Luc Saulnier

Isolation and partial characterization of feruloylated oligosaccharides from maize bran.

Maize bran contains phenolic acids [approximately 4% dry matter; mainly ferulic acid (Fe) and also diferulic acid], heteroxylans (approximately 50%), and cellulose (approximately 20%), but is devoid of lignin. Treatment of maize pericarp with 0.05 M trifluoroacetic acid at 100 degrees C for 2 h released approximately 90% of the heteroxylans and approximately 90% of the ferulic acid and its esters. After fractionation of the products with Amberlite XAD-2 and Sephadex LH-20 three main feruloylated oligosaccharides (F3-F7) were isolated. They represented approximately 30% of the ferulic acid, and approximately 2% of the neutral sugars contained in the hydrolysis supernatant. The compositions of F7, F6, and F3 were Fe-Ara (1:1), Fe-Ara-Xyl (1:1:1), and Fe-Ara-Xyl-Gal (1:1:1:1), respectively. The structures of the three oligomers were determined using chemical methods (methylation, acetalation, reduction) and 13C NMR spectroscopy: F7 was 5-O-(trans-feruloyl)-L-Araf;F6 was O-beta-D-Xyl p-(1-->2)-[5-O-(trans-feruloyl)-L-Araf]; and F3 was O-L-Gal p-(1-->4)-O-D-Xyl p-(1-->2)-[5-O-(trans-feruloyl)-L- Araf]. F7 has been previously isolated from other monocots especially from wheat bran and soluble arabinoxylans from wheat flour; this is the first report of feruloylated oligosaccharides F6 and F3. Our results suggest that these oligomers are side-chain constituents of heteroxylans in maize bran. Ferulic acid is probably partly responsible for the insolubility of heteroxylans by coupling polysaccharide chains through ferulic acid dimers.

Cell wall polysaccharide interactions in maize bran

Sequential extractions with alkali have been carried out in order to study the nature of linkages which hold heteroxylans in maize bran cell walls. Treatment with 0.5 m sodium hydroxide at 30 °C for 2 h released all the phenolic acids (p-coumaric, ferulic, and diferulic) but extracted only ~30% of heteroxylans (S1); further treatment with 1.5 m potassium hydroxide at 100 °C for 2 h released the remaining heteroxylans (S2). The heteroxylans from S1 and S2 had a similar neutral sugar composition and structure, but their weight average molecular weights were 270 kDa (MwMn = 2) and 370 kDa (MwMn = 2.8), respectively. Proteins (5%) are found with polysaccharides from S1 and S2, with different amino acid composition. The results suggest that covalent linkages through phenolic acids are only partly responsible for the associations of maize bran heteroxylans in the cell wall and that linkages to structural cell wall proteins were probably the main cause of heteroxylan insolubility.

Isolation and structural determination of two 5,5′-diferuloyl oligosaccharides indicate that maize heteroxylans are covalently cross-linked by oxidatively coupled ferulates

Large amounts of ferulic acid (∼2.6% w/w) and dehydrodiferulic acids (∼1.3% total, w/w) were detected in saponified extracts of maize bran. After treatment of maize bran by mild acid hydrolysis and fractionation of the solubilised products by liquid chromatography, two dehydrodiferuloylated oligosaccharides (F8 and F9) were isolated. The compositions of F8 and F9 were shown to be 1:2:1 5,5′-diferulic acid (5,5′-diFA)–arabinose–xylose and 1:2 5,5′-diFA–arabinose, respectively. Their structure was determined using chromatography and LCMS and confirmed by 1H and 13C NMR spectroscopy. Fraction F9 was composed of 5,5′-diFA esterified at both carboxylic groups by a 5-O-l-arabinofuranoside moiety, while F8 was similar but with one of the arabinofuranosyl residues further substituted with a (1→2)-linked xylopyranosyl residue. These results provide direct evidence that the heteroxylans in maize cell walls are covalently cross-linked through dehydrodiferulates.

