Wheat Storage Proteins Research Articles

Allele-specific PCR Markers for Distinguishing High Molecular Weight Glutenin Subunit <i>Dtx5</i> of <i>Aegilops tauschii</i> from <i>Dx5</i> of Common Wheat

High molecular weight (HMW) glutenin, one of the important storage proteins of wheat, mainly confers end-use quality of wheat grain. Among the loci of HMW-GS, the locus D1 determines the breaking quality of bread wheat dough mostly. Synthetic hexaploid wheat (SHW) inherits vast genetic diversity in the D genome of Aegilops tauschii and has gradually been used as a bridge-tool for wheat improvement. Therefore, more and more HMW-GS subunit types of Ae. tauschii have been introgressed into the common wheat with irresistible application of SHW in wheat breeding. However, the traditional SDS-PAGE couldn’t distinguish Dtx5 of Ae. tauschii from Dx5 of common wheat. In this study, we developed two AS-PCR markers based on three SNPs in the HMW-GS D1 sequences of Ae. tauschii and common wheat. The sharp and stable fragments amplified by designed AS-PCR primers indicated their availability and convenience in Dtx5 and Dx5 identification. And Dx5 of common wheat obtained the amplified fragments by AS-PCR primers, while no fragment was amplified for Dtx5 of Ae. tauschii. And the amplified results were consistent in both pairs of AS-PCR primers. Moreover, Dtx5 is more highly homologous with Dx2 than Dx5 from their protein sequences.

Molecular characterization and phylogenetic analysis of one Omega-gliadin gene from Aegilops speltoidesl

Gliadins, as the major components of wheat storage proteins, determine the extensibility properties of dough and have important effects on flour processing quality. Wheat related species carries potential storage protein gene resources for quality improvement. In this study, we isolated and characterized the first complete ?-gliadin gene Omega-AS from Aegilops speltoides L. (2n = 2x = 14, SS) by allelic-specific PCR and investigated its phylogenetic relationships among Triticum and Aegilopsspecies. Molecular structure showed that Omega-AS gene consisted of 1122 bp encoding 373 amino acid residues with deduced molecular mass 41379.21 Da. Omega-AS gene was exceptionally rich in prolines and glutamines with fewer methionine and no cysteine. Sequence characterization and epitope analysis showed that three epitopes QQPIPVQPQQ, TQPQQPTPIQ and IQPQQPFPQQ were absent in Omega-AS gene encoded protein, indicating its potential value for wheat quality improvement with less toxic, or no toxic peptides. Phylogenetic analysis revealed that Omega-AS was closely related to gliadin genes of wheat and related species and its divergence from bread wheat was more recently (less than 1.243 MYA). Heterologous expression showed that Omega-AS gene could successfully express with a high level in E. coli under the control of T7promoter. The transcription expression pattern of Omega-AS gene during grain development detected by qRT-PCR revealed that the highest expression level occurred at 17 days post an thesis.

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 significanceThe 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.

Antibody reactivity in patients with IgE-mediated wheat allergy to various subunits and fractions of gluten and non-gluten proteins from ω-gliadin-free wheat genotypes.

[/b] The observed differentiation of immunodetection spectra allows to model highly specific IgE-binding profiles of allergenic wheat proteins attributed to individual patients with symptoms of gluten intolerance. Highly immunoreactive subunits and fractions among GP and NGP were identified. The observed immunoreactivity of 49 kDa NGP is worth to emphasize, as it has never been reported as wheat allergenic protein before.

