferol) and some of their conjugates (quercetin-3-rhamnoglucoside, quercetin-3-sophoroside, quercetin-3-sulphate, quercetin-3-glucuronide, kaempferol-3-sophoroside, kaempferol-3-glucuronide) and of several hydroxycinnamic acids (caffeic acid, ferulic acid, 5-5 diferulic acid, 8-O-4 diferulic acid and 8-8 diferulic acid) were studied. Superoxide anions were generated non-enzymatically in a phenazine methosulphate-NADH system and assayed by reduction of nitro-blue tetrazolium. Among the flavonols examined, the most effective scavengers of superoxide anions were the sophoroside, glucuronide and rhamnoglucoside conjugates. Conversely, quercetin-3-sulphate and the flavonol aglycones, exhibited some pro-oxidant activity at the range of concentrations tested (0.5-10 microM). These results show that conjugation has a marked effect on the scavenging capacity of flavonols and that the type of conjugate at the 3-position determines the final superoxide scavenging capacity. Caffeic acid and ferulic acid showed no effect on the generation of superoxide anions by phenazine methosulphate-NADH. However, dimerization of ferulic acid enhanced the superoxide scavenging capacity of this hydroxycinnamic acid, but this depended on the type of linkage between the monomers. The order, from highest to lowest, of superoxide radical scavenging capacity for the dimers of ferulic acid was: 5-5-diferulic acid > 8-O-4-diferulic acid > 8-8-diferulic acid.     

Occurrence,properties, and applications of feruloyl esterases

Feruloyl esterases hydrolyze the ester linkages of ferulic and diferulic acids present in plant cell walls. This interesting group of enzymes also has a potentially broad range of applications in the pharmaceutical and agri-food industries. An overview of the current knowledge of fungal feruloyl esterases focusing on the diverse of substrate specificity and potential applications is presented in this review. Furthermore, biological functions of ferulic acid are discussed.     

Incorporation of proline and aromatic amino acids into cell walls of maize coleoptiles

Sections excised from maize coleoptiles incorporated radioactivity from proline, tyrosine, and phenylalanine into structural components of the cell wall. Only about 2% of radioactivity from proline taken up by sections was incorporated into cell wall; about 24% of that incorporated was in hydroxyproline and the rest remained in proline. In contrast, as much as 40% of the radioactivity from phenylalanine and 30% from tyrosine was incorporated into cell wall material. Most of this radioactivity was in saponifiable ferulic acid. Small amounts of p-coumaric and diferulic acid were found, but only a small fraction of the hemicellulose can possibly be immobilized directly through cross-linking of diferulic esters. Substantial amounts of radioactivity from aromatic amino acids remained insoluble after strong alkali extractions of wall material, and a large fraction of polysaccharide was solubilized by dilute alkali following oxidation of phenolics by acidic NaClO₂. Hence, hemicellulosic material in the cell walls of maize coleoptiles may be organized and cross-linked primarily through alkali-resistant etherified aromatics.     

Involvement of Phenolic Esters in Cell Aggregation of Suspension-Cultured Rice Cells

Fluorescence microscopy of rice (Oryza sativa L.) callus sections showed that all of the walls fluoresced blue in water (pH 5.8) and green in ammonia (pH 10.0), both characteristics of feruloyl esters. Such fluorescence in the walls of cells cultured in Gamborg's B5 medium was much stronger than that in amino acid (AA) medium. Laser scanning microscopy showed that the level of fluorescence was higher in the intercellular layer, especially at corner junctions between cells, suggesting that ferulic acid ester derivatives are located in the middle lamella as well as in the wall. Extracellular polysaccharides appearing during cultivation in AA medium were more highly feruloylated than those in B5 medium during cultivation. Both the levels of ferulic and diferulic acid and the relative proportion of diferulic acid in the walls of cells increased on transfer of the cells cultured in AA medium to B5 medium. The walls of cells cultured in B5 medium maintained constant levels and proportions of the phenolic acids. Removal of phenolic acids from wall preparations by carboxylesterase facilitated the solubilization of noncellulosic polysaccharides. Treatment of the cell aggregates grown in AA medium with an enzyme that hydrolyzes feruloyl esters decreased the size of the aggregates to between 20 and 500 [mu]m, compared with an original size between 200 and 1000 [mu]m. These findings suggest that feruloyl and diferuloyl esters between polysaccharides are involved in the aggregation of cultured rice cells.     

