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.     

Ferulic Acid: An Antioxidant Found Naturally in Plant Cell Walls and Feruloyl Esterases Involved in its Release and Their Applications

ABSTRACT

Ferulic acid is the most abundant hydroxycinnamic acid in the plant world and maize bran with 3.1% (w/w) ferulic acid is one of the most promising sources of this antioxidant. The dehydrodimers of ferulic acid are important structural components in the plant cell wall and serve to enhance its rigidity and strength. Feruloyl esterases are a subclass of the carboxylic acid esterases that hydrolyze the ester bond between hydroxycinnamic acids and sugars present in plant cell walls and they have been isolated from a wide range of microorganisms, when grown on complex substrates such as cereal brans, sugar beet pulp, pectin and xylan. These enzymes perform a function similar to alkali in the deesterification of plant cell wall and differ in their specificities towards the methyl esters of cinnamic acids and ferulolylated oligosaccharides. They act synergistically with xylanases and pectinases and facilitate the access of hydrolases to the backbone of cell wall polymers. The applications of ferulic acid and feruloyl esterase enzymes are many and varied. Ferulic acid obtained from agricultural byproducts is a potential precursor for the production of natural vanillin, due to the lower production cost.     

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.     

Influence of various soil factors on the effects of ferulic acid on leaf expansion of cucumber seedlings

Summary Cucumber seedlings were grown in a Portsmouth soil-sand system to study how varying soil clay and organic matter content might modify cucumber seedling response to ferulic acid, a reported allelopathic agent. Leaf area expansion of cucumber seedlings, soil respiration, and soil solution concentrations of ferulic acid were monitored. Leaf area, mean absolute rates of leaf expansion, and shoot dry weight of cucumber seedlings were significantly reduced by ferulic acid concentrations ranging from 10 to 70 μg/g dry soil. Ferulic acid was applied every other day, since it rapidly disappeared from soil solution as a result of retention by soil particles, utilization by microbes and/or uptake by roots. The amount of ferulic acid retained (i.e., adsorbed, polymerized,etc.) by soil particles appeared to be secondary to microbial utilization and/or uptake by roots. Varying clay (5.3 to 9.8 g/cup) and organic matter (2.0 to 0.04g/cup) contents of the soil appeared to have little impact on the disappearance of ferulic acid from soil solution under “ideal” growth conditions for cucumber seedlings unless larger amounts of ferulic acid were added to the soil; in this case 200 μg/g. The addition of ferulic acid to the soil materials substantially increased the activity of the soil microbes. This latter conclusion is based on recovery of ferulic acid from soil solution and soil respiration measurements. Paper No. 10347 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, N C 27695-7601. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of the product named, nor criticism of similar ones not mentioned.     

The ferulic acid esterases of Chrysosporium lucknowense C1: purification, characterization and their potential application in biorefinery

Three ferulic acid esterases from the filamentous fungus Chrysosporium lucknowense C1 were purified and characterized. The enzymes were most active at neutral pH and temperatures up to 45 °C. All enzymes released ferulic acid and p-coumaric acid from a soluble corn fibre fraction. Ferulic acid esterases FaeA1 and FaeA2 could also release complex dehydrodiferulic acids and dehydrotriferulic acids from corn fibre oligomers, but released only 20% of all ferulic acid present in sugar beet pectin oligomers. Ferulic acid esterase FaeB2 released almost no complex ferulic acid oligomers from corn fibre oligomers, but 60% of all ferulic acid from sugar beet pectin oligomers. The ferulic acid esterases were classified based on both, sequence similarity and their activities toward synthetic substrates. The type A ferulic acid esterases FaeA1 and FaeA2 are the first members of the phylogenetic subfamily 5 to be biochemically characterized. Type B ferulic acid esterase FaeB2 is a member of subfamily 6.     

Investigating the roles of phenylpropanoids in the growth and development of Zea mays L.