Cloning and characterization of novel γ-gliadin genes from Aegilops markgrafii in relation to evolution and wheat breeding

Gliadins are the major components of storage proteins in wheat and play an important role in determining the extensibility properties of dough. In the present work, six novel full-length γ-gliadin genes were cloned from the C genome of Aegilops markgrafii using a PCR-based strategy. Analysis of the deduced amino acid sequences showed that the cloned genes had primary structures that were similar, but not identical, to published γ-gliadins from other wheat-related species. The lengths of the open reading frames (ORFs) ranged from 909 to 963bp, and the repetitive and glutamine-rich domains were mainly responsible for the size of the proteins. An extra cysteine residue was present in the repetitive domain of sequence JX566513. All amino acid sequences of γ-gliadin genes from Ae. markgrafii were searched for the five peptides identified as T cell stimulatory epitopes in celiac disease (CD) patients. Peptide Gliγ-3 was present in sequences JX566513 and JX566514. Peptide Gliγ-5 was present only in JX566513. The other γ-gliadins contained no toxic epitopes. These results provide information to better understand the use of Ae. markgrafii in wheat breeding and the evolutionary relationship of the γ-gliadin genes in Ae. markgrafii and other Triticeae species.

Novel insights into the effect of nitrogen on storage protein biosynthesis and protein body development in wheat caryopsis

Molecular and cytological mechanisms concerning the effects of nitrogen on wheat (Triticum aestivum L.) storage protein biosynthesis and protein body development remain largely elusive. We used transcriptome sequencing, proteomics techniques, and light microscopy to investigate these issues. In total, 2585 differentially expressed genes (DEGs) and 57 differentially expressed proteins (DEPs) were found 7 days after anthesis (DAA), and 2456 DEGs and 64 DEPs were detected 18 DAA after nitrogen treatment. Gene ontology terms related to protein biosynthesis processes enriched these numbers by 678 and 582 DEGs at 7 and 18 DAA, respectively. Further, 25 Kyoto Encyclopedia of Genes and Genomes pathways were involved in protein biosynthesis at both 7 and 18 DAA. DEPs related to storage protein biosynthesis contained gliadin and glutenin subunits, most of which were up-regulated after nitrogen treatment. Quantitative real-time PCR analysis indicated that some gliadin and glutenin subunit encoding genes were differentially expressed at 18 DAA. Structural observation revealed that wheat endosperm accumulated more and larger protein bodies after nitrogen treatment. Collectively, our findings suggest that nitrogen treatment enhances storage protein content, endosperm protein body quantity, and partial processing quality by altering the expression levels of certain genes involved in protein biosynthesis pathways and storage protein expression at the proteomics level.

Impact of Elevated CO<sub>2</sub> on Wheat Growth and Yield under Free Air CO<sub>2</sub> Enrichment

Impact of elevated CO2 (free air CO2 enrichment) was studied on wheat (Triticum aestivum L. var Kundan) growth, yield and proteome. Elevated CO2 significantly impacted both underground (+24%) and aboveground (+15%) biomass. Grain weight/plant and harvest index were increased by 35% and 11.4%, respectively under high CO2. On the other hand, seed protein content was decreased by 19% under CO2 enrichment while seed starch and soluble sugar contents were increased by 8% and 23%, respectively. Wheat leaf proteomics revealed that 50 proteins were showing differential expression. Twenty proteins were more abundant while 30 were less abundant. Thirty two proteins were identified by MALDI TOF TOF. More abundant proteins were related to defense, photosynthesis, energy metabolism etc. While less abundant proteins were related to glycolysis and gluconeogenesis. Wheat grain proteomics revealed that out of 49 differentially abundant proteins, 24 were more in abundance and 25 were less in abundance in wheat grains under eCO2 condition. Thirty three proteins were identified and functionally characterized. They were found to be involved mainly in carbon metabolism, storage, defence and proteolysis. Gluten proteins are the major component of wheat storage proteins. Our results showed that both high and low molecular weight glutenins were more in eCO2 wheat seeds while there was no change in gliadin evels. This might alter wheat dough strength. Concentration of grain Cr and As was increased at eCO2 while that of Fe, Cu, Zn and Se were found to be decreased. Dynamics of carbon utilization and metabolic abilities of soil microbes under eCO2 were significantly altered. Our study showed that altered wheat seed composition is cause for concern vis-a-vis nutrition and health and for industries which may have implications for agriculturally dominated country like India.

