Y-type Subunits Research Articles

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.

Isolation and Sequence Analysis of HMW Glutenin Subunit 1Dy10.1 Ecoding Gene from Xinjiang Wheat ( Triticum petropavlovskyi Udacz. et Migusch)

A novel HMW glutenin subunit gene 1Dy10.1 was isolated and characterized from Xinjiang wheat ( Triticum petropavlovskyi. Udacz. et Migusch) accession Daomai 2. The complete open reading frame (ORF) of 1Dy10.1 was 1965 bp, encoding 655 amino acids. The numbers and distribution of cysteines in 1Dy10.1 were similar to those of 1Dy10 and other y-type subunits. In the N-terminal of 1Dy10.1, an amino acid was changed from L (leucine) to P (proline) at position 55. The repetitive domain of 1Dy10.1 differed from those of known HMW subunits by substitutions, insertions or/and deletions involving single or more amino acid residues. In the repetitive domain of subunit 1Dy10.1, the deletion of tripeptide GQQ in the consensus unit PGQGQQ resulted in the appearance of the motif PGQ that have not been observed in other known y-type HMW subunits. In comparison with the subunit 1Dy12, a deletion of dipeptide GQ, which occurred in subunit 1Dy10, was also observed in subunit 1Dy10.1. The cloned 1Dy10.1 gene had been successfully expressed in Escherichia coli, and the expressed protein had the identical mobility with the endogenous subunit 1Dy10.1 from seed.

An approach for isolating high-molecular-weight glutenin subunit genes using monoclonal antibodies

High-molecular-weight glutenin subunits (HMW-GSs) play an important role in the breadmaking quality of wheat flour. In China, cultivars such as Triticum aestivum 'Xiaoyan No. 6' carrying the 1Bx14 and 1By15 glutenin subunits usually have attributes that result in high-quality bread and noodles. HMW-GS 1Bx14 and 1By15 were isolated by preparative sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and used as an antigen to immunize BALB/c mice. A resulting monoclonal antibody belonging to the IgG1 subclass was shown to bind to all HMW-GSs of Triticum aestivum cultivars, but did not bind to other storage proteins of wheat seeds in a Western blot analysis. After screening a complementary DNA expression library from immature seeds of 'Xiaoyan No. 6' using the monoclonal antibody, the HMW-GS 1By15 gene was isolated and fully sequenced. The deduced amino acid sequence showed an extra stretch of 15 amino acid repeats consisting of a hexapeptide and a nonapeptide in the repetitive domain of this y-type HMW subunit. Bacterial expression of a modified 1By15 gene, in which the coding sequence for the signal peptide was removed and a BamHI site eliminated, gave rise to a protein with mobility identical to that of HMW-GSs extracted from seeds of 'Xiaoyan No. 6' via SDS-PAGE. This approach for isolating genes using specific monoclonal antibody against HMW-GS genes is a good alternative to the extensively used polymerase chain reaction (PCR) technology based on sequence homology of HMW-GSs in wheat and its relatives.

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.

A novel high-molecular-weight glutenin subunit gene Ee1.5 from Elytrigia elongata (Host) Nevski

The St genome, which is present in nearly half of all Triticeae species, originates from the genus Pseudoroegneria. However, very little is known about the high molecular weight (HMW) subunits of glutenin which are encoded by the St genome. In this paper, we report the isolation from Pd. libanotica of four sequences encoding HMW subunits of glutenin. The four genes were all small compared to standard glutenin genes. All four sequences resemble y-type glutenins rather than x-types. However, their N-terminal domains contain a glutamine residue which is present in all x-type, but very few y-type subunits, and their central repetitive domains included some irregular motifs. The indication is therefore that the Glu-1St genes evolved earlier than other modern day homoeologues, so that they represent an intermediate state in the divergence between x- and y-type subunits. No x-type Glu-1St subunit genes were identified.

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.

Allelic variation of the HMW glutenin subunits in Aegilops tauschii accessions detected by sodium dodecyl sulphate (SDS-PAGE), acid polyacrylamide gel (A-PAGE) and capillary electrophoresis

Variability of high molecular weight glutenin subunits (HMW-GS) was studied in198 accessions of Ae. Tauschii (2n=2x=14, DD) by sodium dodecyl sulphate(SDS-PAGE) and acid polyacrylamide gel electrophoresis (A-PAGE) and capillary electrophoresis (CE). A high allelic variation of HMW-GS, including some novel x- and y-type subunits and variable subunit combinations were observed. One accession(TD159) showed a x-type null form. The results by A-PAGE analysis revealed that the subunits Dx5 t and Dy10 t encoded by Glu-D t 1 locus in Ae. tauschii were different in relative mobilities in comparison with the subunits Dx5 and Dy10 found in bread wheats, whereas they had the same mobilities, respectively, when separated by SDS-PAGE. The higher resolution of Ae. tauschii HMW-GS separated by CE method showed two clear peaks in accordance with x- and y-type subunits, respectively,except the accession TD151 which possessed only subunit Dy12.1*t. The electro elution time of the x-type and y-type subunits were about 13–14 and 7–8minutes, respectively. Characterization of wheat HMW-GS was facilitated by using CE which provides high resolution and increases the speed of analysis in conjunction with the traditional gel electrophoretic methods. A total of 42HMW-GS alleles were identified, among which were several alleles not presently detected in bread wheats. Hence Ae. tauschii is potentially a valuable genetic resource for quality improvement of bread wheat.

