Wheat Storage Proteins Research Articles

The role of starch digestion in the brewing of gluten-free beers

Celiac disease affects an estimated 1% of the global population, and symptoms are triggered by the consumption of gluten in sensitized individuals. Other gluten sensitivities exist but are poorly characterized or often under-diagnosed, leading a number of people to pursue a gluten-free or gluten-reduced diet even in the absence of a diagnosis. Gluten is a complex suite of proteins from the prolamin fraction of storage proteins in barley, wheat, and rye, meaning that foods or beverages that are produced from these cereals will also contain gluten. Beer is one such beverage that is traditionally produced from malted barley and is thus incompatible with a gluten-free lifestyle. The simplest option to produce a gluten-free beer would seem to be to use alternative, gluten-free, grains such as millet, sorghum, teff, rice, corn or other starchy materials. However, barley has thousands of years of development as a brewing agent behind it and these alternative ingredients cannot be incorporated into the brewing process without substantial alterations. Particularly, these ingredients generally have much lower endogenous starch degrading activity and much higher starch gelatinization temperatures as compared to barley which makes generating sufficient fermentable sugars for yeast fermentation a challenge. In this review we will further elucidate these challenges as well as the solutions that are being developed to bring beer to the gluten-free masses.

Influence of pH and lipid membrane on the liquid–liquid phase separation of wheat γ-gliadin in aqueous conditions

Protein body (PB) formation in wheat seeds is a critical process influencing seed content and nutritional quality. In this study, we investigate the potential mechanisms governing PB formation through an in vitro approach, focusing on γ-gliadin, a key wheat storage protein. We used a microfluidic technique to encapsulate γ-gliadin within giant unilamellar vesicles (GUVs) and tune the physicochemical conditions in a controlled and rapid way. We examined the influence of pH and protein concentration on LLPS and protein-membrane interactions using various microscopy and spectroscopy techniques. We showed that γ-gliadin encapsulated in GUVs can undergo a pH-triggered liquid–liquid phase separation (LLPS) by two distinct mechanisms depending on the γ-gliadin concentration. At low protein concentrations, γ-gliadins phase separate by a nucleation and growth-like process, while, at higher protein concentration and pH above 6.0, γ-gliadin formed a bi-continuous phase suggesting a spinodal decomposition-like mechanism. Fluorescence and microscopy data suggested that γ-gliadin dense phase exhibited affinity for the GUV membrane, forming a layer at the interface and affecting the reversibility of the phase separation.

Detection of gluten peptides in human duodenal fluids with immuno-liquid chromatography-mass spectrometry.

Gluten proteins are storage proteins in wheat that exhibit a certain resistance to gastrointestinal digestion. To explore solutions to cope with accidental ingestion of gluten in individuals suffering from gluten-related disorders, it is essential to monitor the fate of gluten peptides in biological samples, i.e., gastrointestinal juices, blood plasma or urine. In this work, we aimed at developing a mass spectrometry (MS)-based method for measuring gluten peptides in human duodenal fluids. Seven gluten peptides, including the well-documented 33-mer gluten peptide (LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF), were selected after a literature review and characterization of a gluten-containing product. Isotopically labelled peptides were used as references and a targeted liquid chromatography (LC) MS assay based on high resolution parallel reaction monitoring (PRM) was designed. Despite iterative and fine tuning of the LC-PRM-MS method, the low level of endogenous gluten peptides in human duodenal fluid samples precluded their direct detection. Thus, an initial immunoprecipitation (IP) step was included. Several antibodies were tested, and one proved reliable for the enrichment of the 33-mer gluten peptide as well as a few additional gluten peptides. Figures-of-merits of the immuno-LC-PRM-MS assay were assessed with a focus on quantification trueness and precision. We have developed an MS-based method for measuring the 33-mer gluten peptide in human duodenal fluids. Based on isotopic dilution, the method relies on the combination of IP and LC-PRM-MS analysis. Measurements were shown to be sensitive, quantitative, and reproducible.

Wheat GSPs and Processing Quality Are Affected by Irrigation and Nitrogen through Nitrogen Remobilisation.