Microbial production,characterization and applications of feruloyl esterases

Feruloyl esterases (FAEs) act synergistically with xylanases to hydrolyze ester-linked ferulic (FA) and diferulic (diFA) acid from cell wall material and therefore play a major role in the degradation of plant biomass. The potential applications of these enzymes with reference to agriculture, food and pharmaceutical industries, are discussed in this review. FAE activities produced by different microorganisms are compared for both submerged and solid state fermentations. In addition, their physicochemical properties and molecular biology are presented.     

Polyethylene glycol treatment promotes metabolic events associated with maize callus morphogenic competence

Metabolic changes were studied, which accompanied the conversion of 6 month old HiII maize non-regenerable (NR) calli into regenerable (R) calli when cultured for 63 days with 10% polyethylene glycol (PEG) (3350 MW) in culture medium.The conversion of 6 month old NR to R callus morphotype caused by PEG application decreased cell wall contents in callus dry mass and changed cell wall phenolics making their profile similar to that of R callus by reduction of lignin and ester- and ether-bound phenolic concentrations, including p-coumaric acid and ester- and ether-bound diferulates and by increase of the ratios of ester- and ether-bound ferulic acid/coumaric acid and ferulic acid/diferulic acid in cell walls of NR callus. Some similar changes of cell wall phenolics caused by PEG application were also found in 48 month old NR callus, that changed the morphology, but did not regenerate plants. However, there were no changes in the old callus in levels of total ester and ether-bound cell wall phenolics and substantially smaller decreases were found in ratios of ester- and ether-bound ferulic acid/coumaric acid and ferulic acid/diferulic acid, as well as in diferulate concentrations compared to young NR callus cultured with PEG. Remarkably, application of PEG also changed the primary metabolism of young NR callus tissues, so that they acquired metabolic features of highly regenerable callus. These data clearly suggest that PEG alters metabolism of NR calli, so they acquire biochemical characteristics of R calli, and that adaptive osmotic adjustments vary in different types of callus tissues.     

A cinnamoyl esterase from Aspergillus niger can break plant cell wall cross-links without release of free diferulic acids.

A cinnamoyl esterase, ferulic acid esterase A, from Aspergillus niger releases ferulic acid and 5-5- and 8-O-4-dehydrodiferulic acids from plant cell walls. The breakage of one or both ester bonds from dehydrodimer cross-links between plant cell wall polymers is essential for optimal action of carbohydrases on these substrates, but it is not known if cinnamoyl esterases can break these cross-links by cleaving one of the ester linkages which would not release the free dimer. It is difficult to determine the mechanism of the reaction on complex substrates, and so we have examined the catalytic properties of ferulic acid esterase A from Aspergillus niger using a range of synthetic ethyl esterified dehydrodimers (5-5-, 8-5-benzofuran and 8-O-4-) and two 5-5-diferulate oligosaccharides. Our results show that the esterase is able to cleave the three major dehydrodiferulate cross-links present in plant cell walls. The enzyme is highly specific at hydrolysing the 5-5- and the 8-5-benzofuran diferulates but the 8-O-4-is a poorer substrate. The hydrolysis of dehydrodiferulates to free acids occurs in two discrete steps, one involving dissociation of a monoesterified intermediate which is negatively charged at the pH of the reaction. Although ferulic acid esterase A was able to release monoesters as products of reactions with all three forms of diesters, only the 5-5- and the 8-O-4-monoesters were substrates for the enzyme, forming the corresponding free diferulic acids. The esterase cannot hydrolyse the second ester bond from the 8-5-benzofuran monoester and therefore, ferulic acid esterase A does not form 8-5-benzofuran diferulic acid. Therefore, ferulic acid esterase A from Aspergillus niger contributes to total plant cell wall degradation by cleaving at least one ester bond from the diferulate cross-links that exist between wall polymers but does not always release the free acid product.     

Intestinal release and uptake of phenolic antioxidant diferulic acids.

Diferulic acids are potent antioxidants and are abundant structural components of plant cell walls, especially in cereal brans. As such, they are part of many human and animal diets and may contribute to the beneficial effect of cereal brans on health. However, these phenolics are ester-linked to cell wall polysaccharides and cannot be absorbed in this form. This study provides the first evidence that diferulic acids can be absorbed via the gastrointestinal tract. The 5-5-, 8-O-4-, and 8-5-diferulic acids were identified in the plasma of rats after oral dosing with a mixture of the three acids in oil. Our study also reveals that human and rat colonic microflora contain esterase activity able to release 5-5-, 8-O-4-, and 8-5-diferulic acids from model compounds and dietary cereal brans, hence providing a mechanism for release of dietary diferulates prior to absorption of the free acids. In addition, cell-free extracts from human and rat small intestine mucosa exhibited esterase activity towards diferulate esters. Hence, we have shown that esterified diferulates can be released from cereal brans by intestinal enzymes, and that free diferulic acids can be absorbed and enter the circulatory system. Our results suggest that the phenolic antioxidant diferulic acids are bioavailable.     