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.

Polymorphism of High MrGlutenin Subunits in Triticum tauschii: Characterisation by Chromatography and Electrophoretic Methods

A large collection of accessions of the wild wheat progenitor Triticum tauschii, the donor of the D genome of Triticum aestivum, was evaluated for the variability of high molecular weight (Mr) glutenin subunits by electrophoretic and chromatographic methods. A large range of allelic variation at theGlu-Dt1 locus was found in this collection and some novel subunits were observed in both x- and y-type glutenin subunits, including x- or y-type null forms. A few accessions showed three bands in the high Mrglutenin subunit region. However, only two subunits were observed when monomeric proteins were removed before SDS-PAGE analysis of polymeric proteins. The presence of monomeric proteins in this region is discussed. Characterisation of these subunits was also carried out by reversed phase-high performance liquid chromatography (RP-HPLC). Very different surface hydrophobicities were observed between x- and y-type subunits and in some cases it was possible to identify glutenin subunits with the same apparent molecular weight but different surface hydrophobicity. Differences in elution times that were detected when the same subunit was either reduced or reduced and alkylated were related to the number of cysteine residues present in each glutenin subunit. The newGlu-Dt1 glutenin subunits have the potential to enhance the genetic variability available for improving the quality of bread wheat (T. aestivum).

Polymorphism of high molecular weight glutenin subunits in three species of Aegilops

A collection of 136 accessions of Aegilops umbellulata (39), Ae. comosa (75) and Ae. markgrafii (22) was analysed for high-molecular-weight (HMW) glutenin subunits composition. The homogeneity of the accessions was studied and 55.1% of the collection was homogeneous for HMW glutenin subunits (29 Ae. umbellulata, 33 Ae. comosa and 14 Ae. markgrafii). The HMW glutenin subunits of Ae. umbellulata are encoded by the Glu-U1 locus; in Ae. comosa results showed that this proteins are encoded at the 1M chromosome, and the locus was named Glu-M1. In Ae. markgrafii it was assumed that HMW glutenin subunits were encoded by an homoeologous locus and it was named Glu-C1. All the accessions of Ae. umbellulata and Ae. markgrafii expressed both, x-type and y-type subunits. Among the Ae. comosa accessions, only one expressed an x-type subunit alone. All the accessions of Ae. umbellulata and some of Ae. comosa had x-type glutenins of higher molecular weights than those commonly present in bread wheat. A total of 8 alleles were detected at the Glu-U1 locus, 11 at the Glu-M1 and 4 at the Glu-C1. The new HMW glutenin variation found in this work suggests their possible utilisation in breeding for wheat quality.

Deficiency of individual high molecular weight glutenin subunits affords flexibility in breeding strategies for bread-making quality in wheat Triticum aestivum L.

High-molecular-weight (HMW) glutenin subunits in wheat Triticumaestivum L., allelic variation for which affects bread-making quality, areencoded by Glu-1 homoeoloci located on the homoeologous group1 chromosomes. Many alleles at Glu-B1 and Glu-D1 producetwo subunits, an x-type of low electrophoretic mobility in polyacrylamidegels, and a y-type of high mobility. In the current study, a combination ofnear isogenic lines of cultivar `Sicco' has been used to characterise theeffect upon quality of the absence of individual subunits 7 (Glu-B1x-type), 12 (Glu-D1 y-type) and, assuming an additive model ofsubunit action, 2 (Glu-D1 x-type). Absence of subunit 7 gave amoderate reduction in SDS-sedimentation volume, indicating its associationwith lower gluten strength (confirmed by Farinogram and Extensogramstudies), yet, from a full mixing input bake, a moderate increase in loafvolume and a considerable improvement in loaf score (an overall evaluationof loaf quality). Absence of subunit 12 gave a slightly larger reduction inSDS-volume, yet no change in loaf volume or score. Absence of bothsubunits 2+12 gave a larger reduction again in SDS-volume, a moderatereduction in loaf volume and a large reduction in loaf score. Absence ofsubunit 2 alone is therefore predicted to reduce SDS-volume, loaf volumeand score such that loss of this x-type subunit would lead to larger changesin quality parameters than loss of y-type subunit 12. A general conclusionof the study is that, while deficiency for HMW glutenin subunits generallyleads to reduced gluten strength and viscoelasticity, the resultantintermediate gluten strength may on occasions lead to improvements in loafperformance in situations where the base gluten strength is high. Theremay, then, be contexts in breeding programmes where selection fordeficiency would be a possible strategy for improving bread-making quality,adding to the flexibility available to the breeder. Somewhat unexpectedly,additional analysis found that, in the genetic background of cultivar `Sicco'used in this study, subunits 7+8 at Glu-B1 were indistinguishablefrom their allelic counterparts subunits 7+9 for virtually all characters, andthat subunits 2+12 at Glu-D1, while inferior in performance formixing properties compared to subunits 5+10, were associated with goodloaf characteristics.

