Peptidomics analysis of in vitro digested wheat breads: Effect of genotype and environment on protein digestibility and release of celiac disease and wheat allergy related epitopes.

Wheat proteins can trigger immunogenic reactions due to their resistance to digestion and immunostimulatory epitopes. Here, we investigated the peptidomic map of partially digested bread samples and the fingerprint of epitope diversity from 16 wheat genotypes grown in two environmental conditions. Flour protein content and composition were characterized; gastric and jejunal peptides were quantified using LC-MS/MS, and genotypes were classified into high or low bread protein digestibility. Differences in flour protein content and peptide composition distinguish high from low digestibility genotypes in both growing environments. No common peptide signature was found between high- and low-digestible genotypes; however, the celiac or allergen epitopes were noted not to be higher in low-digestible genotypes. Overall, this study established a peptidomic and epitope diversity map of digested wheat bread and provided new insights and correlations between weather conditions, genotypes, digestibility and wheat sensitivities such as celiac disease and wheat allergy.

Sulfur in determining seed protein composition: present understanding of its interaction with abiotic stresses and future directions.

Improving and stabilizing the quality of seed proteins are of growing interest in the current food and agroecological transitions. Sulfur is a key determinant of this quality since it is essential for the synthesis of sulfur-rich proteins in seeds. A lack of sulfur provokes drastic changes in seed protein composition, negatively impacting the nutritional and functional properties of proteins, and leading in some cases to diseases or health problems in humans. Sulfur also plays a crucial role in stress tolerance through the synthesis of antioxidant or protective molecules. In the context of climate change, questions arise regarding the trade-off between seed yield and seed quality with respect to sulfur availability and use by crops that represent important sources of proteins for human nutrition. Here, we review recent work obtained in legumes, cereals, as well as in Arabidopsis, that present major advances on: (i) the interaction between sulfur nutrition and environmental or nutritional stresses with regard to seed yield and protein composition; (ii) metabolic pathways that merit to be targeted to mitigate negative impacts of environmental stresses on seed protein quality; and (iii) the importance of sulfur homeostasis for the regulation of seed protein composition and its interplay with seed redox homeostasis.

Bread wheat (T. aestivum) variability: Phenotypic and genotypic data from 75 varieties.

Most bread wheat is consumed after processing, which mainly depends on the quantity and quality of protein in the grain. Storage protein content and composition particularly influence the end use quality of milled grain products. Storage proteins are components of the gluten network that confer dough viscoelasticity, an essential property for processing. To explore grain storage protein diversity, 75 bread wheat accessions were grown with two replicates each at two locations. Grains were harvested at maturity and samples were phenotyped for each site and each replicate plant. Grain hardness, thousand-kernel weight and grain nitrogen content were measured. The protein composition of flour from each replicate was characterised by reverse phase-high performance liquid chromatography (RP-HPLC). The molecular distribution of flour polymers was determined by asymmetric flow field-flow fractionation (AF4) and dough technological properties were assessed using a Glutomatic system and a Chopin alveograph. In addition, the 75 accessions were genotyped by the BreedWheat 35k genotyping array (Axiom TaBW35K) containing 34,746 single nucleotide polymorphism markers (SNPs). The dataset produced by this work includes six files with raw data, two files with protocols and figures. Data show the genotypic and phenotypic variabilities of the material used and can be used to explore genetic and environmental effects on traits involved in grain protein quality. This dataset is associated to the research article "Differences in bread protein digestibility traced to wheat cultivar traits" [1].

Omics Data Reveal Putative Regulators of Einkorn Grain Protein Composition under Sulfur Deficiency.

Understanding the molecular mechanisms controlling the accumulation of grain storage proteins in response to nitrogen (N) and sulfur (S) nutrition is essential to improve cereal grain nutritional and functional properties. Here, we studied the grain transcriptome and metabolome responses to postanthesis N and S supply for the diploid wheat einkorn (Triticum monococcum). During grain filling, 848 transcripts and 24 metabolites were differentially accumulated in response to N and S availability. The accumulation of total free amino acids per grain and the expression levels of 241 genes showed significant modifications during most of the grain filling period and were upregulated in response to S deficiency. Among them, 24 transcripts strongly responded to S deficiency and were identified in coexpression network analyses as potential coordinators of the grain response to N and S supply. Sulfate transporters and genes involved in sulfate and Met metabolism were upregulated, suggesting regulation of the pool of free amino acids and of the grain N-to-S ratio. Several genes highlighted in this study might limit the impact of S deficiency on the accumulation of grain storage proteins.

Proteomic Data Integration Highlights Central Actors Involved in Einkorn (Triticum monococcum ssp. monococcum) Grain Filling in Relation to Grain Storage Protein Composition.