Changes in Dehydrodiferulic Acids and Peroxidase Activity against Ferulic Acid Associated with Cell Walls during Growth of Pinus pinaster Hypocotyl

Hydroxycinnamic acids associated with hypocotyl cell walls of dark-grown seedlings of Pinus pinaster Aiton were extracted with 1 N NaOH and identified by gas chromatography-mass spectrometry. The main hydroxycinnamic acid found was ferulic acid. Diferulic acid dehydrodimers were also found, with the 8,8-coupled isomer (compound 11) being the dehydrodiferulate present in the highest amount. However, the 5,5-coupled isomer, commonly referred to referred to as diferulic acid, was not detected. Two truxillic acids, 4-4[prime]-dihydroxy-3-3[prime]-dimethoxy-[alpha]-truxillic acids I and II, were tentatively identified. The 8,8-coupled dehydrodiferulic acid (compound 11) was the phenolic acid that showed the most conspicuous changes with hypocotyl age as well as along the hypocotyl axis. Peroxidase activity against ferulic acid was found in the apoplastic fluid as well as being ionically and covalently bound to the cell walls. The peroxidase activity increased with hypocotyl age as well as from the subapical toward the basal region of the hypocotyls. A key role in the cell-wall stiffening of 8,8 but not 5,5 dimerization of ferulic acid catalyzed by cell-wall peroxidases is proposed.     

Characterization of Miscanthus cell wall polymers

Efficient utilization of lignocellulosic Miscanthus biomass for the production of biochemicals, such as ethanol, is challenging due to its recalcitrance, which is influenced by the individual plant cell wall polymers and their interactions. Lignocellulosic biomass composition differs depending on several factors, such as plant age, harvest date, organ type, and genotype. Here, four selected Miscanthus genotypes (Miscanthus sinensis, Miscanthus sacchariflorus, Miscanthus × giganteus, Miscanthus sinensis × Miscanthus sacchariflorus hybrid) were grown and harvested, separated into stems and leaves, and characterized for their non‐starch polysaccharide composition and structures, lignin contents and structures, and hydroxycinnamate profiles (monomers and ferulic acid dehydrodimers). Polysaccharides of all genotypes are mainly composed of cellulose and low‐substituted arabinoxylans. Ratios of hemicelluloses to cellulose were comparable, with the exception of Miscanthus sinensis that showed a higher hemicellulose/cellulose ratio. Lignin contents of Miscanthus stems were higher than those of Miscanthus leaves. Considering the same organs, the four genotypes did not differ in their Klason lignin contents, but Miscanthus × giganteus showed the highest acetylbromide soluble lignin content. Lignin polymers isolated from stems varied in their S/G ratios and linkage type distributions across genotypes. p‐Coumaric acid was the most abundant ester‐bound hydroxycinnamte monomer in all samples. Ferulic acid dehydrodimers were analyzed as cell wall cross‐links, with 8‐5‐coupled diferulic acid being the main dimer, followed by 8‐O‐4‐, and 5‐5‐diferulic acid. Contents of p‐coumaric acid, ferulic acid, and ferulic acid dimers varied depending on genotype and organ type. The largest amount of cell wall cross‐links was analyzed for Miscanthus sinensis.     

Synergy between xylanases from glycoside hydrolase family 10 and family 11 and a feruloyl esterase in the release of phenolic acids from cereal arabinoxylan

The bioconversion of waste residues (by-products) from cereal processing industries requires the cooperation of enzymes able to degrade xylanolytic and cellulosic material. The type A feruloyl esterase from Aspergillus niger, AnFaeA, works synergistically with (1→4)-β-d-xylopyranosidases (xylanases) to release monomeric and dimeric ferulic acid (FA) from cereal cell wall-derived material. The esterase was more effective with a family 11 xylanase from Trichoderma viride in releasing FA and with a family 10 xylanase from Thermoascus aurantiacus in releasing the 5,5′ form of diferulic acid from arabinoxylan (AX) derived from brewers’ spent grain. The converse was found for the release of the phenolic acids from wheat bran-derived AXs. This may be indicative of compositional differences in AXs in cereals.     

