Y-type High Molecular Weight Glutenin Subunits Research Articles

Effect of High-Molecular Weight Glutenin Subunits (HMW-GSs) on Gluten Polymerization during Biscuit Making: Insights from Experimental and Molecular Dynamics Simulation Study.

The effect of high-molecular weight glutenin subunits (HMW-GSs) on gluten polymerization during biscuit making was investigated using a set of HMW-GS deletion lines. Results showed that the deletion of HMW-GSs improved the biscuit quality compared with the wild type (WT), especially in x-type HMW-GS deletion lines. Slight gluten depolymerization was observed during dough mixing, while progressive gluten polymerization occurred during biscuit baking. The deletion of HMW-GSs suppressed the polymerization of glutenin and gliadin compared with the WT during biscuit baking, especially in x-type HMW-GS deletion lines. These actions resulted in less elevation of the intermolecular β-sheet and ordered α-helix and altering the disulfide (SS) conformation to a less stable conformation in HMW-GS deletion lines compared with the WT during baking. Molecular dynamics simulation analysis further demonstrated that x-type HMW-GSs had higher thermal stability compared with y-type HMW-GSs during heating.

Influence of High-Molecular-Weight Glutenin Subunit on Components and Multiscale Structure of Gluten and Dough Quality in Soft Wheat

A set of high-molecular-weight glutenin subunit (HMW-GS) deletion lines were used to investigate the influences of HMW-GS on wheat gluten, and dough properties were investigated using a set of HMW-GS deletion lines. Results showed that HMW-GS deletion significantly decreased the dough stability time, as well as viscoelastic moduli (G' and G″), compared with the wild type, where the deletion of x-type HMW-GSs (Ax1d, Bx7d, and Dy12d) decreased more than y-type HMW-GSs (By8d and Dy12d). The deletion of HMW-GS significantly decreased HMW-GS contents and increased α-/γ-gliadin contents. A proteomic study showed that the HMW-GS deletion down-regulated the HMW-GS, β-amylase, serpins, and protein disulfide isomerase and up-regulated the LMW-GS, α/γ-gliadin, and α-amylase inhibitor. Meanwhile, HMW-GS deletion significantly decreased contents of β-turn and β-sheet. In addition, less energetically stable disulfide conformations (trans-gauche-gauche and trans-gauche-trans) were abundant in HMW-GS deletion lines. Furthermore, analysis of five HMW-GSs based on amino acid sequences proved that Dx2 and Bx7 had a more stable structure, followed by Ax1, then Dy12, and finally By8.

Effects of Nitrogen Fertilizer on Quality Characteristics of Wheat with the Absence of Different Individual High-Molecular-Weight Glutenin Subunits (HMW-GSs).

High-molecular-weight glutenin subunits (HMW-GSs) are important components of gluten, which determine the grain quality of wheat. In this study, we investigated the effects of nitrogen (N) fertilizer application on the synthesis and accumulation of grain protein and gluten quality in wheat lines with different HMW-GSs absent. The results showed that the absence of the HMW-GS in the wheat variety Ningmai 9 significantly decreased the contents of gluten, glutenin macropolymer (GMP), protein compositions, HMW-GS and HMW-GS/LMW-GS. The reduction in glutenins was compensated to some extent by an increase of gliadins. The absence of x-type HMW-GSs (1, 7 and 2 subunits) had a greater effect on gluten and GMP properties than y-type HMW-GSs (8 and 12 subunits). The content of protein compositions, gluten and GMP increased with an increase of N level; however, the increment in wheat lines with the absence of HMW-GS, especially in Ax1a, Bx7a and Dx2a, was lower than that in the wild type under various N levels. The expression level of genes encoding HMW-GSs, and activities of nitrate reductase (NR) and glutamine synthetase (GS), differed significantly among the investigated wheat lines. The reduction in gene expression and activities in Ax1a and Dx2a may account for the reductions in gluten, GMP, protein compositions, HMW-GS and HMW-GS/LMW-GS.

