The conformation of glutenin polymers in wheat grain: some genetic and environmental factors associated with this important characteristic.

In a previous study we used asymmetric-flow field-flow fractionation to determine the polymer mass (Mw), gyration radius (Rw) and the polydispersity index of glutenin polymers (GPs) in wheat (Triticum aestivum). Here, using the same multi-location trials (4 years, 11 locations, and 192 cultivars), we report the factors that are associated with the conformation (Conf) of the polymers, which is the slope of Log(Rw) versus a function of Log(Mw). We found that Conf varied between 0.285 and 0.740, it had low broad-sense heritability (H2=16.8), and it was significantly influenced by the temperature occurring over the last month of grain filling. Higher temperatures were found to increase Rw and the compactness and sphericity of GPs. Alleles for both high- and low-molecular-weight glutenin subunits had a significant influence on the Conf value. Assuming a Gaussian distribution for Mw, the number of polymers present in wheat grains was computed for different kernel weights and protein concentrations, and it was found to exceed 1012 GPs per grain. Using atomic force microscopy and cryo-TEM, images of GPs were obtained for the first time. Under higher average temperature, GPs became larger and more spherical and consequently less prone to rapid hydrolysis. We propose some orientations that could be aimed at potentially reducing the impact of numerous GPs on people suffering from non-celiac gluten sensitivity.

Association between SNP Markers and 11 Vitamin Contents in Grains of a Worldwide Bread Wheat Core Collection.

The metabolomic profiling analyses of 11 vitamins' statuses of wheat grain in a subsample of 167 accessions from the INRAE worldwide bread wheat core collection planted in two contrasting environments in France (Le Moulon and Clermont-Ferrand) have been evaluated using a high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) procedure. This has allowed us to perform a genome-wide association study (GWAS) for these nutritional traits of interest combining the phenotypic data with the genotypic data derived from the TaBW280K SNP chip. Considering both thresholds (P < 0.0003 and R2 ≥ 8%), the GWAS identified between 1 and 22 marker-trait associations (MTAs) for the individual vitamins at the individual locations, and 12 SNP markers were stable and associated with vitamin contents across two environments. Desirable alleles and superior genotypes identified in the current analysis provide novel genetic data that can be used for future research on the genetics of vitamins and their application in wheat breeding.

Genetic and Environmental Variation in Starch Content, Starch Granule Distribution and Starch Polymer Molecular Characteristics of French Bread Wheat.

This study investigates genetic and environmental variation in starch content and characteristics of 14 French bread cultivars. Understanding the impact of these factors on wheat quality is important for processors and especially bakers to maintain and meet the requirements of industrial specifications. Different traits were evaluated: starch content, distribution of starch granules, percentage of amylose and amylopectin and their molecular characteristics (weight-average molar mass, number-average molar mass, polydispersity and gyration radius). Genetic, environment and their interaction had significant effects on all parameters. The relative magnitude of variance attributed to growth conditions, for most traits, was substantially higher (21% to 95%) than that attributed to either genotype (2% to 73%) or G × E interaction (2% to 17%). The largest environmental contribution (95%) to total variance was found for starch dispersity. The highest genetic influence was found for the percentage of A-type starch granules. G × E interaction had relatively little influence (≈7%) on total phenotypic variance. All molecular characteristics were much more influenced by environment than the respective percentages of amylose and amylopectin were. This huge difference in variance between factors obviously revealed the importance of the effect of growing conditions on characteristics of cultivars.

Molecular Weight Distribution of Polymeric Proteins in Wheat Grains: The Rheologically Active Polymers.

We characterized the molecular weight distribution of polymeric proteins (PP) of bread wheat grains using asymmetric flow field flow fractionation (A4F). The experiment, involving six environmental conditions and 130 cultivars, offered the opportunity to approach the phenotypic values of the polymer characteristics and their contribution of the rheological properties of flours and/or doughs. The contents of high-molecular-weight polymers (MW > 2 × 106 g·mol-1) that can be considered as "rheologically active polymers" (RAPP) for their major contribution to dough baking strength and mixing tolerance were mainly controlled by environmental factors. Under the influence of the growing conditions, at the cellular level, the redox status of non-protein free thiol, such as glutathione, is modified and leads to the formation of polymeric protein-bound glutathione conjugates (PPSSG). The accumulation of these conjugates reduces the formation of the RAPP by limiting the intermolecular interactions between PP in the grain during desiccation. This phenomenon is, therefore, potentially responsible for decreases in the technological properties of the wheat genotypes concerned. These first results invite us to continue our investigations to fully confirm this phenomenon, with emphasis on the behavior of wheat genotypes under various growing conditions.