Albumins and globulins (AGs) of wheat endosperm represent about 20% of total grain proteins. Some of these physiologically active proteins can influence the synthesis of storage proteins (SPs) (gliadins and glutenins) and consequently, rheological properties of wheat flour and processing. To identify such AGs, data, (published by Bonnot et al., 2017) concerning abundance in 352 AGs and in the different seed SPs during grain filling and in response to different nitrogen (N) and sulfur (S) supply, were integrated with mixOmics R package. Relationships between AGs and SPs were first unraveled using the unsupervised method sparse Partial Least Square, also known as Projection to Latent Structure (sPLS). Then, data were integrated using a supervised approach taking into account the nutrition and the grain developmental stage. We used the block.splda procedure also referred to as DIABLO (Data Integration Analysis for Biomarker discovery using Latent variable approaches for Omics studies). These approaches led to the identification of discriminant and highly correlated features from the two datasets (AGs and SPs) which are not necessarily differentially expressed during seed development or in response to N or S supply. Eighteen AGs were correlated with the quantity of SPs per grain. A statistical validation of these proteins by genetic association analysis confirmed that 5 out of this AG set were robust candidate proteins able to modulate the seed SP synthesis. In conclusion, this latter result confirmed that the integrative strategy is an adequate way to reduce the number of potentially relevant AGs for further functional validation.

Grain subproteome responses to nitrogen and sulfur supply in diploid wheat Triticum monococcum ssp. monococcum.

Wheat grain storage proteins (GSPs) make up most of the protein content of grain and determine flour end-use value. The synthesis and accumulation of GSPs depend highly on nitrogen (N) and sulfur (S) availability and it is important to understand the underlying control mechanisms. Here we studied how the einkorn (Triticum monococcum ssp. monococcum) grain proteome responds to different amounts of N and S supply during grain development. GSP composition at grain maturity was clearly impacted by nutrition treatments, due to early changes in the rate of GSP accumulation during grain filling. Large-scale analysis of the nuclear and albumin-globulin subproteomes during this key developmental phase revealed that the abundance of 203 proteins was significantly modified by the nutrition treatments. Our results showed that the grain proteome was highly affected by perturbation in the N:S balance. S supply strongly increased the rate of accumulation of S-rich α/ß-gliadin and γ-gliadin, and the abundance of several other proteins involved in glutathione metabolism. Post-anthesis N supply resulted in the activation of amino acid metabolism at the expense of carbohydrate metabolism and the activation of transport processes including nucleocytoplasmic transit. Protein accumulation networks were analyzed. Several central actors in the response were identified whose variation in abundance was related to variation in the amounts of many other proteins and are thus potentially important for GSP accumulation. This detailed analysis of grain subproteomes provides information on how wheat GSP composition can possibly be controlled in low-level fertilization condition.

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.

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.

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.

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.

Extraction and proteome analysis of starch granule-associated proteins in mature wheat kernel (Triticum aestivum L.).

Starch consists of the two glucose polymers, amylose and amylopectin, and is deposited as semicrystalline granules inside plastids. The starch granule proteome is particularly challenging to study due to the amount of interfering compounds (sugars, storage proteins), the very low starch granule-associated protein content and also the dynamic range of abundant proteins. Here we present the protocol for extraction and 2-DE of wheat starch granule-associated proteins whose most important steps are: (i) washing and sonication to remove interfering compounds (storage proteins) from the surface of the granules, (ii) scanning electron microscopy (SEM) observations to monitor purification and granules swelling, (iii) appropriate protein extraction and solubilization to obtain enough proteins for Coomassie blue staining and proteomic analysis. Our objective was to minimize the amount of contamination by storage proteins and to preserve the structure of the starch and of starch-associated proteins and to maximize the number of polypeptides that can be resolved. For quantitative proteomic analysis of proteins associated with wheat starch granules, we developed a two-step protein extraction protocol including TCA/acetone precipitation and phenol extraction. With this protocol, proteins were extracted from wheat starch granules and solubilized and satisfactory blue-stained 2-DE protein maps were obtained. The majority of the spots associated with starch granules were identified by peptide mass fingerprinting and MS/MS and functionally classified into carbohydrate metabolism and stress defense.

Effect of the 1BL.1RS translocation on the wheat endosperm, as revealed by proteomic analysis.

The introduction of the 1RS chromosome of rye into wheat made wheat more resistant to several pathogens. Today, this resistance has been overcome but the 1BL.1RS translocation remains interesting because of the improved yield and despite the lower rheological properties it produces. Nothing has been reported yet on the impact of rye chromatin introgression on the grain proteome of wheat. The comparison of the 2-DE profiles of 16 doubled haploid lines, with or without the 1BL.1RS translocation, revealed quantitative and qualitative proteic variations in prolamins and other endosperm proteins. Eight spots were found specifically in lines having the 1BL.1RS translocation; 16 other spots disappeared from the same lines. Twelve spots, present in both genotypes, met the criteria for up- or down-regulated spots. In translocated genotypes, a highly overexpressed spot, identified as a gamma-gliadin with nine cysteine residues, suggests that the lack of LMW-GS induced by 1BL.1RS is counterbalanced by an overexpression of a relatively similar prolamin. Moreover, a spot that was absent from 1BL.1RS genotypes was identified as a dimeric alpha-amylase inhibitor. It was considered to be a valuable candidate to explain the sticky dough associated with translocated cultivars.