Molecular characterization and phylogenetic analysis of active y-type high molecular weight glutenin subunit genes at Glu-A1 locus in wheat

An increased number of high molecular weight glutenin subunits (HMW-GSs) is advantageous to the formation of glutenin macropolymer, the major determinant of end-product quality. Out of the six HMW-GS genes, Glu-1Ay is typically inactive in most hexaploid wheat cultivars. In the current study, we characterized two active Glu-1Ay genes Glu-1AyN11 and Glu-1AyC422 from a hexaploid wheat cultivar N11 and a durum wheat cultivar C422, respectively. The molecular masses of the 1Ay subunit of N11 (Glu-1AyN11) and C422 (Glu-1AyC422) were 61.1 kDa and 63.9 kDa, respectively, measured by matrix-assisted laser desorption/ionization time of flight mass spectrometry. Molecular characterization of Glu-1AyN11 and Glu-1AyC422 showed their complete coding regions were 1764 bp and 1827 bp in length, respectively. The deduced amino acid sequence of both Glu-1AyN11 and Glu-1AyC422 has a similar typical primary structure as the other y-type HMW-GSs, but in comparison to Glu-1By and Glu-1Dy subunits, the Glu-1Ay subunit has one less conserved cysteine residue in the repetitive domain. Phylogenetic analysis revealed that the inactive Glu-1Ay had a closer evolutionary relationship with Glu-1AyC422 than with Glu-1AyN11. The divergence time among Glu-1AyN11, Glu-1AyC422, and other y-type subunit genes was also estimated.

Molecular Cytogenetic Characterization of New Wheat-Dasypyrum breviaristatum Introgression Lines for Improving Grain Quality of Wheat.

As an important relative of wheat (Triticum aestivum L), Dasypyrum breviaristatum contains novel high molecular weight glutenin subunits (HMW-GSs) encoded by Glu-1Vb genes. We identified new wheat—D. breviaristatum chromosome introgression lines including chromosomes 1Vb and 1VbL.5VbL by fluorescence in situ hybridization (FISH) combined with molecular markers. We found that chromosome changes occurred in the wheat—D. breviaristatum introgression lines and particularly induced the deletion of 5BS terminal repeats and formation of a new type of 5B-7B reciprocal translocation. The results imply that the D. breviaristatum chromosome 1Vb may contain genes which induce chromosomal recombination in wheat background. Ten putative high molecular weight glutenin subunit (HMW-GS) genes from D. breviaristatum and wheat—D. breviaristatum introgression lines were isolated. The lengths of the HMW-GS genes in Dasypyrum were significantly shorter than typical HMW-GS of common wheat. A new y-type HMW-GS gene, named Glu-Vb1y, was characterized in wheat—D. breviaristatum 1Vb introgression lines. The new wheat—D. breviaristatum germplasm displayed reduced plant height, increased tillers and superior grain protein and gluten contents, improved gluten performance index. The results showed considerable potential for utilization of D. breviaristatum chromosome 1Vb segments in future wheat improvement.

Genetic analyses of Glu-1S sh in wheat/Aegilops sharonensis hybrid progenies and development of alien HMW-GSs gene-specific markers

Aegilops sharonensis is a diploid relative of wheat and has novel high molecular weight glutenin subunits (HMW-GSs) encoded by Glu-1Ssh. Hybrids of tetraploid wheat species and Ae. sharonensis have been successfully created by wild crosses. In our study, we conducted backcrosses and introduced the novel HMW-GSs of Ae. sharonensis into hexaploid wheat species. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and cytogenetics indicated that lines carrying the HMW-GSs encoded by Glu-1Ssh had varied chromosome numbers ranging from 35 to 50. Among these, three lines with 42 chromosomes (25-8, 33-1, and 42-2) were identified by visual examination. Except for possessing the alien HMW-GSs of Ae. sharonensis, the agricultural traits of the three lines were similar to those of common wheat, indicating their potential for wheat breeding. Additionally, we developed two molecular markers specific to the x- and y-type HMW-GSs genes of Ae. sharonensis. These two molecular markers were validated in backcross progenies and were proven to accurately trace and distinguish Ae. sharonensisGlu-1Ssh from Glu-1A, Glu-1B, and Glu-1D of wheat. Therefore, the Glu-1Ssh HMW-GSs lines with 42 chromosomes, together with gene-specific markers, will be valuable for molecular-assisted selection in alien genes utilization and wheat breeding.

