Weight Glutenin Subunits Of Wheat Research Articles

Multiple elements controlling the expression of wheat high molecular weight glutenin paralogs.

Analysis of gene expression data generated by high-throughput microarray transcript profiling experiments coupled with cis-regulatory elements enrichment study and cluster analysis can be used to define modular gene programs and regulatory networks. Unfortunately, the high molecular weight glutenin subunits of wheat (Triticum aestivum) are more similar than microarray data alone would allow to distinguish between the three homoeologous gene pairs. However, combining complementary DNA (cDNA) expression libraries with microarray data, a co-expressional network was built that highlighted the hidden differences between these highly similar genes. Duplex clusters of cis-regulatory elements were used to focus the co-expressional network of transcription factors to the putative regulatory network of Glu-1 genes. The focused network helped to identify several transcriptional gene programs in the endosperm. Many of these programs demonstrated a conserved temporal pattern across the studied genotypes; however, few others showed variance. Based on this network, transient gene expression assays were performed with mutated promoters to inspect the control of tissue specificity. Results indicated that the interactions of the ABRE│CBF cluster with distal promoter regions may have a dual role in regulation by both recruiting the transcription complex as well as suppressing it in non-endosperm tissue. A putative model of regulation is discussed.

A reassessment of the electrophoretic mobility of high molecular weight glutenin subunits of wheat

The high molecular weight subunits of wheat glutenin (HMW-GS) are important for bread-making quality. Their composition is routinely identified by Tris-glycine SDS-PAGE after reduction of glutenin disulfide bonds. However, the relation between their molecular weight and, hence, their primary structure, and their mobility in Tris-glycine SDS-PAGE has proven to be ambiguous. We demonstrate a Bis-Tris SDS-PAGE procedure with a neutral, instead of alkaline, pH in the gel and running buffers. In this method commonly occurring HMW-GS from wheat migrated in the order 5 > 2 ≈ 3 > 1 > 6 ≈ 2* > 7 > 8 > 9 > 12 > 10, which is different from the order obtained in the Tris-glycine system. HMW-GS were identified by N-terminal sequencing after isolation with RP-HPLC. Protein sequences of HMW-GS were further confirmed by LC-MS/MS analyses of chymotryptic peptides after comparing the MS data to amino acid sequences in protein databases. The numbers of amino acids of HMW-GS reflected well the mobility order in Bis-Tris SDS-PAGE. The results indicate that Bis-Tris SDS-PAGE may not only be used to identify HMW-GS, but also to estimate the length of their polypeptide chain, as such avoiding previously observed anomalies in migration order.

Characterization of a Glutenin-Specific Serine Proteinase of Sunn Bug Eurygaster integricepts Put.

Glutenin hydrolyzing proteinases (GHPs) have been purified, by affinity chromatography, from wheat seeds damaged by the Sunn bug Eurygaster integriceps (Hemiptera, Scutelleridae). A 28 kDa protein was partially sequenced by mass spectrometry and Edman degradation which showed homology to serine proteases from various insects. Three full length clones were obtained from cDNA isolated from Sunn bug salivary glands using degenerate PCR based on the sequences obtained. The cleavage site of the protease was determined using recombinant and synthetic peptides and shown to be between the consensus hexapeptide and nonapeptide repeat motifs present in the high molecular weight subunits of wheat glutenin (PGQGQQ∧GYYPTSLQQ). Homology models were generated for the three proteinases identified in this study using the high resolution X-ray structure of a crayfish (Pontastacus leptodactylus) trypsin complexed with a peptide inhibitor as template (PDB accession 2F91). The novel specificity of this protease may find applications in both fundamental and applied studies.