Protein extraction from cereal seeds.

Seeds may contain different components such as starch and complex carbohydrates that can seriously reduce protein extraction. The proteins in cereal seeds are usually classified in four groups according to their solubility criteria: albumins, globulins, prolamins, and glutelins. They can be specifically extracted. A general procedure for extracting the proteins present in green seeds or immature cereal kernels is given. Then several procedures mostly adapted to cereal seeds are reported for: (1) the whole storage proteins (mostly prolamins and glutelins); (2) the albumins-globulins extracted using salt buffer; (3) the amphiphilic proteins extracted using a phase partitioning process; and (4) the proteins strongly attached to or within the starch granules of the seed endosperm. These procedures have been used for 2-D electrophoresis and proteomic analyses.

Proteomic analysis of aneuploid lines in the homeologous group 1 of the hexaploid wheat cultivar Courtot.

Three monosomic lines (MSLs) and three nullisomic lines (NSLs) of the homeologous group 1 and one euploid line of the bread wheat Triticum aestivum cultivar Courtot were used in a proteomic approach to investigate the effects of zero, one or two doses of chromosomes 1A, 1B and 1D on the amount of endosperm proteins. Polypeptides whose amounts changed significantly between each aneuploid line and the euploid line were identified using image analyses of two-dimensional gel electrophoresis patterns resulting from specific endosperm protein extractions. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry and electrospray ionization tandem mass spectrometry were also used for protein identification. Removing one chromosome or a chromosome pair allowed varying responses to be observed for the remaining endosperm protein genes. Compensation phenomena for the high molecular weight glutenin subunits (HMW-GS) were detected only in the MSLs. Subunits Bx7, By8 and Dy12 were the only HMW-GS overexpressed (from 152-737%) when chromosomes 1A or 1B or 1D were at hemizygous state. Thirteen new protein spots were detected only in the NSL1D, and seven were identified as HMW-GS analogs. These seven new spots may result from the expression of inactive genes. The HMW-GS were of significantly higher volume in MSLs, whereas the low molecular weight glutenin subunits and the gamma-gliadins were of lower volume in aneuploid lines. Most of the down-regulated proteins in the MSLs were storage proteins encoded at loci located on another chromosome pair. Complex regulations between chromosomes and loci of the homeologous groups 1 and 6 in bread wheat are discussed.

Proteomic analysis of the effect of heat stress on hexaploid wheat grain: characterization of heat-responsive proteins from non-prolamins fraction.

The effect of heat stress on hexaploid wheat grain proteome was recently analyzed in our previous works. Proteomic tools allowed the characterization of heat-responsive proteins of total endosperm, composed mainly of prolamins. The present work completes this study; our aim was to analyze the effect of heat stress on the water-soluble fraction, composed essentially of albumins and globulins. These proteins were separated by two-dimensional electrophoresis (2-DE), visualized by Coomassie Brilliant Blue (CBB) staining and analyzed by Melanie-3 software. Of the 43 heat-changed proteins, 24 were found to be up-regulated whereas 19 spot proteins were down-regulated. All of these proteins were subjected to matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) followed by database searching which allowed the identification of 42 spots. Of these, some were enzymes involved in different metabolic pathways of plants, such as granule-bound starch synthase and glucose-1-phosphate adenyltransferase, involved in the starch synthesis pathway; beta-amylase, involved in carbohydrate metabolism, and the ATP synthase beta-chain that was related to four heat-decreased proteins. Moreover, five heat up-regulated proteins showed similarities with small heat shock proteins while three other spots were related to elongation factors or eucaryotic translation initiation factors. Proteins involved in abiotic stresses or in plant defense mechanism were also identified and are discussed.

Proteomic analysis of the effect of heat stress on hexaploid wheat grain: Characterization of heat-responsive proteins from total endosperm.

High temperatures during grain filling have been reported to be one of the factors that can affect the dough properties and quality characteristics of wheat. Responses to high temperature have been related to changes in protein composition at both quantitative and qualitative levels. The present study was conducted to determine the influence of high temperature during grain filling on the protein composition of bread wheat evaluated by proteomic tools. Plants were grown in the field and transferred to cabinets soon after flowering. They were subjected to two thermal regimes 18 degrees C/10 degrees C (day/night) and 34 degrees C/10 degrees C. Total proteins were extracted from control grains and treated plants at three different post-anthesis stages. The proteins were separated by two-dimensional gel electrophoresis and analysed by Melanie 3 software. Of the total number of mature wheat grain proteins, 37 were identified as significantly changed by heat treatment. Analysis by matrix-assisted laser desorption/ionization mass spectrometry and tandem mass spectrometry coupled with database searching allowed the characterization of 25 heat-induced proteins and only one heat-decreased protein spot. To learn more about the function of the identified proteins, we examined their expression during treatment.