Characterization of a novel y-type HMW-GS with eight cysteine residues from Triticum monococcum ssp. monococcum

The composition and number of high-molecular-weight glutenin subunits (HMW-GSs) play important roles in determining the grain-processing quality of common wheat. The Glu-1Ay allele is silent in common wheat. In this study, an active y-type HMW-GS allele termed 1Ay8.2 (GenBank No. KP137569) was identified from Triticum monococcum L. ssp. monococcum (AmAm, 2n=2x=14), a species with a genome related to the A-genome of common wheat. Compared with previously reported active 1Ay subunits, this novel subunit contained an extra cysteine residue at position 103 of the amino acid sequence in the N-terminal region, in addition to the six cysteines in the N- and C-terminal regions found in most active 1Ay subunits and the one in the repetitive region that appears in only a few 1Ay alleles. This subunit was expressed in an amphiploid (AAAmAmBB, 2n=6x=42) between Triticum turgidum L. ssp. dicoccon and T. monococcum ssp. monococcum. This amphiploid could be used as a bridge to transfer 1Ay8.2 into common wheat cultivars. Replacing the silenced 1Ay in common wheat with the active 1Ay8.2 allele harboring an extra cysteine residue is expected to improve the quality by increasing the number of HMW-GSs and promoting the formation of covalent interactions through disulfide bonds with the extra cysteine residue.

Characterization of novel high-molecular-weight glutenin subunits and their coding sequences in Aegilops markgrafii

Wheat's wild relatives are potential resources for the genetic improvement of common wheat quality. Novel high-molecular-weight glutenin subunits (HMW-GS) from Aegilops markgrafii were identified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Three complete coding sequences of novel HMW-GS were cloned and sequenced. The lengths of these sequences, encoding 881, 847, and 644 amino acid residues, were 2649, 2571, and 1938 bp (designated as Cx1, Cx3, and Cy3), respectively. Sequence comparison showed that added SNPs and InDels were responsible for major variations in the new genes. Analysis of the deduced amino acid sequences revealed that the primary structures of the x- and y-type HMW-GS of Ae. markgrafii were similar to those of previously published HMW-GS. In particular, all new Cx subunits had higher molecular weights than the previously reported HMW-GS in Ae. markgrafii, which have potential value in improving bread quality. Phylogenetic analysis supported the information on the origin of the B genome in wheat. In conclusion, our study not only broadens our understanding of the genetics and phylogenetic knowledge of the HMW-GS protein family but also provides new insight into wheat breeding for end-use quality improvement.

Molecular characterisation and evolution of HMW glutenin subunit genes in Brachypodium distachyon L.

Brachypodium distachyon, a small wild grass within the Pooideae family, is a new model organism for exploring the functional genomics of cereal crops. It was shown to have close relationships to wheat, barley and rice. Here, we describe the molecular characterisation and evolutionary relationships of high molecular weight glutenin subunits (HMW-GS) genes from B. distachyon. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), high performance capillary electrophoresis (HPCE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses demonstrated that there was no HMW-GS expression in the Brachypodium grains due to the silencing of their encoding genes. Through allele-specific polymerase chain reaction (AS-PCR) amplification and cloning, a total of 13 HMW-GS encoding genes from diploid, tetraploid and hexaploid Brachypodium species were obtained, and all of them had typical structural features of y-type HMW-GS genes from common wheat and related species, particularly more similar to the 1Dy12 gene. However, the presence of an in-frame premature stop codon (TAG) at position 1521 in the coding region resulted in the conversion of all the genes to pseudogenes. Further, quantitative real-time PCR (qRT-PCR) analysis revealed that HMW-GS genes in B. distachyon displayed a similar trend, but with a low transcriptional expression profile during grain development due to the occurrence of the stop codon. Phylogenetic analysis showed that the highly conserved Glu-1-2 loci were presented in B. distachyon, which displayed close phylogenetic evolutionary relationships with Triticum and related species.