Towards gliadin nanofoams

Bio-derived, fully biodegradable closed-cell micro- and nanocellular foams were generated from gliadin. Gliadin, an abundantly available wheat storage protein, was extracted with aqueous ethanol from wheat gluten. For the newly developed foaming process, dry gliadin powder was plasticized by equal volumes of water and ethanol resulting in a viscoelastic paste that was then subjected to supercritical carbon dioxide in a high-pressure cell. After tempering, a sudden release of pressure and cooling resulted in closed-cell micro- or nanocellular foams. Depending on the processing parameters such as the foaming temperature, pressure, pressure drop rate, plasticizer concentration, and pre- and past-expansion treatment, foam morphology was tunable. Accordingly, cell size and porosity can be varied as desired. The resulting foams and intermediate states were analyzed by scanning electron microscopy, and a foaming mechanism is suggested. The foams prepared via the proposed procedure exhibit the smallest cell sizes and the highest porosity ever reported for foaming of wheat proteins. For example, we found mean cell size of 6.5 ± 1.7 μm at a porosity of ϕ = 0.97, 1.4 ± 0.5 μm at ϕ = 0.90, 720 ± 340 nm at ϕ = 0.77, and 270 ± 110 nm at ϕ = 0.62. The procedure presented is clean, environment friendly, simple, and low cost, possibly lending itself to a technical realization. The promising new approach may have potential in short purpose packaging or insulation at almost zero environmental cost, because the materials are easily and environmentally friendly recyclable and even edible.

Influence of glycosylation of deamidated wheat gliadin on its interaction mechanism with resveratrol.

Gliadin is a main composition of wheat storage protein with unique characteristics. Polyphenol with health benefits tends to form complex with protein. In this study, glycosylation of deamidated wheat gliadin (gliadin) was carried out. Fluorescence quenching was applied to evaluate their binding mechanisms with resveratrol. Results showed that glycosylation could increase the solubility and decrease the surface hydrophobicity of gliadin. Both gliadin and glycosylated gliadin have strong affinity with resveratrol. The thermodynamic parameters of binding process indicated that complexation of resveratrol with gliadin was mainly driven by hydrophobic interaction, while by hydrogen bonds with glycosylated gliadin. The hydrosolubility of resveratrol was dramatically increased especially in the presence of glycosylated gliadin. This was consistent with the higher binding constant of glycosylated gliadin with resveratrol. Therefore, gliadin and glycosylated gliadin are both effective to carry resveratrol or other bioactive compounds, and their binding mechanisms are different due to structural difference.

Dipeptidyl peptidase 4 – An important digestive peptidase in Tenebrio molitor larvae

Dipeptidyl peptidase 4 (DPP 4) is a proline specific serine peptidase that plays an important role in different regulatory processes in mammals. In this report, we isolated and characterized a unique secreted digestive DPP 4 from the anterior midgut of a stored product pest, Tenebrio molitor larvae (TmDPP 4), with a biological function different than that of the well-studied mammalian DPP 4. The sequence of the purified enzyme was confirmed by mass-spectrometry, and was identical to the translated RNA sequence found in a gut EST database. The purified peptidase was characterized according to its localization in the midgut, and substrate specificity and inhibitor sensitivity were compared with those of human recombinant DPP 4 (rhDPP 4). The T. molitor enzyme was localized mainly in the anterior midgut of the larvae, and 81% of the activity was found in the fraction of soluble gut contents, while human DPP 4 is a membrane enzyme. TmDPP 4 was stable in the pH range 5.0–9.0, with an optimum activity at pH 7.9, similar to human DPP 4. Only specific inhibitors of serine peptidases, diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, suppressed TmDPP 4 activity, and the specific dipeptidyl peptidase inhibitor vildagliptin was most potent. The highest rate of TmDPP 4 hydrolysis was found for the synthetic substrate Arg-Pro-pNA, while Ala-Pro-pNA was a better substrate for rhDPP 4. Related to its function in the insect midgut, TmDPP 4 efficiently hydrolyzed the wheat storage proteins gliadins, which are major dietary proteins of T. molitor.