The rheological properties and end-use qualities of many foods are mainly determined by the types and levels of grain storage proteins (GSPs) in wheat. GSP levels are influenced by various factors, including tillage management, irrigation, and fertiliser application. However, the effects of irrigation and nitrogen on GSPs remain unclear. To address this knowledge gap, a stationary split-split block design experiment was carried out in low- and high-fertility (LF and HF) soil, with the main plots subjected to irrigation treatments (W0, no irrigation; W1, irrigation only during the jointing stage; W2, irrigation twice during both jointing and flowering stages), subplots subjected to nitrogen application treatments (N0, no nitrogen application; N180, 180 kg/ha; N240, 240 kg/ha; N300, 300 kg/ha), and cultivars tested in sub-sub plots (FDC5, the strong-gluten cultivar Fengdecun 5; BN207, the medium-gluten cultivar Bainong 207). The results showed that GSP levels and processing qualities were significantly influenced by nitrogen application (p < 0.01), N240 was the optimal nitrogen rate, and the influence of irrigation was dependent on soil fertility. Optimal GSP levels were obtained under W2 treatment at LF conditions, and the content was increased by 17% and 16% for FDC5 and BN207 compared with W0 under N240 treatment, respectively. While the optimal GSP levels were obtained under W1 treatments at HF conditions, and the content was increased by 3% and 21% for FDC5 and BN207 compared with W0 under N240 treatment, respectively. Irrigation and nitrogen application increased the glutenin content by increasing Bx7 and Dy10 levels in FDC5, and by increasing the accumulation of Ax1 and Dx5 in BN207. Gliadins were mainly increased by enhancing α/β-gliadin levels. Correlation analysis indicated that a higher soil nitrate (NO3-N) content increased nitrogen remobilisation in leaves. Path analysis showed that Dy10, Dx5, and γ-gliadin largely determined wet glutenin content (WGC), dough stability time (DST), dough water absorption rate (DWR), and sedimentation value (SV). Therefore, appropriate irrigation and nitrogen application can improve nitrogen remobilisation, GSP levels, and processing qualities, thereby improving wheat quality and production.

Unlocking the genetic potential of Ethiopian durum wheat landraces with high protein quality: Sources to be used in future breeding for pasta production

AbstractThe content and composition of the grain storage proteins in wheat determine to a high extent its end‐use quality for pasta and bread production. This study aimed to evaluate the content and composition of the grain storage proteins in Ethiopian landraces and cultivars to contribute to future breeding toward improved pasta quality. Thus, 116 landraces and 34 cultivars originating from Ethiopia were grown in three locations, and the protein parameters were analyzed using size exclusion‐high performance liquid chromatography (SE‐HPLC). A considerable variation in the amount of the analyzed protein parameters was found. The genotypes, environments, and interactions contributed significantly (p &lt; 0.001) to the differences obtained. The broad‐sense heritability was high (0.75–0.98) for all protein parameters except for unextractable small monomeric protein (uSMP). Using the principal component analysis (PCA) to evaluate the impact of protein parameters and using either PCA or unweighted pair group method with arithmetic mean (UPGMA) to assess the impact of the genetic composition, the cultivar group was found to form a separate cluster. This indicates that durum wheat improvement in Ethiopia has relied on exotic materials, which might result from a narrow genetic base. Unlike most landraces, most released cultivars showed a high and stable gluten strength across environments. Two landraces, G057 and G107, were found genetically distinct from the released cultivars but with high and stable gluten. The two selected landraces might be of extremely high value for future use in durum wheat breeding programs, as they might be adapted to wide‐ranging Ethiopian growing conditions, they might carry genes of relevance to withstand abiotic and biotic stresses, and they seem to hold essential protein properties, which might result in high‐quality grains for industrial processes.