The Poaceae includes some of the most important food, fiber, and bio-fuel crops. While there have been many studies investigating the function of phenylpropanoids in this family, most of our understanding is based on correlative data rather than experimental evidence. The current study was conducted to evaluate the roles of phenylpropanoids in the growth and development of Zea mays and to develop an experimental model for further investigations. Z. mays seedlings were grown in vitro with various concentrations of the competitive phenylalanine ammonia lyase inhibitor, 2-aminoindane-2-phosphonic acid (AIP). Ferulic acid, a downstream biosynthetic product, was added to determine if it could rescue the induced phenotypes. At lower concentrations of AIP, plants exhibited elongated roots and shoots, but at higher concentrations, growth was extremely stunted. At the cellular level, the epidermal cells of roots cultured with AIP exhibited a loss of intercellular adhesion and organization, and their cell walls were more readily degraded by enzymatic digestion. These characteristics were accompanied by significant reductions in primary cell wall autofluorescence, indicating that less ferulic acid and other phenolics were incorporated in the cell wall. The majority of these symptoms could be partially or entirely rescued by ferulic acid, providing further evidence that these differences were due to the inhibition of phenylpropanoid biosynthesis. This study provides experimental evidence supporting and expanding upon hypothesized functions of phenylpropanoids in the growth and development of Z. mays and provides an experimental system for further investigations in the Poaceae and other taxonomic groups.     

Isolation of diferulic bridges ester-linked to arabinan in sugar beet cell walls

After degradation of sugar beet cell walls with Driselase and fractionation of the solubilised products by hydrophobic interaction chromatography, a dehydrodiferuloylated oligoarabinan was isolated. Its structure was assigned to two dimers of (1-->5)-linked arabinose units esterified by a central 8-O-4' ferulic dimer. These results provide the first direct evidence that pectic arabinans in sugar beet cell walls may be covalently cross-linked through dehydrodiferulates.     

Ferulic acid: an antioxidant found naturally in plant cell walls and feruloyl esterases involved in its release and their applications

Ferulic acid is the most abundant hydroxycinnamic acid in the plant world and maize bran with 3.1% (w/w) ferulic acid is one of the most promising sources of this antioxidant. The dehydrodimers of ferulic acid are important structural components in the plant cell wall and serve to enhance its rigidity and strength. Feruloyl esterases are a subclass of the carboxylic acid esterases that hydrolyze the ester bond between hydroxycinnamic acids and sugars present in plant cell walls and they have been isolated from a wide range of microorganisms, when grown on complex substrates such as cereal brans, sugar beet pulp, pectin and xylan. These enzymes perform a function similar to alkali in the deesterification of plant cell wall and differ in their specificities towards the methyl esters of cinnamic acids and ferulolylated oligosaccharides. They act synergistically with xylanases and pectinases and facilitate the access of hydrolases to the backbone of cell wall polymers. The applications of ferulic acid and feruloyl esterase enzymes are many and varied. Ferulic acid obtained from agricultural byproducts is a potential precursor for the production of natural vanillin, due to the lower production cost.     

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.     

川芎晒干过程中阿魏酸和阿魏酸松柏酯含量的变化

为了解新鲜川芎采后干燥过程中阿魏酸和阿魏酸松柏酯含量的动态变化规律,采用高效液相色谱法测定了川芎晒干过程中总阿魏酸、游离阿魏酸和阿魏酸松柏酯的含量。结果显示,在整个晒干过程中(30 d),总阿魏酸、游离阿魏酸和阿魏酸松柏酯含量呈先升高后下降的变化趋势,晾晒第3 d时总阿魏酸含量最高(0.23%),因此在晾晒的第3 d利用快速干燥技术能较好地保留川芎药材中总阿魏酸含量,使其发挥更佳的药效。川芎药材中的阿魏酸松柏酯能水解产生阿魏酸,因此研究川芎干燥过程中的生理响应与含水量的关系对阿魏酸积累有重要意义。由于川芎在用药过程中是以总阿魏酸含量发挥药效的,所以以总阿魏酸含量作为川芎药材质量控制指标更加科学。     

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.     

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.     

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.     