Molecular characterization of the IgE-binding epitopes in the fast ω-gliadins of Triticeae in relation to wheat-dependent, exercise-induced anaphylaxis

Fast ω-gliadins were minor components of wheat storage proteins but a major antigen triggering allergy to wheat. Sixty-six novel full-length fast ω-gliadin genes with unique characteristics were cloned and sequenced from wheat and its relative species using a PCR-based strategy. Their coding regions ranged from 177bp to 987bp in length and encoded 4.28kDa to 37.56kDa proteins. On the base of first three deduced amino acids at the N-terminal, these genes could be classified into the six subclasses of SRL-, TRQ-, GRL-, NRL-, SRP- and SRM-type ω-gliadin genes. Compared by multiple alignments, these genes were significantly different from each other, due to the insertion or deletion at the repetitive domain. An analysis of the IgE-binding epitopes of the 66 deduced fast ω-gliadins demonstrated that they contained 0–24 IgE-binding epitopes. The phylogenetic tree demonstrated that the fast ω-gliadins and slow ω-gliadins were separated into two groups and their divergence time was 21.64millionyears ago. Sequence data of the fast ω-gliadin genes assist in the study of the origins and evolutions of the different types of ω-gliadins while also providing a basis for the synthesis of monoclonal antibodies to detect wheat antigen content.

Quantification and In Vitro Bioaccessibility of Selenium from Osborne Fractions of Selenium‐Rich Cereal Grains

Cereal crops cultivated in the seleniferous belt of Punjab, India, were observed to hyperaccumulate a significantly high concentration of selenium (20–123 µg/g). Selenium concentration (µg/g) in storage proteins of wheat, maize, and rice, namely, albumin (401, 280, and 29, respectively), globulin (264, 192, and 242, respectively), glutelin (563, 359, and 178, respectively), and prolamin (629, 339, and 257, respectively) indicated variable selenium levels, with prolamin contributing significantly higher levels of selenium when compared with other proteins with reference to the total concentration of the protein fraction. The simulated gastric and gastrointestinal digestion studies indicated better accessibility of selenium during intestinal digestion, with variability across proteins and cereal types. The observations provide an insight into the bioavailability of selenium in selenium‐rich cereal grains, used in the study, and their potential use as source for selenium supplementation to deficient populations, or as “bioactive” selenium‐rich nutraceutical blends for health benefits.

Comprehensive Identification and Bread-Making Quality Evaluation of Common Wheat Somatic Variation Line AS208 on Glutenin Composition.

High molecular weight glutenin subunits (HMW-GSs) are important seed storage proteins in wheat (Triticum aestivum) that determine wheat dough elasticity and processing quality. Clarification of the defined effectiveness of HMW-GSs is very important to breeding efforts aimed at improving wheat quality. To date, there have no report on the expression silencing and quality effects of 1Bx20 and 1By20 at the Glu-B1 locus in wheat. A wheat somatic variation line, AS208, in which both 1Bx20 and 1By20 at Glu-B1 locus were silenced, was developed recently in our laboratory. Evaluation of agronomic traits and seed storage proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and reversed-phase high performance liquid chromatography (RP-HPLC) indicated that AS208 was highly similar to its parental cultivar Lunxuan987 (LX987), with the exception that the composition and expression of HMW-GSs was altered. The 1Bx20 and 1By20 in AS208 were further identified to be missing by polymerase chain reaction (PCR) and quantitative real-time RT-PCR (qRT-PCR) assays. Based on the PCR results for HMW-GS genes and their promoters in AS208 compared with LX987, 1Bx20 and 1By20 were speculated to be deleted in AS208 during in vitro culture. Quality analysis of this line with Mixograph, Farinograph, and Extensograph instruments, as well as analysis of bread-making quality traits, demonstrated that the lack of the genes encoding 1Bx20 and 1By20 caused various negative effects on dough processing and bread-making quality traits, including falling number, dough stability time, mixing tolerance index, crude protein values, wet gluten content, bread size, and internal cell structure. AS208 can potentially be used in the functional dissection of other HMW-GSs as a plant material with desirable genetic background, and in biscuit making industry as a high-quality weak gluten wheat source.