New DNA markers for high molecular weight glutenin subunits in wheat

End-use quality is one of the priorities of modern wheat (Triticum aestivum L.) breeding. Even though quality is a complex trait, high molecular weight (HMW) glutenins play a major role in determining the bread making quality of wheat. DNA markers developed from the sequences of HMW glutenin genes were reported in several previous studies to facilitate marker-assisted selection (MAS). However, most of the previously available markers are dominant and amplify large DNA fragments, and thus are not ideal for high throughput genotyping using modern equipment. The objective of this study was to develop and validate co-dominant markers suitable for high throughput MAS for HMW glutenin subunits encoded at the Glu-A1 and Glu-D1 loci. Indels were identified by sequence alignment of allelic HMW glutenin genes, and were targeted to develop locus-specific co-dominant markers. Marker UMN19 was developed by targeting an 18-bp deletion in the coding sequence of subunit Ax2* of Glu-A1. A single DNA fragment was amplified by marker UMN19, and was placed onto chromosome 1AL. Sixteen wheat cultivars with known HMW glutenin subunits were used to validate marker UMN19. The cultivars with subunit Ax2* amplified the 362-bp fragment as expected, and a 344-bp fragment was observed for cultivars with subunit Ax1 or the Ax-null allele. Two co-dominant markers, UMN25 and UMN26, were developed for Glu-D1 by targeting the fragment size polymorphic sites between subunits Dx2 and Dx5, and between Dy10 and Dy12, respectively. The 16 wheat cultivars with known HMW glutenin subunit composition were genotyped with markers UMN25 and UMN26, and the genotypes perfectly matched their subunit types. Using an Applied Biosystems 3130xl Genetic Analyzer, four F(2) populations segregating for the Glu-A1 or Glu-D1 locus were successfully genotyped with primers UMN19, UMN25 and UMN26 labeled with fluorescent dyes.

The toxicity of high molecular weight glutenin subunits of wheat to patients with coeliac disease

The ability of the gliadin fraction of wheat gluten to exacerbate coeliac disease is well documented. We investigated the possible toxicity of high molecular weight glutenin subunits (HMW-GS) in coeliac disease in vitro using gluten-sensitive T cells, and in vivo with challenge studies in patient volunteers. A mixture of four HMW-GS was chemically separated from wheat flour and checked for purity by HPLC, SDS-PAGE and ELISA. T-cell lines, grown up from small intestinal biopsies from coeliac patients (n=17), were tested for their reactivity to HMW-GS. Adults with coeliac disease and who were on a gluten-free diet (n=3) underwent in-vivo challenges with HMW-GS. Duodenal biopsies, taken prior to the challenge and at intervals up to 6 h afterwards, were assessed for morphology, intra-epithelial lymphocyte count, and interleukin 15 (IL-15) expression, by immunohistochemistry. T-cell lines from 11 of 17 patients were stimulated by HMW-GS. There was a significant change in small intestinal morphology 4 h after commencing infusions with HMW-GS in all three subjects. For example villus height to crypt depth ratios were reduced in the three patients from 3.0+/-0.5 to 1.29+/-0.2, 2.53+/-0.7 to 0.81+/-0.6 and 3.0+/-0.7 to 1.85+/-0.3, P<0.0001 in all cases. There was increased expression of IL-15 in the small intestine from 2 h after the HMW-GS challenges. Mixed HMW-GS stimulate T-cell lines from some coeliac patients and exacerbate coeliac disease in vivo, inducing expression, within 2 h, of IL-15. This suggests an innate immune response to these proteins.

Comparison of Repetitive Sequences Derived from High Molecular Weight Subunits of Wheat Glutenin, an Elastomeric Plant Protein

A strategy has been developed to create repetitive peptides incorporating substitutions in the PGQGQQGYYPTSLQQ consensus repeat sequence of high molecular weight subunits in order to investigate natural sequence variations in elastomeric proteins of wheat gluten. After introduction of glutamic and aspartic acid residues, the peptide behaved similarly to the unmodified form at low pH, but became readily water soluble at pH > 6. Substitution of Gln for Leu at position 13 resulted in only small changes to the secondary structure of the water-insoluble peptides, as did Tyr8His and Thr11Ala. The effects of proline substitutions depended on their location: Leu13Pro substitution had little effect on solubility and structure, but Gln6Pro substitution resulted in dramatic changes. Peptides with two Gln6Pro substitutions had similar properties to the water-insoluble parental peptide, but those with 6 or 10 substitutions were readily soluble. The results indicated that specific sequences influence noncovalent intermolecular interactions in wheat gluten proteins.