A complete enzymatic recovery of ferulic acid from corn residues with extracellular enzymes from Neosartorya spinosa NRRL185

An economic ferulic acid recovery from biomass via biological methods is of interest for a number of reasons. Ferulic acid is a precursor to vanillin synthesis. It is also a known antioxidant with potential food and medical applications. Despite its universal presence in all plant cell wall material, the complex structure of the plant cell wall makes ferulic acid recovery from biomass a challenging bioprocess. Previously, without pretreatment, very low (3-13%) recovery of ferulic acid from corn residues was achieved. We report here the discovery of a filamentous fungus Neosartorya spinosa NRRL185 capable of producing a full complement of enzymes to release ferulic acid and the development of an enzymatic process for a complete recovery of ferulic acid from corn bran and corn fibers. A partial characterization of the extracellular proteome of the microbe revealed the presence of at least seven cellulases and hemicellulases activities, including multiple iso-forms of xylanase and ferulic acid esterase. The recovered ferulic acid was bio-converted to vanillin, demonstrating its potential application in natural vanillin synthesis. The enzymatic ferulic acid recovery accompanied a significant release of reducing sugars (76-100%), suggesting much broader applications of the enzymes and enzyme mixtures from this organism.     

Synthesized esters of ferulic acid induce release of cytochrome c from rat testes mitochondria

Ferulic acid plays a chemopreventive role in cancer by inducing tumor cells apoptosis. As mitochondria play a key role in the induction of apoptosis in many cells types, here we investigate the mitochondrial permeability transition (MPT) and the release of cytochrome c induced by ferulic acid and its esters in rat testes mitochondria, in TM-3 and MLTC-1 cells. While ferulic acid, but not its esters, induced MPT and cytochrome c release in rat testes isolated mitochondria, in TM-3 cells we found that both ferulic acid and its esters induced cytochrome c release from mitochondria in a dose-dependent manner, suggesting a potential target of these compounds in the induction of cell apoptosis. The apoptosis induced by ferulic acid is therefore associated with the mitochondrial pathway involving cytochrome c release and caspase-3 activation. Cione and Tucci have equally contributed to this article.     

Effects of cell wall components on the functionality of wheat gluten

Normal white wheat flours and especially whole meal flour contain solids from the inner endosperm cell walls, from germ, aleurone layer and the outer layers of cereal grains. These solids can prevent either gluten formation or gas cell structure. The addition of small amounts of pericarp layers (1–2%) to wheat flour had a marked detrimental effect on loaf volume. Microstructural studies indicated that in particular the epicarp hairs appeared to disturb the gas cell structure. The detrimental effects of insoluble cell walls can be prevented by using endoxylanases. It has been shown that some oxidative enzymes, naturally present in flour or added to the dough, will oxidise water-extractable arabinoxylans via ferulic acid bridges, and the resulting arabinoxylan gel will hinder gluten formation. The negative effects of water-unextractable arabinoxylans on gluten yield and rheological properties can be compensated by the addition of ferulic acid. Free ferulic acid can probably prevent arabinoxylan cross-linking via ferulic acid.     

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.     

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.     

Effects of ferulic acid on L-malate oxidation in isolated soybean mitochondria

The effects of ferulic acid on L-malate oxidation in mitochondria isolated from soybean (Glycine max L.) seedlings were investigated. Oxygen uptake and the products of L-malate oxidation were measured under two conditions: pH 6.8 and 7.8. At acidic pH, the activity of the NAD+-linked malic enzyme (L-malate:NAD+oxidoreductase [decarboxylating] EC 1.1.1.39) was favoured, whereas at alkaline pH a predominance of the L-malate dehydrogenase activity (L-malate:NAD+oxidoreductase EC 1.1.1.37) was apparent. Ferulic acid inhibited basal and coupled respiration during L-malate oxidation either at acidic or alkaline pH, reducing also the amounts of pyruvate or oxaloacetate produced. The results suggest that the site of ferulic acid action is situated at some step that precedes the respiratory chain. An interference with the L-malate entry into the mitochondria could be an explanation for the effects of ferulic acid, but the possibility of a direct inhibition of both enzymes involved in L-malate oxidation cannot be ruled out. This revised version was published online in August 2006 with corrections to the Cover Date.