Structure, variation and expression analysis of glutenin gene promoters from Triticum aestivum cultivar Chinese Spring shows the distal region of promoter 1Bx7 is key regulatory sequence

In this study, ten glutenin gene promoters were isolated from model wheat (Triticum aestivum L. cv. Chinese Spring) using a genomic PCR strategy with gene-specific primers. Six belonged to high-molecular-weight glutenin subunit (HMW-GS) gene promoters, and four to low-molecular-weight glutenin subunit (LMW-GS). Sequence lengths varied from 1361 to 2554bp. We show that the glutenin gene promoter motifs are conserved in diverse sequences in this study, with HMW-GS and LMW-GS gene promoters characterized by distinct conserved motif combinations. Our findings show that HMW-GS promoters contain more functional motifs in the distal region of the glutenin gene promoter (> −700bp) compared with LMW-GS. The y-type HMW-GS gene promoters possess unique motifs including RY repeat and as-2 box compared to the x-type. We also identified important motifs in the distal region of HMW-GS gene promoters including the 5′-UTR Py-rich stretch motif and the as-2 box motif. We found that cis-acting elements in the distal region of promoter 1Bx7 enhanced the expression of HMW-GS gene 1Bx7. Taken together, these data support efforts in designing molecular breeding strategies aiming to improve wheat quality. Our results offer insight into the regulatory mechanisms of glutenin gene expression.

Molecular cloning, heterologous expression, and phylogenetic analysis of a novel y-type HMW glutenin subunit gene from the G genome of Triticum timopheevi

A novel y-type high molecular weight (HMW) glutenin subunit gene from the G genome of Triticum timopheevi (2n=4x=28, AAGG) was isolated and characterized. Genomic DNA from accession CWI17006 was amplified and a 2200 bp fragment was obtained. Sequence analysis revealed a complete open reading frame including N- and C-terminal ends and a central repetitive domain encoding 565 amino acid residues. The molecular weight of the deduced subunit was 77,031, close to that of the x-type glutenin subunits. Its mature protein structure, however, demonstrated that it was a typical y-type HMW subunit. To our knowledge, this is the largest y-type subunit gene among Triticum genomes. The molecular structure and phylogenetic analysis assigned it to the G genome and it is the first characterized y-type HMW glutenin subunit gene from T. timopheevi. Comparative analysis and secondary structure prediction showed that the subunit possessed some unique characters, especially 2 large insertions of 45 (6 hexapeptides and a nonapeptide) and 12 (2 hexapeptides) amino acid residues that mainly contributed to its higher molecular weight and allowed more coils to be formed in its tertiary structure. Additionally, more alpha-helixes in the repeat domain of the subunit were found when compared with 3 other y-type subunits. We speculate that these structural characteristics improve the formation of gluten polymer. The novel subunit, expressed as a fusion protein in E. coli, moved more slowly in SDS-PAGE than the subunit Bx7, so it was designated Gy7*. As indicated in previous studies, increased size and more numerous coils and alpha-helixes of the repetitive domain might enhance the functional properties of HMW glutenins. Consequently, the novel Gy7* gene could have greater potential for improving wheat quality.

