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    ABSTRACT: Arabinoxylans (AX) and (1→3),(1→4)-β-glucans (BG) are the major components of wheat grain cell walls. Although incompletely described at the molecular level, it is known that the chemical and distributional heterogeneity of these compounds impacts the quality and use of wheat. In this work, an emerging technique based on MALDI mass spectrometry imaging (MSI) was employed to map variations in the quantity, localization, and structure of these polysaccharides in the endosperm during wheat maturation. MALDI MSI couples detailed structural information with the spatial localization observed at the micrometer scale. The enzymic hydrolysis of AX and BG was performed directly on the grain sections, resulting in the efficient formation of smaller oligosaccharides that are easily measurable through MS, with no relocation across the grain. The relative quantification of the generated oligosaccharides was achieved. The method was validated by confirming data previously obtained using other analytical techniques. Furthermore, in situ analysis of grain cell walls through MSI revealed previously undetectable intense acetylation of AX in young compared to mature grains, together with findings concerning the feruloylation of AX and different structural features of BG. These results provide new insights into the physiological roles of these polysaccharides in cell walls and the specificity of the hydrolytic enzymes involved.
    Journal of Experimental Botany 03/2014; · 5.79 Impact Factor
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    ABSTRACT: Bread is one of the main sources of starch of the French diet. Starch is the principal source of energy for humans; the glycemic index (GI) allows to classify the glycemic response to different types of food. Consumption of low GI food is generally recognized as beneficial for human health. Bread GI varies quite largely (40–85) according to the processes that determine the structure of baked products, especially density and crumb texture. For very different baked products such as sandwich bread and French bread, the production of denser bread leads to a lower GI. The densification of bread improves its nutritional profile but largely impacts its sensorial features. Consumer's acceptability of denser products depends on the bread texture; products with an irregular crumb structure, like traditional French bread, tend to be better appreciated.
    Cahiers de Nutrition et de Diététique 01/2014;
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    Innovations Agronomiques. 01/2014; 36:97-110.
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    ABSTRACT: The development of enzyme-mediated glycosynthesis using glycoside hydrolases is still an inexact science, because the underlying molecular determinants of transglycosylation are not well understood. In the framework of this challenge, this study focused on the family GH51 α-l-arabinofuranosidase from Thermobacillus xylanilyticus, with the aim to understand why the mutation of position 344 provokes a significant modification of the transglycosylation/hydrolysis partition. Detailed kinetic analysis (kcat, KM, pKa determination and time-course NMR kinetics) and saturation transfer difference nuclear magnetic resonance spectroscopy was employed to determine the synthetic and hydrolytic ability modification induced by the redundant N344 mutation disclosed in libraries from directed evolution. The mutants N344P and N344Y displayed crippled hydrolytic abilities, and thus procured improved transglycosylation yields. This behavior was correlated with an increased pKa of the catalytic nucleophile (E298), the pKa of the acid/base catalyst remaining unaffected. Finally, mutations at position 344 provoked a pH-dependent product inhibition phenomenon, which is likely to be the result of a significant modification of the proton sharing network in the mutants. Using a combination of biochemical and biophysical methods, we have studied TxAbf-N344 mutants, thus revealing some fundamental details concerning pH modulation. Although these results concern a GH51 α-l-arabinofuranosidase, it is likely that the general lessons that can be drawn from them will be applicable to other glycoside hydrolases. Moreover, the effects of mutations at position 344 on the transglycosylation/hydrolysis partition provide clues as to how TxAbf can be further engineered to obtain an efficient transfuranosidase.