Gluten subfractions of wheat storage proteins are affected by high night temperature during grain formation

AbstractGluten (gliadin + glutenin) protein in wheat flour is affected by high temperature (day and/or night) resulting in undesirable consequences on dough quality. A study was conducted with early and late‐maturing wheat genotypes, to assess the spatial (superior‐ central and inferior‐ apical and basal spikelets) variation in the composition of gluten subfractions in the developing ear under high night temperature (HNT). We hypothesised that protein content in the superior and inferior grains may show a differential quantitative and qualitative response to HNT. HNT resulted in a significant increase in protein content which exhibited a strong (r = −0.44*) negative correlation with sedimentation volume (SV) that determines baking quality. The late‐maturity genotypes were more responsive to HNT with changes in ω‐5 and γ gliadin subfractions of both superior and inferior spikelets, though a consistent trend was not established. The proportion of high molecular weight (HMW) glutenins increased, whereas low molecular weight (LMW) glutenins reduced in most of the genotypes under HNT. Both HMW and LMW glutenins revealed significant positive (r = 0.43* and r = 0.81***, respectively) correlation with SV. The expression analysis of genes for gluten subfractions showed a significant decrease in transcript abundance of α, ω‐5, γ, HMW, and LMW fractions under HNT.

Efficient proteome-wide identification of transcription factors targeting Glu-1: A case study for functional validation of TaB3-2A1 in wheat.

High-molecular-weight glutenin subunits (HMW-GS), a major component of seed storage proteins (SSP) in wheat, largely determine processing quality. HMW-GS encoded by GLU-1 loci are mainly controlled at the transcriptional level by interactions between cis-elements and transcription factors (TFs). We previously identified a conserved cis-regulatory module CCRM1-1 as the most essential cis-element for Glu-1 endosperm-specific high expression. However, the TFs targeting CCRM1-1 remained unknown. Here, we built the first DNA pull-down plus liquid chromatography-mass spectrometry platform in wheat and identified 31 TFs interacting with CCRM1-1. TaB3-2A1 as proof of concept was confirmed to bind to CCRM1-1 by yeast one hybrid and electrophoretic mobility shift assays. Transactivation experiments demonstrated that TaB3-2A1 repressed CCRM1-1-driven transcription activity. TaB3-2A1 overexpression significantly reduced HMW-GS and other SSP, but enhanced starch content. Transcriptome analyses confirmed that enhanced expression of TaB3-2A1 down-regulated SSP genes and up-regulated starch synthesis-related genes, such as TaAGPL3, TaAGPS2, TaGBSSI, TaSUS1 and TaSUS5, suggesting that it is an integrator modulating the balance of carbon and nitrogen metabolism. TaB3-2A1 also had significant effects on agronomic traits, including heading date, plant height and grain weight. We identified two major haplotypes of TaB3-2A1 and found that TaB3-2A1-Hap1 conferred lower seed protein content, but higher starch content, plant height and grain weight than TaB3-2A1-Hap2 and was subjected to positive selection in a panel of elite wheat cultivars. These findings provide a high-efficiency tool to detect TFs binding to targeted promoters, considerable gene resources for dissecting regulatory mechanisms underlying Glu-1 expression, and a useful gene for wheat improvement.

Impact of Fusarium Head Blight on Wheat Flour Quality: Examination of Protease Activity, Technological Quality and Rheological Properties