Inhibition of Gibberellin-Induced Elongation Growth of Rice by Feruloyl Oligosaccharides

The biological activity of cell wall-derived feruloyl oligosaccharideswas investigated using a modified micro-drop bioassay. A feruloylarabinoxylan trisaccharide (FAXX) and a feruloyl arabinoxylantetrasaccharide (FAXXX) were found to inhibit the gibberellin-inducedelongation of dwarf rice (Oryza sativa L., cv, Tan-ginbozu)that had been treated with uniconazole (S-3307), an inhibitorof the biosynthesis of gibberellins. An arabinoxylan trisaccharide(AXX) was ineffective. The growth-inhibitory effecf of feruloyloligosaccharides depended on their feruloyl and glycosyl moieties.The amount of esterified diferulic acid residues in the cellwalls of the second leaf sheath of rice seedlings that had beentreated with FAXX was almost same as that of controls. Feruloyloligosaccharides did not inhibit the incorporation of [¹⁴C]leucineinto acid-precipitable proteins by suspension-cultured maizecells, whereas cinnamic acid and its derivatives strongly inhibitedsuch incorporation. (Received May 10, 1995; Accepted August 23, 1995)     

Phenolic components and degradability of cell walls of grass and legume species

Cell walls separated from the aerial parts of Lolium multiflorum, Lolium perenne and Phleum pratense contained bound cis and trans ferulic and p-coumaric acids and diferulic acid which were released from the walls by treatment with sodium hydroxide. The total content of these acids in L. multiflorum ranged from 5 to 16.8 mg/g of wall, the trans-ferulic acid content varying between 2.8 and 8.9 mg/g of wall. In addition, small amounts of p-hydroxybenzoic acid were released from senescent leaf blade plus sheath parts. Cell walls from legume species gave much smaller amounts of the acids, the total content of aerial parts of Trifolium pratense being <0.8 mg/g of wall. The degra dability of the cell walls with a commercial cellulase preparation was determined and the water-soluble phenolic compounds released were estimated by UV absorption spectroscopy.     

Light-induced increase in the contents of ferulic and diferulic acids in cell walls of Avena coleoptiles: its relationship to growth inhibition by light

White fluorescent light (5 W m⁻²) inhibited Avena coleoptile growth. Light caused in increase in minimum stress relaxation time and a decrease in extensibility (strain/load) of coleoptile cell walls. Light increased the contents of ferulic acid (FA) and diferulic acid (DFA) ester-linked to the hemicellulose I in cell walls. These changes in the phenolic contents correlated with those of the mechanical properties of cell walls, suggesting that light stimulates the formation of DFA in hemicellulose I, making cell walls rigid, and thus results in growth inhibition. The ratio of DFA to FA was almost constant in the dark, but decreased in light, although it was almost constant in Oryza coleoptiles either in the dark or in light (Tan et al. 1992). From this fact, it is speculated that in the light condition, the formation of DFA in cell walls is limited in the step of the peroxidase catalyzed coupling reaction to produce DFA, while in the dark it is limited in the step of the feruloylation of hemicellulose I.     

Arabinoxylan gels: impact of the feruloylation degree on their structure and properties

Arabinoxylan (AX) samples of decreasing ferulic acid (FA) contents were chemically prepared from water-extractable wheat arabinoxylans without affecting their other structural properties. Gels were obtained from these partially feruloylated WEAX (PF-WEAX) by enzymatic covalent cross-linking of FA leading to the formation of diferulic (di-FA) and tri-ferulic acid (tri-FA). WEAX gelling ability was found related to the WEAX FA content whereas the gel structure and properties depended on the density of newly formed covalent cross-links. FA content of WEAX ranging from 1.4 to 2.3 microg/mg AX gave gels with di-FA cross-links contents from 0.20 to 0.43 microg/mg AX and G' values from 5 to 44 Pa. For WEAX gels with initial FA contents from 1.6 to 2.3 microg/mg AX, average mesh size ranging from 331 to 263 nm were calculated from swelling experiments. Cross-linking densities of gels, determined from swelling experiments, were higher than those that could be theoretically estimated from the di-FA and tri-FA content of WEAX gels. This result suggests that, in addition to di-FA and tri-FA, higher ferulate cross-linking and physical entanglements would contribute to the final WEAX gel structure.