Structural studies of pectic substances from the pulp of grape berries

Abstract The structure of the neutral sugar side-chains of pectic substances extracted with water (WSP), dilute HCl (HP), and endopectinlyase (AIR/PEL) from an alcohol-insoluble residue (AIR) from the pulp of grape berries was investigated by using chemical methods (methylation, carboxyl-reduction, and periodate oxidation), enzymic degradation with an α- l -arabinofuranosidase, and 13 C-n.m.r. spectroscopy. The side chains of WSP are essentially a type II arabinogalactan, whereas those of HP and AIR/PEL are arabinan-like structures associated with minor proportions of type I and II arabinogalactans. The side chains are ∼80% degraded by the α- l -arabinofuranosidase, indicating that the α- l -arabinofuranosyl residues are mainly 3-linked to the (1→6)-galactan side-chains of the type II arabinogalactan. The three pectic fractions contain minor amounts of proteins rich in hydroxyproline and serine.

Alkaline extraction and characterisation of heteroxylans from maize bran

Abstract Response surface methodology was used to study the alkaline extraction of heteroxylans from maize bran. The ratio volume of alkali/weight of bran and the particle size had no effect on extraction yield, whereas the yield increased significantly with the temperature and time of extraction and the concentration of the alkali. The variation in the yield depended on the nature of the alkali, and empirical second-order models were built to fit the results obtained by extraction with KOH and Ca(OH)2. Comparison of the compositional and structural features of the heteroxylans obtained by extraction with 0·8 m KOH at 85 °C, saturated Ca(OH)2 at 95 °C and 0·5 m KOH at 65 °C with one obtained by industrial lime-cooking of maize kernels showed that all four samples were very similar and that a very high extraction yield (87 %) was achieved.

Experimental evidence for a semi-flexible conformation for arabinoxylans.

Purified water-soluble arabinoxylans from wheat flour were deferuloylated and fractionated into six fractions by graded ethanol precipitation. Further fractionation by HPSEC on Sephacryl S500 resulted in 48 subfractions with low polydispersity index. Conformational characteristics (persistence length q, hydrodynamic parameter v and Mark-Houwink exponent a) were similar among all subfractions and fitted with a semi-flexible conformation, whatever their structural characteristics. Substitution degree of the xylan backbone by arabinose residues has no influence on the conformation of arabinoxylans.

Down-Regulation of the CSLF6 Gene Results in Decreased (1,3;1,4)-β-d-Glucan in Endosperm of Wheat

(1,3;1,4)-β-d-Glucan (β-glucan) accounts for 20% of the total cell walls in the starchy endosperm of wheat (Triticum aestivum) and is an important source of dietary fiber for human nutrition with potential health benefits. Bioinformatic and array analyses of gene expression profiles in developing caryopses identified the CELLULOSE SYNTHASE-LIKE F6 (CSLF6) gene as encoding a putative β-glucan synthase. RNA interference constructs were therefore designed to down-regulate CSLF6 gene expression and expressed in transgenic wheat under the control of a starchy endosperm-specific HMW subunit gene promoter. Analysis of wholemeal flours using an enzyme-based kit and by high-performance anion-exchange chromatography after digestion with lichenase showed decreases in total β-glucan of between 30% and 52% and between 36% and 53%, respectively, in five transgenic lines compared to three control lines. The content of water-extractable β-glucan was also reduced by about 50% in the transgenic lines, and the Mr distribution of the fraction was decreased from an average of 79 to 85 × 104 g/mol in the controls and 36 to 57 × 104 g/mol in the transgenics. Immunolocalization of β-glucan in semithin sections of mature and developing grains confirmed that the impact of the transgene was confined to the starchy endosperm with little or no effect on the aleurone or outer layers of the grain. The results confirm that the CSLF6 gene of wheat encodes a β-glucan synthase and indicate that transgenic manipulation can be used to enhance the health benefits of wheat products.