Deciphering carbohydrate metabolism during wheat grain development via integrated transcriptome and proteome dynamics.

Grain development of Triticum aestivum is being studied extensively using individual OMICS tools. However, integrated transcriptome and proteome studies are limited mainly due to complexity of genome. Current study focused to unravel the transcriptome-proteome coordination of key mechanisms underlying carbohydrate metabolism during whole wheat grain development. Wheat grains were manually dissected to obtain grain tissues for proteomics and transcriptomics analyses. Differentially expressed proteins and transcripts at the 11 stages of grain development were compared. Computational workflow for integration of two datasets related to carbohydrate metabolism was designed. For CM proteins, output peptide sequences of proteomic analyses (via LC-MS/MS) were used as source to search corresponding transcripts. The transcript that turned out with higher number of peptides was selected as bona fide ribonucleotide sequence for respective protein synthesis. More than 90% of hits resulted in successful identification of respective transcripts. Comparative analysis of protein and transcript expression profiles resulted in overall 32% concordance between these two series of data. However, during grain development correlation of two datasets gradually increased up to ~ tenfold from 152 to 655 °Cd and then dropped down. Proteins involved in carbohydrate metabolism were divided in five categories in accordance with their functions. Enzymes involved in starch and sucrose biosynthesis showed the highest correlations between proteome-transcriptome profiles. High percentage of identification and validation of protein-transcript hits highlighted the power of omics data integration approach over existing gene functional annotation tools. We found that correlation of two datasets is highly influenced by stage of grain development. Further, gene regulatory networks would be helpful in unraveling the mechanisms underlying the complex and significant traits such as grain weight and yield.

Genetic and Environmental Factors Associated to Glutenin Polymer Characteristics of Wheat.

The polymers of wheat glutenins are studied here using asymmetric flow field flow fractionation (A4F). Molecular mass (Mw), gyration radius (Rw), and the polydispersity index (PI) of polymers were measured over a four-year, multi-local wheat trial in France. The experiment, involving 11 locations and 192 cultivars, offered the opportunity to approach the genetic and environmental factors associated with the phenotypic values of the polymer characteristics. These characteristics, which were all highly influenced by environmental factors, exhibited low broad-sense heritability coefficients and were not influenced by grain protein content and grain hardness. The 31 alleles encoding the glutenin subunits explained only 17.1, 25.4, and 16.8% of the phenotypic values of Mw, Rw, and PI, respectively. The climatic data revealed that a 3.5 °C increase between locations of the daily average temperature, during the last month of the grain development, caused an increase of more than 189%, 242%, and 434% of the Mw, Rw, and PI, respectively. These findings have to be considered in regard to possible consequences of global warming and health concerns assigned to gluten. It is suggested that the molecular characteristics of glutenins be measured today, especially for research addressing non-celiac gluten sensitivity (NCGS).

SNP markers for early identification of high molecular weight glutenin subunits (HMW-GSs) in bread wheat.

KEY MESSAGE: A set of eight SNP markers was developed to facilitate the early selection of HMW-GS alleles in breeding programmes. In bread wheat (Triticum aestivum), the high molecular weight glutenin subunits (HMW-GSs) are the most important determinants of technological quality. Known to be very diverse, HMW-GSs are encoded by the tightly linked genes Glu-1-1 and Glu-1-2. Alleles that improve the quality of dough have been identified. Up to now, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of grain proteins is the most widely used for their identification. To facilitate the early selection of HMW-GS alleles in breeding programmes, we developed DNA-based molecular markers. For each accession of a core collection (n = 364 lines) representative of worldwide bread wheat diversity, HMW-GSs were characterized by both genotyping and SDS-PAGE. Based on electrophoresis, we observed at least 8, 22 and 9 different alleles at the Glu-A1, Glu-B1 and Glu-D1 loci, respectively, including new variants. We designed a set of 17 single-nucleotide polymorphism (SNP) markers that were representative of the most frequent SDS-PAGE alleles at each locus. At Glu-A1 and Glu-D1, two and three marker-based haplotypes, respectively, captured the diversity of the SDS-PAGE alleles rather well. Discrepancies were found mainly for the Glu-B1 locus. However, statistical tests revealed that two markers at each Glu-B1 gene and their corresponding haplotypes were more significantly associated with the rheological properties of the dough than were the relevant SDS-PAGE alleles. To conclude, this study demonstrates that the SNP markers developed provide additional information on HMW-GS diversity. Two markers at Glu-A1, four at Glu-B1 and two at Glu-D1 constitute a useful toolbox for breeding wheat to improve end-use value.