Allelic variation of LMW-GS composition in Chinese wheat landraces of the Yangtze-River region detected by MALDI-TOF-MS.

Low molecular weight glutenin subunits are important components of wheat storage proteins, which play an important role in determining end-use quality of common wheat. A newly established matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) procedure was used to analyze 478 landraces of bread wheat collected from the Yangtze-River region in China. Results indicated that 17 alleles at three loci: Glu-A3, Glu-B3 and Glu-D3 were identified, resulting in 87 different allele combinations. Of the 17 alleles detected at all the Glu-3 loci, five belonged to Glu-A3, seven to Glu-B3 and five to Glu-D3 locus. MALDI-TOF-MS indicated Glu-A3a/c was present in 72.8%, Glu-A3b in 8.4%, Glu-A3d in 8.4%, Glu-A3f in 5.2% and Glu-A3e in 3.6% lines. Seven types of alleles were identified at the Glu-B3 locus: Glu-B3d/i (25.5%), Glu-B3b (21.3%), Glu-B3c (16.9%), Glu-B3h (13.8%), Glu-B3f (8.4%), Glu-B3a (8.2%), and Glu-B3g (5.2%). Five types of Glu-D3 alleles were detected: Glu-D3a (58.4%), Glu-D3c (22.6%), Glu-D3d (15.5%), Glu-D3b (3.3%) and Glu-D3f (0.2%). Four new alleles that showed abnormal MALDI-TOF spectrum patterns were identified at the Glu-A3 and Glu-B3 loci. A detailed study is needed to further characterize these alleles and their potential usage for wheat improvement.

Drought stress delays endosperm development and misregulates genes associated with cytoskeleton organization and grain quality proteins in developing wheat seeds

Drought stress is a major yield-limiting factor for wheat. Wheat yields are particularly sensitive to drought stress during reproductive development. Early seed development stage is an important determinant of seed size, one of the yield components. We specifically examined the impact of drought stress imposed during postzygotic early seed development in wheat. We imposed a short-term drought stress on plants with day-old seeds and observed that even a short-duration drought stress significantly reduced the size of developing seeds as well as mature seeds. Drought stress delayed the developmental transition from syncytial to cellularized stage of endosperm. Coincident with reduced seed size and delayed endosperm development, a subset of genes associated with cytoskeleton organization was misregulated in developing seeds under drought-stressed. Several genes linked to hormone pathways were also differentially regulated in response to drought stress in early seeds. Notably, drought stress strongly repressed the expression of wheat storage protein genes such as gliadins, glutenins and avenins as early as 3 days after pollination. Our results provide new insights on how some of the early seed developmental events are impacted by water stress, and the underlying molecular pathways that can possibly impact both grain size and quality in wheat.

Gluten and wheat sensitivities – An overview

Wheat products are important staple foods worldwide. However, a small portion of the population has to avoid wheat-containing foods because of harmful immune responses. Countless studies have demonstrated that the storage (gluten) proteins of wheat are major causative agents for wheat-dependent immune-mediated disorders. The unique structural features of gluten proteins are long repetitive amino acid sequences rich in glutamine and proline. These sequence sections are involved in most wheat sensitivities. Corresponding homologous sequence sections of rye and barley proteins may also be harmful, but relevant studies are rare. Wheat sensitivities can be classified into three main forms: autoimmunogenic sensitivities including coeliac disease, dermatitis herpetiformis, and gluten ataxia; allergic sensitivities including immediate wheat allergy, wheat-dependent exercise-induced anaphylaxis, respiratory allergy, and contact urticaria; and non-coeliac gluten sensitivity. The presented overview summarizes our current knowledge of gluten protein structures related to wheat sensitivities and the epidemiological, clinical, and pathogenic differences between these immune-mediated disorders.