    Biochimica et Biophysica Acta 10/2013; · 4.66 Impact Factor
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    ABSTRACT: The cell walls of wheat starchy endosperm are dominated by arabinoxylan (AX), accounting for 65-70% of the polysaccharide content. Genes within two glycosyl transferase (GT) families, GT43 (IRX9, IRX14) and GT47 (IRX10), have previously been shown to be involved in the synthesis of the xylan backbone in Arabidopsis, and close homologues of these have been implicated in the synthesis of xylan in other species. Here homologues of IRX10 TaGT47_2 and of IRX9 TaGT43_2, which are highly expressed in wheat starchy endosperm cells, were suppressed by RNAi constructs driven by a starchy endosperm-specific promoter. The total amount of AX was decreased by 40-50% and the degree of arabinosylation was increased by 25-30% in transgenic lines carrying either of the transgenes. The cell walls of starchy endosperm in sections of grain from TaGT43_2 and TaGT47_2 RNAi transgenics showed decreased immunolabelling for xylan and arabinoxylan epitopes and ~50% decreased cell wall thickness compared to controls. The proportion of AX that was water-soluble was not significantly affected, but average AX polymer chain length was decreased in both TaGT43_2 and TaGT47_2 RNAi transgenics. However, long AX chains seen in controls were absent in TaGT43_2 RNAi transgenics but still present in TaGT47_2 RNAi transgenics. The results support an emerging picture of IRX9-like and IRX10-like proteins acting as key components in the xylan synthesis machinery in both dicots and grasses. Since AX is the main component of dietary fibre in wheat foods, the TaGT43_2 and TaGT47_2 genes are of major importance to human nutrition.
    Plant physiology 07/2013; · 6.56 Impact Factor
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    ABSTRACT: Arabinoxylans (AX) and (1→3)(1→4)-β-d-glucans (BG) are the main components of the cell walls in the endosperm of wheat grain. The relative occurrence of these two polysaccharides and the fine structure of the AX are highly variable within the endosperm. Films of AX and BG were used as models of the cell wall to study the impact of polymer structure on the hydration and mechanical properties of the cell walls. Effective moisture diffusivities (Deff) of AX and BG films were determined from 0 to 95% relative humidity (RH) at 20°C. Deff was influenced by the water content, and the structure of polysaccharides. Higher Deff was obtained for films made with highly substituted AX compared to values obtained for films made with BG or lowly substituted AX. Proton dipolar second moments M2 and water T2 relaxation times measured by TD-NMR, indicated that the highly branched AX films exhibited a higher nano-porosity, favoring water motions within films. Results from traction tests showed significant different mechanical properties between the AX and BG films. BG films exhibited much higher extensibility than AX films. Strength and extensibility of AX films decreased with increasing arabinose to xylose ratio. Our results show that the water motions and the mechanical properties of AX and BG films can be linked to the polysaccharide chains interactions that modulate the nanostructure of films.
    Carbohydrate polymers. 07/2013; 96(1):31-38.
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    ABSTRACT: This work aimed to elucidate the effect of wheat aleurone integrity on its fermentability, i.e. the formation of short-chain fatty acids (SCFA) and microbial phenolic metabolites, in an in vitro model using human faecal microbiota as an inoculum. The structure of aleurone was modified by mechanical (dry grinding) or enzymatic (xylanase with or without feruloyl esterase) treatments in order to increase its physical accessibility and degrade its complex cell-wall network. The ground aleurone (smaller particle size) produced slightly more SCFA than the native aleurone (102.5 and 101 mmol/L, respectively), but showed a similar colonic metabolism of ferulic acid (FA). The enzymatic treatments of aleurone allowed a higher solubilization of arabinoxylan (up to 82%) and a higher release of FA in its conjugated and free forms (up to 87%). The enzymatic disintegration of aleurone's structure led to a higher concentration and formation rate of the colonic metabolites of FA (especially phenylpropionic acids), but did not change significantly the formation of SCFA (81 mmol/L for enzyme treated vs. 101 mmol/L for the native aleurone).