Wheat infections caused by Fusarium represent a global agricultural problem that reduces grain yield and negatively impacts wheat’s technological and rheological quality. Although fungal proteases or an increase in endogenous proteases due to Fusarium infection could negatively influence wheat storage proteins and dough performance, little research has been performed on either of these topics. The primary objective of this study was to identify the effect of Fusarium infection on protease activity in 25 wheat cultivars grown in two distinct locations in eastern Croatia. Apart from proteolytic activity, this paper describes the impact of Fusarium head blight (FHB) infection on the technological quality parameters of wheat flour and the dough’s rheological properties. The first treatment consisted of naturally grown, healthy wheat without fungicides, while the second treatment utilized wheat varieties subjected to intense FHB infection. Protein and wet gluten content in wheat grain and flour of uninfected cultivars were heavily influenced by testing location, soil type, and quality. Fusarium infection increased the activity of nonspecific proteases by 43% in flour samples from Osijek and 125% in flour samples from Tovarnik. Estimates of effect size showed that FHB infection had twice as big an effect on protease activity in Tovarnik as in Osijek, and a similar trend was found for dough softening. Moreover, the infection significantly impacted wheat cultivars’ extensograph values, indicating a lower resistance to stretching, extensibility, and total stretching energy in infected flour samples, indicating that dough functionality and volume loss can be attributed to exogenous fungal proteases. Still, the magnitude of the effect varied depending on the growth location and the cultivar’s traits. Multivariate data analysis identified three clusters of wheat cultivars, each with varying degrees of the Fusarium infection’s effects. Some cultivars displayed consistent protease activity and flour quality across sites. In contrast, others showed variability in their responses due to environmental conditions. To conclude, genetic resistance could provide adequate control of FHB, guaranteeing the successful protection of wheat quality. However, the possibility of confounding factors influencing genetic and cultivation conditions must be considered, and further research is needed to understand their interaction.

Introgression of Seedling Plant Resistance to Leaf Rust from Agropyron cristatum into Wheat by Induced Homoeologous Recombination

Agropyron cristatum (P genome) is a Triticeae species from the wheat tertiary gene pool which has economic importance as forage and also displays traits beneficial to wheat. Resistance to leaf rust was previously mapped to the short arm of chromosome 1P (1PS) in A. cristatum by the development of a compensating Robertsonian translocation involving chromosome arm 1PS and the long arm of wheat chromosome 1B (1BL). In this study, chromosome arm 1PS was engineered using the ph1b mutation to induce 1BS/1PS homoeologous recombination and to obtain new translocations with shortened fragments of chromosome arm 1PS. Two translocations with different alien fragment sizes were identified by genomic in situ hybridization, wheat 1BS- and 1PS-specific molecular markers and gene-specific markers for glutenin, Glu-B3 and gliadin Gli-B1 seed storage protein. One translocation (called type 1) replaces a proximal segment of 1PS chromatin, and the other (called type 2) replaces a distal 1PS segment and introduces the Glu-B3 and Gli-B1 wheat storage protein loci. Six specific EST-STS markers for chromosome arm 1PS amplified PCR products in the recombinant type 2 translocation line. Resistance analysis showed that the type 2 translocation was highly resistant to a virulent race of leaf rust pathogen. The new wheat–A. cristatum translocations obtained yield material with seedling plant resistance to leaf rust and seed storage protein loci.

Characterization of α-gliadin alleles of Japanese wheat cultivars in relation to flour dough extensibility and celiac disease epitopes

Alpha-gliadins are major seed storage proteins of wheat and are involved in dough extensibility, which is related to the end-use properties of flour. However, α-gliadin causes gluten-related diseases such as celiac disease (CD). To characterize the α-gliadin allele types of Japanese cultivars, PCR markers that detect the presence of each α-gliadin gene in Chinese Spring wheat were used to determine allele-types. We surveyed 95 accessions of the Japanese wheat core collection (JWC) and 22 commercial cultivars and found 2, 10, and 4 allele types at the loci Gli-A2, Gli-B2, and Gli-D2, respectively. According to the allele-type frequency, the α-gliadin alleles of JWC showed diversities comparable to those of foreign cultivars. Allele diversities were compared within four populations of JWC, classified by genome-wide single nucleotide polymorphisms. The degree of diversity of alleles reflected the population structures, except for high diversity of the allele-types at Gli-B2 in the classical varieties. The frequencies of alleles related to end-use were higher in newer varieties than in classical varieties and landraces. Allele types that dramatically decreased and increased CD epitopes were not found in JWC, suggesting that selection(s) of α-gliadin genes was not linked to CD epitopes in Japanese cultivars.