In Situ Gluten-Chitosan Interlocked Self-Assembled Supramolecular Architecture Reduces T-Cell-Mediated Immune Response to Gluten in Celiac Disease.

SCOPE: The prevalence of celiac disease has increased since the last half of the 20th century and is now about 1% in most western populations. At present, people who suffer from celiac disease have to follow a gluten-exclusion diet throughout their lives. Compliance to this restrictive diet is demanding and the development of alternative strategies has become urgent. METHODS AND RESULTS: In this context, it is found that the biocompatible aminopolysaccharide chitosan imposes a different gluten reorganization after gluten redox reaction producing in situ mechanically interlocked supramolecular assemblies between gluten and chitosan. These new structures result in the decrease of gluten digestibility, tissue transglutaminase deamidation activity, and interferon-γ production in intestinal T cell lines generated from biopsy specimens of celiac disease patients. CONCLUSION: Overall, the results demonstrate the potential of this research avenue to celiac disease is problematic, as the reorganization of gluten proteins to a novel supramolecular architecture shows a positive impact on known pathogenesis mechanisms of the disease. At present, the only therapy for celiac disease is adherence to a gluten-free diet. Here, it is shown that chitosan-imposed gluten reorganization to an interlocked self-assembled supramolecular architecture reduces gluten digestibility, R5-reactivity, tissue transglutaminase deamidation activity, and its capacity to stimulate a T-cell-mediated immune response in celiac disease.

Wheat glutenin: the "tail" of the 1By protein subunits.

Gluten-forming storage proteins play a major role in the viscoelastic properties of wheat dough through the formation of a continuous proteinaceous network. The high-molecular-weight glutenin subunits represent a functionally important subgroup of gluten proteins by promoting the formation of large glutenin polymers through interchain disulphide bonds between glutenin subunits. Here, we present evidences that y-type glutenin subunits encoded at the Glu-B1 locus are prone to proteolytic processing at the C-terminus tail, leading to the loss of the unique cysteine residue present at the C-terminal domain. Results obtained by intact mass measurement and immunochemistry for each proteoform indicate that the proteolytic cleavage appears to occur at the carboxyl-side of two conserved asparagine residues at the C-terminal domain start. Hence, we hypothesize that the responsible enzymes are a class of cysteine endopeptidases - asparaginyl endopeptidases - described in post-translational processing of other storage proteins in wheat. Biological significance The reported study provides new insights into wheat storage protein maturation. In view of the importance of gluten proteins on dough viscoelastic properties and end-product quality, the reported C-terminal domain cleavage of high-molecular-weight glutenin subunits is of particular interest, since this domain possesses a unique conserved cysteine residue which is assumed to participate in gluten polymerization.

New insights into wheat toxicity: Breeding did not seem to contribute to a prevalence of potential celiac disease's immunostimulatory epitopes.

Gluten proteins, namely gliadins, are the primary trigger of the abnormal immune response in celiac disease. It has been hypothesised that modern wheat breeding practices may have contributed to the increase in celiac disease prevalence during the latter half of the 20th century. Our results do not support this hypothesis as Triticum aestivum spp. vulgare landraces, which were not subjected to breeding practices, presented higher amounts of potential celiac disease's immunostimulatory epitopes when compared to modern varieties. Furthermore, high variation between wheat varieties concerning the toxic epitopes amount was observed. We carried out quantitative analysis of gliadin types by RP-HPLC to verify its correlation with the amount of toxic epitopes: ω-type gliadins content explain about 40% of the variation of toxic epitopes in tetraploid wheat varieties. This research provides new insights regarding wheat toxicity and into the controversial idea that human practices may have conducted to an increased exposure to toxic epitopes.