    Journal of Agricultural and Food Chemistry 05/2013; · 3.11 Impact Factor
  • A Rakha, L Saulnier, P Aman, R Andersson
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    ABSTRACT: Enzymatic fingerprinting of arabinoxylan (AX) and β-glucan using endo-xylanase and lichenase, respectively, helps determine the structural heterogeneity between different cereals and within genotypes of the same cereal. This study characterised the structural features of AX and β-glucan in whole grains of eight triticale cultivars grown at two locations, 20 barley cultivars/lines with wide variation in composition and morphology and five tritordeum breeding lines. Principal component analysis (PCA) resulted in clear clustering of these cereals. In general, barley and tritordeum had a higher relative proportion of highly branched arabinoxylan oligosaccharides (AXOS) than triticale. Subsequent analysis of triticale revealed two clusters based on growing region along principal component (PC) 1, while PC2 explained the genetic variability and was based on mono-substitution and di-substitution in AX fragments. PCA of β-glucan features separated the three cereals based on β-glucan content. The molar ratio of trisaccharide to tetrasaccharide was 2.5-3.4 in triticale, 2.3-3.3 in barley and 2.8-3.4 in tritordeum. Barley showed a strong positive correlation (r=0.86) between β-glucan content and relative proportion of trisaccharide. The results show that structural features of AX and β-glucan vary between and within triticale, barley and tritordeum grains which might be important determinants of end-use quality of grains.
    Carbohydrate polymers. 10/2012; 90(3):1226-34.
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    ABSTRACT: Immunolabeling can be used to locate plant cell wall carbohydrates or other components to specific cell types or to specific regions of the wall. Some antibodies against xylans exist; however, many partly react with the xylan backbone and thus provide limited information on the type of substituents present in various xylans. We have produced a monoclonal antibody which specifically recognizes glucopyranosyl uronic acid (GlcA), or its 4-O-methyl ether (meGlcA), substituents in xylan and has no cross-reactivity with linear or arabinofuranosyl-substituted xylans. The UX1 antibody binds most strongly to (me)GlcA substitutions at the non-reducing ends of xylan chains, but has a low cross-reactivity with internal substitutions as well, at least on oligosaccharides. The antibody labeled plant cell walls from both mono- and dicotyledons, but in most tissues an alkaline pretreatment was needed for antibody binding. The treatment removed acetyl groups from xylan, indicating that the vicinity of glucuronic acid substituents is also acetylated. The novel labeling patterns observed in the xylem of tree species suggested that differences within the cell wall exist both in acetylation degree and in glucuronic acid content.
    Planta 04/2012; 236(2):739-51. · 3.38 Impact Factor
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    ABSTRACT: A detailed and comprehensive understanding of seed reserve accumulation is of great importance for agriculture and crop improvement strategies. This work is part of a research programme aimed at using Brachypodium distachyon as a model plant for cereal grain development and filling. The focus was on the Bd21-3 accession, gathering morphological, cytological, and biochemical data, including protein, lipid, sugars, starch, and cell-wall analyses during grain development. This study highlighted the existence of three main developmental phases in Brachypodium caryopsis and provided an extensive description of Brachypodium grain development. In the first phase, namely morphogenesis, the embryo developed rapidly reaching its final morphology about 18 d after fertilization (DAF). Over the same period the endosperm enlarged, finally to occupy 80% of the grain volume. During the maturation phase, carbohydrates were continuously stored, mainly in the endosperm, switching from sucrose to starch accumulation. Large quantities of β-glucans accumulated in the endosperm with local variations in the deposition pattern. Interestingly, new β-glucans were found in Brachypodium compared with other cereals. Proteins (i.e. globulins and prolamins) were found in large quantities from 15 DAF onwards. These proteins were stored in two different sub-cellular structures which are also found in rice, but are unusual for the Pooideae. During the late stage of development, the grain desiccated while the dry matter remained fairly constant. Brachypodium exhibits some significant differences with domesticated cereals. Beta-glucan accumulates during grain development and this cell wall polysaccharide is the main storage carbohydrate at the expense of starch.
    Journal of Experimental Botany 01/2012; 63(2):739-55. · 5.79 Impact Factor
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    ABSTRACT: The consumption of wholegrain wheat is associated with a number of health benefits which may relate to the presence of a range of dietary fibre, phytochemical, vitamin and mineral components. Analysis of 150 bread wheat lines within the HEALTHGRAIN programme showed wide variation in content and composition of bioactive components within 150 bread wheat lines. Furthermore, in some cases (notably arabinoxylan in flour and tocols, sterols and alkylresorcinols in wholemeal) this variation was highly heritable and hence accessible to plant breeders. A number of tools are therefore being developed to facilitate the selection of these components in plant breeding programmes including molecular markers, biochemical kits and NIR calibrations.