Bread Wheat Gluten and Its Health Effects

Glutens are the major constituents of bread wheat grain storage proteins and constituting 85% of the total. They are mainly responsible for the processing quality of wheat dough and were among the first proteins isolated and studied by human beings. Different compositions of wheat storage proteins confer different dough physical properties, which are required by different products. Gluten provides dough with unique extensibility and elasticity, which are essential for various wheat products. Gluten is one of the most commonly used proteins in food industry. Its characteristic properties make it an essential ingredient in the preparation of high quality dough, hence it is popular in the baking industry. Some wheat gliadin proteins are strong allergens that can cause various symptoms of food allergies and baker asthma. The most immune reactive ω-5 gliadin fractions are the main allergens in wheat dependent exercise induced anaphylaxis. Apart from their role in dough quality, gluten proteins can affect the health of genetically susceptible individuals. Many gluten proteins contain T-cell stimulatory epitopes that can cause celiac disease. The consumption of gluten proteins can trigger an immune response that damages the small intestine. Therefore, patients with celiac disease (CD) are restricted to a lifelong gluten-free diet

Clustering and cross-linking of the wheat storage protein α-gliadin: A combined experimental and theoretical approach

Our aim was to understand mechanisms for clustering and cross-linking of gliadins, a wheat seed storage protein type, monomeric in native state, but incorporated in network while processed. The mechanisms were studied utilizing spectroscopy and high-performance liquid chromatography on a gliadin-rich fraction, in vitro produced α-gliadins, and synthetic gliadin peptides, and by coarse-grained modelling, Monte Carlo simulations and prediction algorithms. In solution, gliadins with α-helix structures (dip at 205 nm in CD) were primarily present as monomeric molecules and clusters of gliadins (peaks at 650- and 700-s on SE-HPLC). At drying, large polymers (Rg 90.3 nm by DLS) were formed and β-sheets increased (14% by FTIR). Trained algorithms predicted aggregation areas at amino acids 115–140, 150–179, and 250–268, and induction of liquid-liquid phase separation at P- and Poly-Q-sequences (Score = 1). Simulations showed that gliadins formed polymers by tail-to-tail or a hydrophobic core (Kratky plots and Ree = 35 and 60 for C- and N-terminal). Thus, the N-terminal formed clusters while the C-terminal formed aggregates by disulphide and lanthionine bonds, with favoured hydrophobic clustering of similar/exact peptide sections (synthetic peptide mixtures on SE-HPLC). Mechanisms of clustering and cross-linking of the gliadins presented here, contribute ability to tailor processing results, using these proteins.

Wheat Quality Formation and Its Regulatory Mechanism

Elucidation of the composition, functional characteristics, and formation mechanism of wheat quality is critical for the sustainable development of wheat industry. It is well documented that wheat processing quality is largely determined by its seed storage proteins including glutenins and gliadins, which confer wheat dough with unique rheological properties, making it possible to produce a series of foods for human consumption. The proportion of different gluten components has become an important target for wheat quality improvement. In many cases, the processing quality of wheat is closely associated with the nutritional value and healthy effect of the end-products. The components of wheat seed storage proteins can greatly influence wheat quality and some can even cause intestinal inflammatory diseases or allergy in humans. Genetic and environmental factors have great impacts on seed storage protein synthesis and accumulation, and fertilization and irrigation strategies also greatly affect the seed storage protein content and composition, which together determine the final end-use quality of wheat. This review summarizes the recent progress in research on the composition, function, biosynthesis, and regulatory mechanism of wheat storage proteins and their impacts on wheat end-product quality.

Oral Consumption of Bread from an RNAi Wheat Line with Strongly Silenced Gliadins Elicits No Immunogenic Response in a Pilot Study with Celiac Disease Patients.

Celiac disease (CD) is a genetically predisposed, T cell-mediated and autoimmune-like disorder caused by dietary exposure to the storage proteins of wheat and related cereals. A gluten-free diet (GFD) is the only treatment available for CD. The celiac immune response mediated by CD4+ T-cells can be assessed with a short-term oral gluten challenge. This study aimed to determine whether the consumption of bread made using flour from a low-gluten RNAi wheat line (named E82) can activate the immune response in DQ2.5-positive patients with CD after a blind crossover challenge. The experimental protocol included assessing IFN-γ production by peripheral blood mononuclear cells (PBMCs), evaluating gastrointestinal symptoms, and measuring gluten immunogenic peptides (GIP) in stool samples. The response of PBMCs was not significant to gliadin and the 33-mer peptide after E82 bread consumption. In contrast, PBMCs reacted significantly to Standard bread. This lack of immune response is correlated with the fact that, after E82 bread consumption, stool samples from patients with CD showed very low levels of GIP, and the symptoms were comparable to those of the GFD. This pilot study provides evidence that bread from RNAi E82 flour does not elicit an immune response after a short-term oral challenge and could help manage GFD in patients with CD.