Identification of low‐molecular weight glutenin subunits of wheat associated with bread‐making quality

AbstractAlthough it is known that the compositions of low‐molecular weight glutenin subunits (LMW‐GSs) are important factors for bread‐making quality of wheat, it is still not clear which LMW‐GSs confer improved bread‐making quality and how those LMW‐GSs interact with high‐molecular weight (HMW) GSs. Using a hard red winter wheat line with good bread‐making quality and a Japanese wheat cultivar with poor quality as well as their progeny we identified LMW‐GSs associated with the bread‐making quality. One such LMW‐GS, KS2, which had a molecular weight of 42 kDa and was allelic to HS1, was associated with bread‐making quality. Furthermore, by using four recombinant inbred lines with different HMW‐GS and LMW‐GS combinations, KS2 and HMW‐GS 5+10 showed interaction effects on the bread‐making quality. Two‐dimensional polyacrylamide gel electrophoresis (Page) analysis showed that KS2 consists of two protein components and that HS1 is composed of three components. The N‐terminal amino acid sequences of the five components were identical to the most frequently analysed sequence of LMW glutenin components.

Molecular structures and interactions of repetitive peptides based on wheat glutenin subunits depend on chain length

Synthetic and recombinant peptide models of the central repetitive domain of the high molecular weight subunits of wheat glutenin with different numbers of the consensus repeat motifs PGQGQQ + GYYPTSLQQ (21, 45, 110, and 203 residues long) and a recombinant 58,000-Da relative molecular mass (M(r) 58,000) repetitive peptide from a single subunit (1Dx5) are studied using Fourier transform IR spectroscopy. The spectra of the dry peptides are very similar; at low water contents (<76% relative humidity) there is an increase in beta-sheet structures in all peptides. However, on further hydration the content of beta sheets decrease and more beta turns are observed. The changes during the second step of hydration are very marked in the 21 and 45 residue peptides, but they are less apparent in the longer perfect repeat peptides. In the 110 and 203 residue peptides hydration results in increased contents of intermolecular beta-sheets and less beta-turn formation. In contrast, the beta-turn content of the M(r) 58,000 peptide increases during the second hydration step. The decreased extent of structure changes with increasing chain length indicates that cumulative intermolecular interactions, in particular hydrogen bonds, are an important factor in determining the structures in the solid state. The regularity of the perfect repeat sequences in the 21, 45, 110, and 203 residue peptides may favor the formation of larger stretches of intermolecular beta sheets. In contrast, the M(r) 58,000 peptide contains imperfect repeats (in common with native glutenin subunits), which may limit its ability to form intermolecular beta sheets.

The high molecular weight subunits of wheat glutenin and their role in determining wheat processing properties

The high molecular weight subunits of wheat glutenin and their role in determining wheat processing properties

Changes in expression of HMW glutenins of wheat (Triticum aestivum L.) induced by nitrosoethylurea

AbstractThe application of a chemical mutagen, N‐nitroso‐N‐ethylurea, to the grains of wheat (Triticum aestivum L.) cv. ‘Viginta’, provided a mutant line,‘NT‐10′, with an altered composition of high molecular weight (HMW) glutenin subunits. The qualitative mutation was detected in the Glu‐B1 locus by electrophoretic analyses of glutenins. Instead of the HMW glutenin subunits 7 + 9 present in the original genotype, a separate HMW subunit 6 was expressed in the mutant line. The other glutenin and gliadin proteins of the mutant line remained unchanged. The mutant line is also characterized by several changes in morphological and physiological characters—stronger stem, wider leaf, bigger spike and higher grain hardness. This is the first communication of the possibility of changing the composition of high molecular weight subunits of wheat glutenin by means of mutagenesis.

Evidence for the Presence of Only One Cysteine Residue in the D-type Low Molecular Weight Subunits of Wheat Glutenin

D-type low molecular weight subunits of bread wheat glutenin have ω-gliadin type N-terminal amino acid sequences, but are incorporated into the glutenin polymers because of the presence of cysteine residues. In order to determine the number and position of cysteine residues, ID-encoded D-type low molecular weight subunits of wheat glutenin were purified from the cv. Chinese Spring using a procedure that allowed high recovery. Comparison of the molecular weights of alkylated and unalkylated subunits by MALDI mass spectrometry indicated the presence of only one cysteine residue per molecule. This was supported also by the detection of dimers of D subunits in gluten. An internal sequence of 62 amino acids preceding the cysteine was obtained, but it was not possible to identify the cysteine residue, either because it was not within the range of N-terminal sequencing of peptides obtained, or because it was present in one of the two unidentified positions.