    Trends in Food Science & Technology 01/2012; 25:70-77. · 4.14 Impact Factor
  • L. Saulnier
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    ABSTRACT: After a fast reminder on the use of cereals, the biochemical, technological and nutritional characteristics of the various classes of nutrients (starch, proteins, dietary fibres, lipids, vitamins, minerals and phenolic compounds) are presented, in connection with the diversity of composition of the different tissues of the grain and the specificities of main cereals: wheat, corn (maize), rice, rye, barley and oat. Thanks to their richness in starch, cereal grains are an essential part of the diet. They are also a source of dietary fibres and macronutrients (vitamins, minerals, etc.), interesting constituents for health, essentially concentred in the external parts of the kernel. Despite these common characteristics, cereals show various nutritional assets, partly depending on vegetal species, but mostly on technological processes used in flours and foods productions.
    Cahiers de Nutrition et de Diététique 01/2012; 47:S4-S15.
  • L. Saulnier, Valérie Micard
    Carrefours de l'innovation agronomique 2012 "Du champ à l'assiette: nouveaux enjeux pour la filière blé"Carrefours de l'innovation agronomique 2012 "Du champ à l'assiette: nouveaux enjeux pour la filière blé"; 01/2012
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    M.R. Cyran, L. Saulnier
    Food Chemistry. 01/2012; 131:667-676.
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    ABSTRACT: There is an amazing diversity of cell wall composition and cell wall polysaccharide structure in wheat grain that is only partly explained by the complexity of plant tissues and cell type functions present in this organ. This review presents the state of the art on the structure and diversity of cell wall polymers in mature wheat grain and recent knowledge on cell wall metabolism with a focus on development-associated changes in wheat grain cell wall polysaccharides and genes controlling their biosynthesis. The diversity of polysaccharide structure observed in endosperm cell walls is tentatively explained on the basis of polysaccharide properties and cell type function.
    Journal of Cereal Science 01/2012; 56(1):91-108. · 2.09 Impact Factor
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    ABSTRACT: Arabinoxylans (AXs) extracted from wheat flour were characterized by using three different techniques of NMR spectroscopy. Liquid-state (1)H NMR and solid-state (13)C NMR allowed the investigation of the fine structure of the three specific fractions of AXs representative of the structural heterogeneity of AX in wheat tissues. Three pure AX fractions exhibiting an arabinose to xylose ratio of 0.33, 0.53, and 0.73 were compared relative to their substitution feature and also to their assembly into thin films. Measurements of M(2), i.e. the second moment of proton dipolar interactions between the polysaccharide chains, were achieved using time-domain (TD) (1)H NMR at different water contents and temperatures. Transitions of the M(2) values were observed at a certain temperature close to the glass transition temperature T(g) values of AXs in films. Comparison of the different AX films containing various water contents pointed out stronger dipolar interactions for lowly substituted AX. This indicated that, in films, contiguous unsubstituted xylan chains can interact together through hydrogen bonding resulting in a compact structure with small nanopores because of the lower chain motions and the shorter average distances between the lowly substituted AX chains.
    Magnetic Resonance in Chemistry 12/2011; 49 Suppl 1:S85-92. · 1.53 Impact Factor
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    ABSTRACT: The main purpose of this research was to determine the impact of the structure and organisation of polysaccharides on the hydration properties of the cell walls of cereal grains. In order to remodel the lamellar assembly of arabinoxylan (AX) and (1 → 3)(1 → 4)-β-D-glucan (BG) within the endosperm cell walls, films were prepared and analysed using dynamic vapour sorption and time domain nuclear magnetic resonance spectroscopy. The water diffusivities within the AX and BG films were measured at 20 °C by observing the water sorption kinetics within a mathematical model based on Fick's second law. The evolution of spin-spin relaxation times of water protons measured by increasing the temperature is explained by the additional contributions of motion of the protons of polysaccharides and/or rapid chemical exchanges of protons between water and hydroxyl groups of polysaccharides. The difference between patterns of water behaviour within the AX and BG films can be related to the difference in their nanostructures. The smaller nanopores of the BG films cause their nanostructure to be more compact.