Sequence variations and molecular phylogenetic analyses of the HMW-GS genes from different genomes in Triticeae

To investigate the molecular evolution of the high-molecular-weight glutenin subunits (HMW-GSs) in Triticeae, multiple sequence alignment was carried out on the N-terminal sequences of 77 HMW-GSs. The sequence of N-terminal domain showed high homology in both x- and y-type subunits. Sequence alignment of the N-terminal domain of y-type subunits suggested that they shared similar primary structure with each other, not only in the length variation of the N-terminal domain but also they have less variation in amino acids in this domain than those of x-type subunits. Analysis of the x-type HMW-GSs genes revealed that there were three conservative HMW-GSs gene groups with distinct fragments at the beginning of the N-terminal domains of ORFs, defined by the presence or absence of 24 bp fragment, and the presence of 15 bp fragment, respectively. The frequency of mutations in the x-type and y-type HMW-GSs genes was 1.89 and 1.25 out of 10 bases, respectively. Sequence analysis revealed that the y-type HMW-GSs did not contain enough variation for evolutionary analyses. It is obvious that the HMW-GSs encoded by genes of A, B and D genomes in common wheat formed three clusters. The x-type HMW-GSs from Aegilops bicornis, Aegilops comosa, Aegilops cylindrica, Aegilops umbellulata and Aegilops uniaristata were more closely related to the subunits from the D genome rather than those from the B genome.

Characterization of HMW prolamines and their coding sequences from Crithopsis delileana

The high-molecular-weight (HMW) prolamines subunits and their coding sequences from wheat-related diploid species Crithopsis delileana were investigated. Only one HMW prolamine subunit with the similar electrophoresis mobility to the y-type HMW glutenin subunit of hexaploid wheat was observed in two accessions of C. delileana by SDS-PAGE analyses of the total storage protein fractions. It was confirmed by sequencing and expression analysis that this prolamine subunit was an x-type subunit. The amino acid sequence of this subunit had the similar typical structure to those of x-type HMW glutenin genes previously described in wheat. An in-frame stop codon was found in the coding sequences of y-type prolamine subunits. It was found by specifically extraction of HMW prolamines and sequence analysis that the coding regions of Ky prolamine subunit gene is very likely to be not expressed as a full-length protein. Phylogenetic analysis indicated that the Kx subunit could be clustered together with 1Ax1 subunit by an interior paralleled branch, and Ky subunit (inactive) was most closely related to the 1Ay subunit. The coding sequences of Kx subunit could successfully be expressed in bacterial expression system, and the expressed protein had the same electrophoresis mobility as the Kx subunit from the seed of C. delileana. It was the first time that the HMW prolamines subunits encoded by K genome of C. delileana were characterized.

Molecular structure of a novel y-type HMW glutenin subunit gene present in Triticum tauschii

An unusually small y-type high molecular weight (HMW) glutenin subunit gene from Triticum tauschii was sequenced. This gene, encoded at the Glu-D t 1 locus was designated 12.4 t and is the smallest HMW glutenin subunit gene described so far in Triticum species. Oligonucleotide primers based on published sequences of HMW glutenin genes were designed to amplify the encoding region and the central repetitive domain of the gene, which produced fragments of 1.4 and 0.85 kb, respectively. PCR products were cloned and sequenced. The derived amino acid sequence was compared with the amino acid sequences of the HMW glutenin subunits Dy12 t , from T. tauschii, and subunits Dy10 and Dy12 of T. aestivum. The amino acid sequence deduced from the nucleotide sequence demonstrated that deletions of hexapeptides and nonapeptides were responsible for the reduction in the size of this HMW glutenin subunit. The estimated molecular weight of the Dy12.4 t subunit, calculated on the basis of the deduced amino acid sequence, was 45,228 Daltons. There were also single amino acid differences in the N-, C-terminal and central repetitive domains of this gene in comparison to the three other y-type subunits encoded at the Glu-D1 locus. The Dy12.4 t subunit showed the highest similarity to the Dy12 subunit present in the hexaploid wheat Chinese Spring.

Analysis of HMW glutenin subunits and their coding sequences in two diploid Aegilops species.