Influence of Salts and Aggregation of Gluten Proteins on Reduction and Extraction of High Molecular Weight Glutenin Subunits of Wheat

ABSTRACTHigh‐molecular weight glutenin subunits (HMW‐GS) of wheat were extracted by various combinations of reducing agents, salt solutions, and solvents. Preferential extraction of 1D‐encoded HMW‐GS occurred when flours were extracted with Tris‐HCl SDS buffer at pH 6.8 containing 6% mercaptoethanesulfonic acid sodium salt (MESNA) and analyzed by SDS‐PAGE. Similar effects were also found when dithiothreitol or β‐mercaptoethanol were used in conjunction with nonchaotropic salts. If flours were first extracted with 50% 1‐propanol, the extraction procedure yielded all HMW‐GS, even in the presence of MESNA or high levels of salts. Addition of alcohols or chaotropes to the Tris buffer solutions containing MESNA or of solutions containing salt also extracted all HMW‐GS. The HMW‐GS reported most important in baking quality were found preferentially extracted by nonchaotropic salts and reducing agents. This is related to gluten aggregation and the gliadin‐glutenin interaction and structure.

Genetic and biochemical characterization of novel low molecular weight glutenin subunits in wheat (Triticum aestivum L.).

Two novel low molecular weight subunits of glutenin with relative molecular mass (M(r) values) of 30 and 32 kDa were isolated from the seeds of hexaploid wheat and characterized at genetic and biochemical levels. Among 115 Indian bread wheat cultivars analysed, 40 had a narrow doublet of the new protein bands, 69 had a wide doublet, 3 had only the faster moving band of the doublet, and the remaining 3 cultivars had only the slower moving band. These subunits could be seen in the alkylated glutenin preparations only and the genes for the faster (designated Glu-D4) and slower (designated Glu-D5) moving protein bands of the doublet were located on chromosomes 1D and 7D, respectively. Amino acid composition of the two new subunits was quite different from those of the other well-characterized gluten proteins, as the new subunits have lower amounts of proline and relatively higher amounts of glycine, aspartic acid-asparagine, cysteine, and lysine. Polyclonal antibodies raised against these polypeptides cross-reacted strongly with the major low molecular weight subunits of wheat glutenin (Glu-3 subunits), but did not cross-react with the high molecular weight glutenin subunits or monomeric gliadins. Furthermore, preliminary results on the N-terminal amino acid sequences of the new subunits show homology with the major low molecular weight glutenin subunits, suggesting an evolutionary link between the two.

Evidence for the Non-Glycoprotein Nature of High Molecular Weight Glutenin Subunits of Wheat

Abstract An isolation and purification procedure for wheat high molecular weight glutenin subunits (HMW-GS) involving no steps expected to cause deglycosylation of glycoproteins was developed to allow removal of the maximum amount of carbohydrates not covalently linked to HMW-GS. Flour was defatted and then extracted with 50%n-propanol (50%n-PrOH) to remove arabinogalactans and glucose-containing material. HMW-GS were extracted with 50%n-PrOH containing 5% β-mercaptoethanol, precipitated in 60%n-PrOH, alkylated and recovered by precipitation. The partially purified HMW-GS were separated by cation exchange chromatography and resulting fractions purified by RP–HPLC. The purified HMW-GS contained only 0·1% of glucose (corresponding to 51–59 monosaccharide residues per 100 HMW-GS molecules) and no other amino or neutral sugars. The HMW-GS gave a positive reaction in a periodate–digoxigenin glycan detection assay when labelled in solution (with or without periodate oxidation) but not when labelled directly on-the-blot. HMW-GS expressed inEscherichia coli, a micro-organism without glycosylation capabilities, reacted in an identical way. Possible reasons are given for the difference in results obtained after labelling in solution or on-the-blot. No positive reaction between the purified HMW-GS and mannose-specificGalanthus nivalisagglutinin was observed. The presence of N-acetylglucosamine in HMW-GS has been previously reported20. Since neither mannose nor N-acetylgalactosamine were found in the HMW-GS, and these proteins lack the sequon necessary for N-glycosylation, only O-linked GlcNAc moieties are possible. This modification could not be detected using a galactosyltransferase labelling assay. Taken together these results suggest that HMW-GS are not glycosylated.

Determination of the number of cysteine residues in high molecular weight subunits of wheat glutenin.