Selenium in storage proteins of wheat cultivated on selenium impacted soils of Punjab, India

Wheat, an important staple cereal crop cultivated in seleniferous region of India, noted to accumulated significantly high concentrations of Se, was examined for the distribution of selenium in various protein fractions of the grains. Amongst the protein fractions, Se was dominantly (33–37%) present in the albumin fraction in Se rich grains followed by other fractions viz., globulin (20–25%), glutelin (20–25%), and prolamin (17–20%). The observations are important in context of exploring the use of this material as functional foods in formulating Se-enriched diets for Se-deficient population.

IDENTIFICATION OF TECHNOLOGICALLY IMPORTANT GENES AND THEIR PRODUCTS IN THE COLLECTION OF BREAD WHEAT GENOTYPES

Wheat is the second most cultivated crop on the world and is very important plant for feed not only mankind but also animals. Because of this is necessary to develop new varieties with better properties. Bread making quality of wheat grain is one of the most important paramaters for quality evaluation. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of wheat storage proteins and allelic specific polymerase chain reaction (AS-PCR) are analysis suitable for identification, differentiation and characterization of bread wheat (Triticum aestivum L.). There were analysed 16 genotypes of new varieties of bread wheat in our work by SDS-PAGE and obtained results were verified by AS-PCR. Analysed genotypes of bread wheat genotypes were homogenous and single line with very good bread making quality. Our results confirmed hypothesis, that cultivated bread wheat genotypes are uniformed with high production and quality but there is a risk of sensitivity to environmental conditions. SDS-PAGE analyses of wheat grain proteins are fast and not very expensive technique, which provide us information of bread making quality of grains. However, there is possibility of environmental influence on protein synthesis and because of this is necessary to couple these analysis with analysis of DNA.

THE USE OF DIFFERENT PROTEASES TO HYDROLYZE GLIADINS

Gliadins represent alcohol-soluble fraction of wheat storage proteins which is responsible for development of celiac disease. The only and effective treatment for celiac disease is strict adherence to a gluten-free diet excluding any food made with wheat, as well as rye, barley and possibly oat flour. Enzymatic modification of wheat gliadins seems to be an alternative method for decreasing of celiac activity. The aim of our study was a trial of enzymatic modification of wheat gliadins using fungal (Aspergillus sp., Aspergillus oryzae, Aspergillus niger) and bacterial (Bacillus licheniformis, Bacillus stearothermophilus, Bacillus thermoproteolyticus, Streptomyces griseus) proteases. The reaction was performed up to 60 min, stopped by addition of appropriate synthetic inhibitor and products of limited proteolysis were analyzed by SDS-PAGE method. From fungal proteases most effective proteolytic activity was observed using acid proteinase from A. niger since wheat gliadins and low molecular weight peptides were completely degraded. Bacterial proteases form B. licheniformis and B. thermoproteolyticus acted very effective and as the result of hydrolysis, the products of lower molecular weight (

Genetic Diversity of High and Low Molecular Weight Glutenin Subunits in Algerian <i>Aegilops geniculata</i>

Aegilops geniculata Roth is an annual grass relative to cultivated wheat and is widely distributed in North Algeria. Endosperm storage proteins of wheat and its relatives, namely glutenins and gliadins, play an important role in dough properties and bread making quality. In the present study, the different alleles encoded at the four glutenin loci (Glu-M1, Glu-U1, Glu-M3 and Glu-U3) were identified from thirty five accessions of the tetraploid wild wheat A. geniculata collected in Algeria using Sodium dodecyl Sulfate - Polyacrylamide Gel Electrophoresis (SDS-PAGE). At Glu-M1 and Glu-U1 loci, encoding high molecular weight glutenin subunits (HMW-GS) or A-subunits, 15 and 12 alleles were observed respectively, including one new subunit. B-Low molecular weight glutenin subunits zone (B-LMW-GS) displayed a far greater variation, as 28 and 25 alleles were identified at loci Glu-M3 and Glu-U3 respectively. Thirty two subunits patterns were revealed at the C subunits- zone and a total of thirty four patterns resulted from the genetic combination of the two zones (B- and C-zone). The wide range of glutenin subunits variation (high molecular weight glutenin subunits and low molecular weight glutenin subunits) in this species has the potential to enhance the genetic variability for improving the quality of wheat./span>