Considerable progress has been made in understanding the structure, function and genetic regulation of high-molecular-weight (HMW) glutenin subunits in hexaploid wheat. In contrast, less is known about these types of proteins in wheat related species. In this paper, we report the analysis of HMW glutenin subunits and their coding sequences in two diploid Aegilops species, Aegilops umbellulata (UU) and Aegilops caudata (CC). SDS-PAGE analysis demonstrated that, for each of the four Ae. umbellulata accessions, there were two HMW glutenin subunits (designated here as 1Ux and 1Uy) with electrophoretic mobilities comparable to those of the x- and y-type subunits encoded by the Glu-D1 locus, respectively. In our previous study involving multiple accessions of Ae. caudata, two HMW glutenin subunits (designated as 1Cx and 1Cy) with electrophoretic mobilities similar to those of the subunits controlled by the Glu-D1 locus were also detected. These results indicate that the U genome of Ae. umbellulata and the C genome of Ae. caudata encode HMW glutenin subunits that may be structurally similar to those specified by the D genome. The complete open reading frames (ORFs) coding for x- and y-type HMW glutenin subunits in the two diploid species were cloned and sequenced. Analysis of deduced amino acid sequences revealed that the primary structures of the x- and y-type HMW glutenin subunits of the two Aegilops species were similar to those of previously published HMW glutenin subunits. Bacterial expression of modified ORFs, in which the coding sequence for the signal peptide was removed, gave rise to proteins with electrophoretic mobilities identical to those of HMW glutenin subunits extracted from seeds, indicating that upon seed maturation the signal peptide is removed from the HMW glutenin subunit in the two species. Phylogenetic analysis showed that 1Ux and 1Cx subunits were most closely related to the 1Dx type subunit encoded by the Glu-D1 locus. The 1Uy subunit possessed a higher level of homology to the 1Dy-type subunit compared with the 1Cy subunit. In conclusion, our study suggests that the Glu-U1 locus of Ae. umbellulata and the Glu-C1 locus of Ae. caudata specify the expression of HMW glutenin subunits in a manner similar to the Glu-D1 locus. Consequently, HMW glutenin subunits from the two diploid species may have potential value in improving the processing properties of hexaploid wheat varieties.

The Relative Amounts of HMW Glutenin Subunits of OS Wheat Cultivars in Relation to Bread-Making Quality

Qualitative and quantitative variation of high molecular weight (HMW) glutenin subunits is very important for wheat bread-making quality. The aim of this investigation was to estimate the relative amounts (%) of HMW glutenin subunits in OS winter wheat cultivars and their contribution to bread-making quality by SDS-PAGE and scanning densitometry. In this paper 10 winter wheat cultivars created at Agricultural Institute Osijek (OS) and cultivar Divana (Jost-Sjeme, KriŽevci), as Croatian standard improver, were investigated. The wheat quality evaluation was done by indirect parameters, rheological properties of dough and baking test. The quantitative analysis of HMW glutenin subunits have demonstrated that subunits encoded at Glu-D1 locus (Dx2, DylO and Dyl2) were the dominant components. Subunit Axl and subunits encoded at Glu-B1 locus (Bx7, By8 and By9) were the minor ones. The proportion of subunit Dx5 depends on presence of subunit Ax1. When subunit Axl was present (1 7+9 5+10) the proportion of subunit Dx5 was approximately 50% reduced. The positive correlation between the relative amounts of the y-type HMW glutenin subunits and extensographic resistance and energy of dough, loaf volumes and appearance of bread (h/d) was found. That indicates that the y-type HMW glutenin subunits are more important for bread-making quality of investigated cultivars than the x-type. This investigation has indicated that the presence of HMW glutenin subunit 1 and both the presence and proportion of subunits 5+10 contributes to very good and good bread-making quality, while good bread-making quality of cultivars with subunits N 7+8(9) 2+12 is due to higher proportion of subunits at Glu-D 1 locus.

A New Method to Purify y-type High-molecular Weight Glutenin Subunits from Wheat

Il y a deux types de sousunites de haut poids moleculaire de la glutenine: y et x. L'influence des sousunites x et y dans les qualites boulangeres de la farine n'est pas encore eclaircie. Le but de cet article est de presenter une methode de purification des sousunites y en utilisant le materiel basique du laboratoire.