Optimization of large-scale purification of omega gliadins and other wheat gliadins

Among wheat storage proteins, omega-gliadins display a singular amino acid sequence only composed by repetitive sequences. They have been described as a major wheat allergen and also contain T-cell epitopes implicated in coeliac disease. To study the structural and biophysical properties of omega-gliadins and other gliadins (e.g., gluten network formation, allergic response), highly purified and concentrated fractions are needed. In the present work, we used chromatography media screening to improve their fractionation. A S Ceramic Hyper D (Pall) resin was selected for its ability to concentrate omega-gliadins in the first elution peaks. Gamma- and beta-gliadins were then separated by hydrophobic interaction chromatography and eluted with an ethanol gradient. Alpha-gliadins were purified by gel filtration. Each fraction was characterized by electrophoresis and reverse phase HPLC. The developed preparative protocol enabled the purification of several grams of highly purified gliadins to the detriment of the yield.

Dense Phases of γ-Gliadins in Confined Geometries

The binary phase diagram of γ-gliadin, a wheat storage protein, in water was explored thanks to the microevaporator, an original PDMS microfluidic device. This protein, usually qualified as insoluble in aqueous environments, displayed a partial solubility in water. Two liquid phases, a very dilute and a dense phase, were identified after a few hours of accumulation time in the microevaporator. This liquid–liquid phase separation (LLPS) was further characterized through in situ micro-Raman spectroscopy of the dilute and dense protein phases. Micro-Raman spectroscopy showed a specific orientation of phenylalanine residues perpendicular to the PDMS surfaces only for the diluted phase. This orientation was ascribed to the protein adsorption at interfaces, which would act as nuclei for the growth of dense phase in bulk. This study, thanks to the use of both aqueous solvent and a microevaporator, would provide some evidence for a possible physicochemical origin of the gliadin assembly in the endoplasmic reticulum of albumen cells, leading to the formation of dense phases called protein bodies. The microfluidic tool could be used also in food science to probe protein–protein interactions in order to build up phase diagrams.

A Rapid, Reliable RP-UPLC Method for Large-Scale Analysis of Wheat HMW-GS Alleles.

High-molecular-weight glutenin subunits (HMW-GS) account for only 10% of total wheat storage proteins, but play an important role in the processing quality of wheat flour. Therefore, identifying HMW-GS alleles associated with good end-use quality provides important information for wheat breeders. To rapidly, accurately and reproducibly identify HMW-GS, we established an optimized reversed-phase ultra-performance liquid chromatography (RP-UPLC) method. Separation parameters were optimized using an ACQUITY UPLC Protein BEH C4 column and stepwise ACN gradient, and the separation patterns and retention times (RTs) of 22 subunits were comparatively analyzed in 16 standard wheat cultivars. All HMW-GS proteins were well separated within about 5.5 min, and all analyses were complete within 12 min. We distinguished the 16 subunits based on RT, although three subunits in 1Bx (1Bx7/1Bx7OE and 1Bx17) and three subunits in 1By (1By8*, 1By9 and 1By15) had overlapping RTs; these were differentiated by SDS-PAGE. To distinguish 1Bx7 and 1Bx7OE, which differ in protein abundance, RP-UPLC was combined with PCR analysis of DNA junction markers. The optimized method was successfully applied to determine HMW-GS alleles in a large collection of bread wheat germplasm (1787 lines). This protocol is an appropriate option for selecting lines harboring favorable HMW-GS alleles in wheat breeding.