Interruption of magnesium supply at heading influenced proteome of peripheral layers and reduced grain dry weight of two wheat (Triticum aestivum L.) genotypes.

UNLABELLED: Magnesium (Mg), an indispensable mineral for plant growth, is concentrated in the peripheral layers (PLs) of the mature grain of wheat. The supply of Mg was interrupted from plant heading to maturity and a proteomic approach was used to investigate the PLs at three stages of development. Two genotypes with contrasting concentrations of Mg in the grain were studied: Apache (low Mg) and MgHL (high Mg). The concentration of Mg was significantly reduced in the roots (10-21%), straw (18-50%) and grain (24-10%), respectively. Mg deficiency altered enzymes involved in photosynthesis, glycolysis, respiration, amino acid synthesis, cell division, protein degradation and folding at early stages, especially in MgHL. This latter had smaller grain by reducing grain potential size and dry matter accumulation. By contrast in Apache, few proteins were affected at early stages and proteins related to stress/defense and arginine/proline metabolism were up accumulated resulting in lower number of grains per ear (24.9%). This study showed that Mg in PLs plays an important role in cell division, ATP generation, carbohydrate and amino acid metabolism, and hence may influence grain potential size and assimilates in grain, which determines grain weight. These results should help wheat breeders improve Mg content and hence grain yield. BIOLOGICAL SIGNIFICANCE: Magnesium (Mg) is an abundant cation and is involved in many cell activities. Its role in determining wheat productivity remains unclear. This study is the first to investigate how Mg deficiency influences the physiological characters of wheat and dry matter in the grain in two genotypes with contrasting Mg content. Moreover, Mg is concentrated in peripheral layers of grain, which are known to play a critical role in grain development. In this study, we investigated proteins in the peripheral layers expressed differentially in three development stages to identify the mechanism by which Mg influences grain development. This study revealed that the supply of Mg influences grain yield and that Mg regulates proteins related to cell metabolism and stress defense in grain.

The impact of raw materials and baking conditions on Maillard reaction products, thiamine, folate, phytic acid and minerals in white bread.

The aim of this study was to develop a white bread with improved nutrient contents and reduced levels of potentially harmful Maillard reaction products such as N(ε)-carboxymethyllysine (CML) and 5-hydroxymethylfurfural (HMF). Assays were carried out through a full factorial experimental design allowing the simultaneous analysis of four factors at two levels: (1) wheat flour extraction rates (ash content: 0.60%-0.72%), (2) leavening agents (bakers' yeast - bakers' yeast and sourdough), (3) prebaking and (4) baking conditions (different sets of time and temperature). The baking conditions affected HMF and CML as well as certain mineral contents. A reduced baking temperature along with a prolonged heat treatment was found to be favourable for reducing both the CML (up to 20%) and HMF concentrations (up to 96%). The presence of sourdough decreased the formation of CML (up to 28%), and increased the apparent amounts of calcium (up to 8%) and manganese (up to 17.5%) probably through acidification of the dough. The extraction rate of flours as well as interactions between multiple factors also affected certain mineral content. However, compounds like folate, thiamine, copper, zinc, iron and phytic acid were not affected by any of the factors studied.

Identification and molecular characterization of oat peptides implicated on coeliac immune response.

BACKGROUND: Oats provide important nutritional and pharmacological properties, although their safety in coeliac patients remains controversial. Previous studies have confirmed that the reactivity of the anti-33-mer monoclonal antibody with different oat varieties is proportional to the immune responses in terms of T-cell proliferation. Although the impact of these varieties on the adaptive response has been studied, the role of the dendritic cells (DC) is still poorly understood. The aim of this study is to characterize different oat fractions and to study their effect on DC from coeliac patients. METHODS AND RESULTS: Protein fractions were isolated from oat grains and analyzed by SDS-PAGE. Several proteins were characterized in the prolamin fraction using immunological and proteomic tools, and by Nano-LC-MS/MS. These proteins, analogous to α- and γ-gliadin-like, showed reactive sequences to anti-33-mer antibody suggesting their immunogenic potential. That was further confirmed as some of the newly identified oat peptides had a differential stimulatory capacity on circulating DC from coeliac patients compared with healthy controls. CONCLUSIONS: This is the first time, to our knowledge, where newly identified oat peptides have been shown to elicit a differential stimulatory capacity on circulating DC obtained from coeliac patients, potentially identifying immunogenic properties of these oat peptides.