    Journal of the Science of Food and Agriculture 06/2011; 91(14):2601-5. · 1.88 Impact Factor
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    ABSTRACT: Fifty bread wheat (Triticum aestivum L.) cultivars were selected from the HEALTHGRAIN germplasm collection based on variation in their contents of total and water-extractable arabinoxylan. FT-IR spectroscopic mapping of thin transverse sections of grain showed variation in cell wall arabinoxylan composition between the cultivars, from consisting almost entirely of low-substituted arabinoxylan (e.g., T.aestivum 'Claire') to almost entirely of highly substituted arabinoxylan (e.g., T.aestivum 'Manital') and a mixture of the two forms (e.g., T.aestivum 'Hereward'). Complementary data were obtained using endoxylanase digestion of flour followed by HP-AEC analysis of the arabinoxylan oligosaccharides. This allowed the selection of six cultivars for more detailed analysis using FT-IR and (1)H NMR spectroscopy to determine the proportions of mono-, di-, and unsubstituted xylose residues. The results of the two analyses were consistent, showing that variation in the composition and structure of the endosperm cell wall arabinoxylan is present between bread wheat cultivars. The heterogeneity and spatial distribution of the arabinoxylan in endosperm cell walls may be exploited in wheat processing as it may allow the production of mill streams enriched in various arabinoxylan fractions which have beneficial effects on health.
    Journal of Agricultural and Food Chemistry 06/2011; 59(13):7075-82. · 3.11 Impact Factor
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    ABSTRACT: One of the objectives of the malting industry is to reduce the energy cost during kilning without major effect on malt quality. In this study, the impact of a low hydration steeping process on lipid transfer protein (LTP1) modifications and β-glucan breakdown was evaluated in low (LH) and high (HH) hydrated malts. LTP1 modifications analyzed by MS/MS revealed acylation, glycation, and disulfide bond breakage in both LH and HH malts. LTP1 free amine content measurement and fluorescence of Maillard protein adducts revealed no significant difference between LH and HH malts. Immunolabeling of LTP1 during malting highlighted the diffusion of the protein from the aleurone layer to the endosperm at the end of steeping in both LH and HH malts. By contrast, a significant higher amount of β-glucans was measured in LH malts after five days of germination, whereas no significant difference between LH and HH malts was revealed through immunostaining of β-glucans or evaluation of the endosperm integrity after seven days of germination. The possibility to reduce the effects of a low hydration steeping process on β-glucan hydrolysis by increasing germination time was discussed.
    Journal of Agricultural and Food Chemistry 06/2011; 59(15):8256-64. · 3.11 Impact Factor
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    ABSTRACT: Grain dietary fiber content in wheat not only affects its end use and technological properties including milling, baking and animal feed but is also of great importance for health benefits. In this study, integration of association genetics (seven detected loci on chromosomes 1B, 3A, 3D, 5B, 6B, 7A, 7B) and meta-QTL (three consensus QTL on chromosomes 1B, 3D and 6B) analyses allowed the identification of seven chromosomal regions underlying grain dietary fiber content in bread wheat. Based either on a diversity panel or on bi-parental populations, we clearly demonstrate that this trait is mainly driven by a major locus located on chromosome 1B associated with a log of p value >13 and a LOD score >8, respectively. In parallel, we identified 73 genes differentially expressed during the grain development and between genotypes with contrasting grain fiber contents. Integration of quantitative genetics and transcriptomic data allowed us to propose a short list of candidate genes that are conserved in the rice, sorghum and Brachypodium chromosome regions orthologous to the seven wheat grain fiber content QTL and that can be considered as major candidate genes for future improvement of the grain dietary fiber content in bread wheat breeding programs.
    Functional & Integrative Genomics 03/2011; 11(1):71-83. · 3.83 Impact Factor

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