A simple method allowing the determination of the number of cysteine residues of the high molecular weight glutenin subunits (HMW-GS) is presented. The method was adapted from that of Creighton (T. E. Creighton, Nature 1980, 2840, 487-489) with modified reagents for alkylation of the cysteine residues and the electrophoretic system. The acid-urea-polyacrylamide gel electrophoresis (PAGE) method, developed by Morel (M. H. Morel, Cereal Chem. 1994, 713, 238-242), was adopted and mixtures of iodoacetic acid and 4-vinylpyridine were used to alkylate the glutenin subunits. The accuracy of the method was checked with some HMW-GS whose number of cysteine residues was already known from molecular biology approaches. In subunits 5*, 2.2, 2.2* and 4.1 (of Dx types) from cultivars Ben and Fiorello, MG 7249, MG 315, and Kador, respectively, only 4 cysteine residues were demonstrated. Subunit 20 was found to exist as a regular Bx and By subunit pair, with the Bx subunit containing only 2 cysteine residues, which is unusual.

Quantification of individual high molecular weight subunits of wheat glutenin using SDS—PAGE and scanning densitometry

A method was developed for quantifying the individual high molecular weight (HMW) glutenin subunits present in a wholemeal flour of wheat (Triticum aestivum L.) Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) was used to separate the subunits from each other, as well as from the other proteins present in an extract of flour. The extraction and separation of the HMW glutenin subunits were optimized, which was necessary for accurate quantification of the proteins by scanning densitometry, as was the use of a sensitive method for staining the proteins in the polyacrylamide gels, using a colloidal suspension of Coomassie Brilliant Blue. The stained bands were quantified by scanning densitometry. The coefficient of variation (CV) of the staining intensity of individual HMW glutenin subunits was 12·4 % for lanes of the same gel; variability between gels used on the same or different days introduced an additional CV of 7 % each. The method developed has several important advantages over the quantification of the subunits by reversed-phase high-performance liquid chromatography (RP-HPLC). First, in contrast to RP-HPLC, a crude extract containing all the storage proteins can be used. Second, the proteins can be quantified and identified in the same analysis.

Quantitative variation of total and individual high molecular weight glutenin subunits of wheat in relation to variation in environmental conditions

AbstractThe total amount of the high molecular weight glutenin (HMWg) subunits, and the amount of the individual subunits, present in kernels of wheat (Triticum aestivum L) were studied in relation to differences in growing conditions. The amounts of individual HMWg subunits in four varieties, each grown at six sites in The Netherlands, differed considerably between the sites. Only to a limited extent (0‐66%, depending on variety) could this variation be ascribed to differences in protein content of the samples. The cause of the rest of the variation is unknown. Accordingly, the total amount of the HMWg subunits, relative to the total amount of proteins present in a wholemeal flour of a variety, differs between the samples produced at different sites. Thus the preference for synthesis of a certain protein fraction may vary depending on the environment. A second experiment showed that variation in protein contents of samples of two varieties grown on the same trial field accounted for about 90% of the variation in the total amount of HMWg subunits. The samples of each variety differed in protein content as a result of differences in the amount of N fertiliser applied. Within each variety, the ratio between the individual HMWg subunits was less dependent on variations in environmental conditions than on the actual amount, indicating that the levels of expression of the individual HMWg subunit genes are regulated by a common mechanism.The implications of the results for the selection possibilities of wheat with desired bread‐making quality and for studies concerning genetic Variation in the amount of HMWg subunits are discussed.

The Structures of Barley and Wheat Prolamins and their Genes

Summary Our current knowledge of the structures of wheat and barley prolamins and their genes is summarised using as examples the high molecular weight subunits of wheat glutenin and B1 and C hordeins of barley. Recent experiments on the regulation of expression of the B1 hordein genes are also described. These studies are providing a sound basis for attempts to manipulate grain protein quality by genetic engineering.

Biochemical properties of some high molecular weight subunits of wheat glutenin

Five high molecular weight subunits (A subunits) of glutenin were isolated from wheat flour by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE). Their overall amino acid compositions were practically identical. The cysteine residues accessible to 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) and alkylating reagents after reduction with various concentrations of 2-mercaptoethanol were determined. With low concentration of 2-mercaptoethanol there were two accessible cysteine residues in subunits 2, 3 and 5, and six in subunits 10 and 11. These values were substantially lower than those calculated from the overall amino acid compositions. Cleavage at cysteine residues showed that in subunits 2, 3 and 5, cysteine residues are located close to one or both ends of the polypeptide chains, in subunits 10 and 11 some of the cysteine residues are also located within the central regions of the polypeptide chains.