Efficient chemo-enzymatic gluten detoxification: reducing toxic epitopes for celiac patients improving functional properties.

Protein engineering of gluten, the exogenous effector in celiac disease, seeking its detoxification by selective chemical modification of toxic epitopes is a very attractive strategy and promising technology when compared to pharmacological treatment or genetic engineering of wheat. Here we present a simple and efficient chemo-enzymatic methodology that decreases celiac disease toxic epitopes of gluten proteins improving its technological value through microbial transglutaminase-mediated transamidation of glutamine with n-butylamine under reducing conditions. First, we found that using low concentrations of amine-nucleophile under non-reducing conditions, the decrease in toxic epitopes is mainly due to transglutaminase-mediated cross-linking. Second, using high amine nucleophile concentrations protein cross-linking is substantially reduced. Third, reducing conditions increase 7-fold the transamidation reaction further decreasing toxic epitopes amount. Fourth, using n-butylamine improves gluten hydrophobicity that strengthens the gluten network. These results open the possibility of tailoring gluten for producing hypoallergenic flours while still taking advantage of the unique viscoelastic properties of gluten.

Changes in the nuclear proteome of developing wheat (Triticum aestivum L.) grain.

Wheat grain end-use value is determined by complex molecular interactions that occur during grain development, including those in the cell nucleus. However, our knowledge of how the nuclear proteome changes during grain development is limited. Here, we analyzed nuclear proteins of developing wheat grains collected during the cellularization, effective grain-filling, and maturation phases of development, respectively. Nuclear proteins were extracted and separated by two-dimensional gel electrophoresis. Image analysis revealed 371 and 299 reproducible spots in gels with first dimension separation along pH 4-7 and pH 6-11 isoelectric gradients, respectively. The relative abundance of 464 (67%) protein spots changed during grain development. Abundance profiles of these proteins clustered in six groups associated with the major phases and phase transitions of grain development. Using nano liquid chromatography-tandem mass spectrometry to analyse 387 variant and non-variant protein spots, 114 different proteins were identified that were classified into 16 functional classes. We noted that some proteins involved in the regulation of transcription, like HMG1/2-like protein and histone deacetylase HDAC2, were most abundant before the phase transition from cellularization to grain-filling, suggesting that major transcriptional changes occur during this key developmental phase. The maturation period was characterized by high relative abundance of proteins involved in ribosome biogenesis. Data are available via ProteomeXchange with identifier PXD002999.

Proteomic Approach to Identify Nuclear Proteins in Wheat Grain.

The nuclear proteome of the grain of the two cultivated wheat species Triticum aestivum (hexaploid wheat; genomes A, B, and D) and T. monococcum (diploid wheat; genome A) was analyzed in two early stages of development using shotgun-based proteomics. A procedure was optimized to purify nuclei, and an improved protein sample preparation was developed to efficiently remove nonprotein substances (starch and nucleic acids). A total of 797 proteins corresponding to 528 unique proteins were identified, 36% of which were classified in functional groups related to DNA and RNA metabolism. A large number (107 proteins) of unknown functions and hypothetical proteins were also found. Some identified proteins may be multifunctional and may present multiple localizations. On the basis of the MS/MS analysis, 368 proteins were present in the two species, and in two stages of development, some qualitative differences between species and stages of development were also found. All of these data illustrate the dynamic function of the grain nucleus in the early stages of development.

Proteome evolution of wheat (Triticum aestivum L.) aleurone layer at fifteen stages of grain development.