Arabinoxylan and (1→3),(1→4)-β-glucan deposition in cell walls during wheat endosperm development

Arabinoxylans (AX) and (1→3),(1→4)-β-glucans are major components of wheat endosperm cell walls. Their chemical heterogeneity has been described but little is known about the sequence of their deposition in cell walls during endosperm development. The time course and pattern of deposition of the (1→3) and (1→3),(1→4)-β-glucans and AX in the endosperm cell walls of wheat (Triticum aestivum L. cv. Recital) during grain development was studied using specific antibodies. At approximately 45°D (degree-days) after anthesis the developing walls contained (1→3)-β-glucans but not (1→3),(1→4)-β-glucans. In contrast, (1→3),(1→4)-β-glucans occurred widely in the walls of maternal tissues. At the end of the cellularization stage (72°D), (1→3)-β-glucan epitopes disappeared and (1→3),(1→4)-β-glucans were found equally distributed in all thin walls of wheat endosperm. The AX were detected at the beginning of differentiation (245°D) in wheat endosperm, but were missing in previous stages. However, epitopes related to AX were present in nucellar epidermis and cross cells surrounding endosperm at all stages but not detected in the maternal outer tissues. As soon as the differentiation was apparent, the cell walls exhibited a strong heterogeneity in the distribution of polysaccharides within the endosperm.

The simultaneous repression of CCR and CAD, two enzymes of the lignin biosynthetic pathway, results in sterility and dwarfism in Arabidopsis thaliana.

Cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) catalyze the last steps of monolignol biosynthesis. In Arabidopsis, one CCR gene (CCR1, At1g15950) and two CAD genes (CAD C At3g19450 and CAD D At4g34230) are involved in this pathway. A triple cad c cad d ccr1 mutant, named ccc, was obtained. This mutant displays a severe dwarf phenotype and male sterility. The lignin content in ccc mature stems is reduced to 50% of the wild-type level. In addition, stem lignin structure is severely affected, as shown by the dramatic enrichment in resistant inter-unit bonds and incorporation into the polymer of monolignol precursors such as coniferaldehyde, sinapaldehyde, and ferulic acid. Male sterility is due to the lack of lignification in the anther endothecium, which causes the failure of anther dehiscence and of pollen release. The ccc hypolignified stems accumulate higher amounts of flavonol glycosides, sinapoyl malate and feruloyl malate, which suggests a redirection of the phenolic pathway. Therefore, the absence of CAD and CCR, key enzymes of the monolignol pathway, has more severe consequences on the phenotype than the individual absence of each of them. Induction of another CCR (CCR2, At1g80820) and another CAD (CAD1, At4g39330) does not compensate the absence of the main CCR and CAD activities. This lack of CCR and CAD activities not only impacts lignification, but also severely affects the development of the plants. These consequences must be carefully considered when trying to reduce the lignin content of plants in order to facilitate the lignocellulose-to-bioethanol conversion process.

Brachypodium distachyon grain: characterization of endosperm cell walls

The wild grass Brachypodium distachyon has been proposed as an alternative model species for temperate cereals. The present paper reports on the characterization of B. distachyon grain, placing emphasis on endosperm cell walls. Brachypodium distachyon is notable for its high cell wall polysaccharide content that accounts for ∼52% (w/w) of the endosperm in comparison with 2-7% (w/w) in other cereals. Starch, the typical storage polysaccharide, is low [<10% (w/w)] in the endosperm where the main polysaccharide is (1-3) (1-4)-β-glucan [40% (w/w) of the endosperm], which in all likelihood plays a role as a storage compound. In addition to (1-3) (1-4)-β-glucan, endosperm cells contain cellulose and xylan in significant amounts. Interestingly, the ratio of ferulic acid to arabinoxylan is higher in B. distachyon grain than in other investigated cereals. Feruloylated arabinoxylan is mainly found in the middle lamella and cell junction zones of the storage endosperm, suggesting a potential role in cell-cell adhesion. The present results indicate that B. distachyon grains contain all the cell wall polysaccharides encountered in other cereal grains. Thus, due to its fully sequenced genome, its short life cycle, and the genetic tools available for mutagenesis/transformation, B. distachyon is a good model to investigate cell wall polysaccharide synthesis and function in cereal grains.