HMW glutenin subunits in multiploidAegilops species: composition analysis and molecular cloning of coding sequences

TheAegilops genus contains species closely related to wheat. In common with wheat,Aegilops species accumulate high molecular weight (HMW) glutenin subunits in their endospermic tissue. In this study, we investigated the composition of HMW glutenin subunits in four multiploidAegilops species using SDS-PAGE analysis. Furthermore, by working withAe. ventricosa, we established an efficient genomic PCR condition for simultaneous amplification of DNA sequences coding for either x-or y-type HMW glutenin subunits from polyploidAegilops species. Using the genomic PCR condition, we amplified and subsequently cloned two DNA fragments that may code for HMW glutenin subunits inAe. ventricosa. Based on an analysis of the deduced amino acid sequences, we concluded that the two cloned sequences encode one x- and one y-type of HMW glutenin subunit, respectively.

A Recombinant Protein of Two High Molecular Weight Glutenins Alters Gluten Polymer Formation in Transgenic Wheat

Wheat high molecular weight glutenin subunits (HMW-GS) are the most important determinants of its superiority for making leavened bread. Following synthesis, these proteins are sequestered into the endoplasmic reticulum and assemble into extremely large elastic polymers, linked by noncovalent and intermolecular disulfide bonds. To study the structural requirements for the assembly of HMW-GS, we have expressed in transgenic wheat a recombinant protein between two cognate x- and y-type subunits. In contrast to the natural polymerized x- and y-type HMW-GS, a significant amount of the recombinant subunit remained monomeric. Nonreducing SDS-polyacrylamide gel electrophoresis, coupled with limited proteolysis, showed that the monomeric form of the recombinant subunit contained an unusual intramolecular disulfide bond, linking an N-terminal cysteine to the single C-terminal cysteine residue. In addition, sucrose gradient analysis revealed that this intramolecular disulfide bond impeded the ability of the recombinant subunit to assemble into polymers. Despite of its altered assembly, a notable amount of the overexpressed recombinant subunit was also present in glutenin polymers. Moreover, its presence significantly altered the subunit composition of the polymer. Our results show that it is possible to modify gluten assembly and properties by expressing recombinant HMW-GS in transgenic wheat, and have a major implication for the improvement of wheat bread-making quality.

Allelic variation at the Gli-A1m, Gli-A2m and Glu-A1m loci and breadmaking quality in diploid wheat Triticum monococcum

SummaryFifty-six accessions of Triticum monococcum and one accession each of T. beoticum and T. sinskajae were analysed for their storage protein compositions and breadmaking quality as determined by the SDS-sedimentation test. In total 30 different alleles at the Glu-A1m locus coding for high-molecular-weight glutenin subunits (HMW-GS), 25 alleles at the Gli-A1m locus coding for ω- and γ-gliadins and 45 alleles at the Gli-A2m locus controlling the synthesis of α/β-gliadins were detected. Most accessions contained one x-type and one y-type HMW-GS and two genotypes were null for both types of subunits. Two polypeptides within the mobility range of HMW-GS in SDS-PAGE were shown to be ω-type gliadins encoded by genes on the short arm of chromosome 1 A. T. sinskajae and several ‘monococcum’ accessions were shown to share the same alleles at Gli-A1m, Gli-A2m and Glu-A1m, confirming sinskajae as a subspecies of T. monococcum. The SDS-sedimentation volumes of most accessions were very low (11–35 ml), a few accessions showing mean sedimentation volumes as high as 90–93 ml. Through the comparison between biotypes occurring in some accessions of ‘monococcum’, good bread-making quality was found to be associated with the presence of alleles y, c and i at the Gli-A1m locus. All accessions were resistant to leaf rust and rich in protein (≥ 16·5%), and most of them showed resistance to powdery mildew.