The aleurone layer (AL) is the grain peripheral tissue; it is rich in micronutrients, vitamins, antioxidants, and essential amino acids. This highly nutritive part of the grain has been less studied partly because its isolation is so laborious. In the present study, the ALs of Triticum aestivum (variety Récital) were separated manually at 15 stages of grain development. A total of 327 proteins were identified using 2-DE LC-MS/MS. They were classified in six main groups and 26 sub-groups according to their biochemical function. Proteomic analysis revealed seven different profiles distributed among three main development stages: (i) early AL development, with proteins involved in intense metabolic activities in the growth and development of the cell wall compounds; (ii) the intermediate stage, characterized by oxidative stress and defense proteins (65%) linked with loss of water in peripheral layers during grain filling; and (iii) AL maturation, involving the production of amino acids and the control of reactive oxidative species to enable the accumulation and maturation of globulins within the AL. The present study provides the first insights into developing proteome in the AL. We describe the numerous AL enzymes involved in the accumulation of storage protein and in the protection of the endosperm over time. BIOLOGICAL SIGNIFICANCE: The hand dissection of wheat aleurone layer (AL) was carried in this study for the first time on fifteen developmental stages from cell differentiation to grain maturity. Three major phases were revealed over AL development: cell division activities, globulins storage, and grain protection. Enzymes related to metabolites and vitamins were abundantly expressed during the two first phases. In parallel to the progressive globulins accumulation, the final phase was characterized by key enzyme synthesis involved in energy production, amino-acids and antioxidant synthesis plus others to face hypoxia and dehydration of grain tissues.

Development of a LC-MS/MS method for the simultaneous screening of seven water-soluble vitamins in processing semi-coarse wheat flour products.

Wheat is the second largest crop cultivated around the world and constitutes a major part of the daily diet in Europe. It is therefore important to determine the content of micronutrient in wheat and wheat-based food products to define the contribution of wheat-based foods to the nutrition of the consumers. The aim of the present work was to develop a simple and rapid method based on liquid chromatography tandem mass spectrometry (LC-MS/MS) for the simultaneous determination of seven water-soluble vitamins in various wheat-based food materials. The vitamins present in the test material were separated in less than 15 min by using a reverse-phase C18 column, and analyzed by positive ion electrospray selected reaction monitoring MS/MS. The MS response for all the vitamins was linear over the working range (0.05 to 9 µg/mL) with correlation coefficients ranging between 0.991 and 1. Limits of quantification in the different food materials ranged from 0.09 to 3.5 µg/g. Intra-day and inter-day precision were found satisfactory. The developed method was applied for the simultaneous analysis of the water-soluble vitamin natural content of different semi-coarse wheat flours and in their corresponding baking products.

One hundred years of grain omics: identifying the glutens that feed the world.

Glutens, the storage proteins in wheat grains, are a major source of protein in human nutrition. The protein composition of wheat has therefore been an important focus of cereal research. Proteomic tools have been used to describe the genetic diversity of wheat germplasms from different origins at the level of polymorphisms in alleles encoding glutenin and gliadin, the two main proteins of gluten. More recently, proteomics has been used to understand the impact of specific gluten proteins on wheat quality. Here we review the impact of proteomics on the study of gluten proteins as it has evolved from fractionation and electrophoretic techniques to advanced mass spectrometry. In the postgenome era, proteomics is proving to be essential in the effort to identify and understand the interactions between different gluten proteins. This is helping to fill in gaps in our knowledge of how the technological quality of wheat is determined by the interaction between genotype and environment. We also collate information on the various storage protein alleles identified and their prevalence, which makes it possible to infer the effects of wheat selection on grain protein content. We conclude by reviewing the more recent use of transgenesis aimed at improving the quality of gluten.

Proteogenomic Characterization of Novel x-Type High Molecular Weight Glutenin Subunit 1Ax1.1.

Analysis of Portuguese wheat (Triticum aestivum L.) landrace 'Barbela' revealed the existence of a new x-type high molecular weight-glutenin subunit (HMW-GS) encoded at the Glu-A1 locus, which we named 1Ax1.1. Using one-dimensional and two-dimensional electrophoresis and mass spectrometry, we compared subunit 1Ax1.1 with other subunits encoded at the Glu-A1 locus. Subunit 1Ax1.1 has a theoretical molecular weight of 93,648 Da (or 91,508 Da for the mature protein) and an isoelectric point (pI) of about 5.7, making it the largest and most acidic HMW-GS known to be encoded at Glu-A1. Specific primers were designed to amplify and sequence 2601 bp of the Glu-A1 locus from the 'Barbela 28' wheat genome. A very high level of identity was found between the sequence encoding 1Ax1.1 and those encoding other alleles of the locus. The major difference found was an insertion of 36 amino acids in the central repetitive domain.