Effects of water deficit and high N fertilization on wheat storage protein synthesis, gluten secondary structure, and breadmaking quality

The content and composition of wheat storage proteins are the major determinants of dough rheological properties and breadmaking quality and are influenced by cultivation conditions. This study aimed to investigate the effects of water deficit and high N-fertilizer application on wheat storage protein synthesis, gluten secondary structure, and breadmaking quality. Reverse-phase ultrahigh-performance liquid chromatography analysis showed that storage protein and gluten macropolymer accumulation was promoted under both independent applications and a combination of water-deficit and high N-fertilizer treatments. Fourier-transform infrared spectroscopy showed that water deficit and high N-fertilizer treatments generally improved protein secondary structure formation and lipid accumulation, and reduced flour moisture. In particular, high N-fertilizer application increased β-sheet content by 10.4% and the combination of water-deficit and high N-fertilizer treatments increased random coil content by 7.6%. These changes in gluten content and secondary structure led to improved dough rheological properties and breadmaking quality, including superior loaf internal structure, volume, and score. Our results demonstrate that moderately high N-fertilizer application under drought conditions can improve gluten accumulation, gluten secondary structure formation, and baking quality.

Some intra-varietal non-uniformities in Triticum aestivum can be explained as repeated spontaneous mutations at the Gli loci

Intra-varietal non-uniformity, presumably caused by spontaneous mutations at the Gli loci, was detected by means of seed-by-seed electrophoretic analysis (APAGE) of wheat storage protein, gliadin, in two independent sets of grain samples of Triticum aestivum world-wide cultivars registered during the 20th century (in total, 12,035 seeds studied). The frequency of null alleles, which caused a loss of all gliadin electrophoretic bands encoded by one Gli locus, was found to be strongly disproportional among six Gli loci; thus, the Gli-B1null occurred 20 times more often than the Gli-B2null. The presence of the null mutation at a Gli locus was frequently accompanied by the over-production (visible as a remarkable increase of staining intensity of some APAGE bands) of gliadins encoded by other Gli loci. Nulls at the Gli-B1 locus were, as a rule, accompanied by the over-production of ω-gliadins encoded by the Gli-D1. At the Gli-B1 locus, mutants were discovered that lacked more than one gliadin, but not all gliadins composing a block. The loss of one APAGE band, as well as an alteration of the electrophoretic mobility (EM) of one band in the electrophoregram of a given cultivar, was also observed. Alteration of the EM of one APAGE band occurred mainly among gliadins encoded at the Gli-2 loci, particularly at the Gli-B2. A frequency (obviously under-estimated in our work) of occurrence of about 1% was observed for seeds carrying any presumed spontaneous mutations at Gli loci.

Response of Wheat Storage Proteins and Breadmaking Quality to Dimethylpyrazole-Based Nitrification Inhibitors under Different Nitrogen Fertilization Splitting Strategies.

Improving fertilizer nitrogen (N) use efficiency is essential to increase crop productivity and avoid environmental damage. This study was conducted during four crop cycles of winter wheat under humid Mediterranean conditions (Araba, northern Spain). The effects of N-fertilization splitting and the application of the nitrification inhibitors (NIs) 3,4-dimethylpyrazole phosphate (DMPP) and 2-(3,4-dimethyl-1H-pyrazol-1-yl) succinic acid isomeric mixture (DMPSA) as strategies to improve grain quality were examined. The hypothesis of this study was to test if the partial ammonium nutrition and the reduction of fertilizer losses presumably induced by the application of NIs can modify the grain gliadin and glutenin protein contents and the breadmaking quality (dough rheological properties). Among both NIs assayed, only DMPP showed a slight effect of decreasing the omega gliadin fraction, following splitting either two or three times, although this effect was dependent on the year and was not reflected in terms of dough extensibility. The slight decreases observed in grain quality in terms of dough strength and glutenin content induced by DMPP suggest that DMPSA is more promising in terms of maintaining grain quality. Nonetheless, these poor effects exerted by NI application on grain quality parameters did not lead to changes in the quality parameters defining the flour aptitudes for breadmaking.