Comparative Studies of High MrSubunits of Rye and Wheat. III. Localisation of Cysteine Residues

The high Mrsubunit fraction of rye glutelin was isolated from flour (cultivar Danko) by a specific extraction/precipitation procedure. The Cys content of the rye fraction (1·9 mol-%) was increased by a factor of 2–3 in comparison with wheat high Mrsubunits. To identify the positions of the Cys residues within the sequences, the high Mrsubunit fraction of rye was partially hydrolysed with chymotrypsin and the digest was separated by covalent chromatography on Thiopropylsepharose 6B. The chymotryptic digest was separated into a minor bound portion (4%) and a major non-bound portion (96%). The Cys content of the bound portion (13·2 mol-%) was strongly increased compared with the total digest (2·3%). The Cys-containing peptides of the bound portion were separated by RP-HPLC, alkylated with 4-vinylpyridine, purified by RP-HPLC and sequenced by automated Edman degradation. Altogether, 51 Cys peptides were sequenced; 24 of them could be assigned to known sequences of wheat highMr subunits. The other peptides were either derived from alpha -amylase or trypsin inhibitors or fromalpha -chymotrypsin. Four peptides could not be assigned. The peptides of the high Mrsubunits of rye represented six different Cys residues within the amino acid sequences. Five of these Cys residues corresponded to known Cys residues of wheat subunits: Ca, Cband Cdfrom domain A, Cyfrom domain B and Czfrom domain C. Three Cys residues present in wheat subunits could not be detected in rye, the adjacent Cc1Cc2from domain A of y-type subunits and Ceat the beginning of domain B of x-type subunit 5. One additional Cys residue of rye subunits (Cz*) located in domain C was found, which is not present in wheat highMr subunits. Thus, rye subunits could have a second Cys residue besides Czin domain C. This second Cys residue opens the possibility that high Mrsubunits of rye containing residue Cz*are chain terminators.

Characterization of a monoclonal antibody specific for HMW subunits of glutenin and its use to investigate glutenin polymers.

A monoclonal antibody, IFRN 1602, has been developed to a synthetic peptide based on the sequence (94)GSVTCPQQV(101) of HMW subunit 1Dx5. The antibody bound strongly to the synthetic peptide based on the cognate sequence of HMW subunit 1Dx2 which contains a serine instead of a cysteine residue. However, it recognized the immunizing peptide by enzyme-linked immunosorbent assay (ELISA) only poorly, probably because the peptide exists as a disulfide-bonded dimer under the assay conditions. From immunoblotting studies against a wide range of wheat varieties, IFRN 1602 was shown to primarily recognize x-type HMW subunits of glutenin encoded on chromosomes 1A and 1D, cross-reacting weakly with the 1A and 1D y-type subunits. It did not bind to any of the 1B-encoded subunits. The Mab also recognized a small number of polypeptides of greater mobility than HMW subunits which were not visible on the stained gels and occurred only in the presence of specific 1A and 1D x-type HMW subunits. Such polypeptides were not present in a preparation of recombinant subunit 2, suggesting that they are modified forms of the subunits which arise in the seed perhaps by processing of the associated subunits. When used to probe partially reduced glutenin, IFRN 1602 bound to 1Dx5-1Dy10 dimers. As the Mab reacted primarily with Cys(97) of 1Dx5 in a reduced form, these data suggest that this residue is not involved in either intra- or intermolecular disulfide bond in the HMW subunit dimers. Thus, Cys(97) of 1Dx5 may be present in gluten in a reduced form, involved in intramolecular disulfide bonds, or linking of the HMW subunit dimers into larger polymers.

Conformational Polymorphism of High MrGlutenin Subunits Detected by Transverse Urea Gradient Gel Electrophoresis

Unfolding behaviour of different allelic variants of high Mrglutenin subunits has been studied by transverse urea gradient gel electrophoresis, a system in which a gradient of urea (from 0 to 8 M) is generated perpendicularly to the direction of migration. A different unfolding pattern is observed between x- and y-type subunits. The former type of subunits generally present a broad unfolding pattern without any clear transition, as urea concentration increases, suggesting the existence of several conformational intermediates. On the contrary most of the y-type subunits present an unfolding pattern showing an abrupt decrease in mobility, with a single inflection point at the midpoint of the transition indicative of a protein unfolding in a single, cooperative, two-state transition. Native and bacterial expressed subunits, differing for the size of the repetitive domain, were used to assess the role of variation in size of such region on unfolding behaviour. Free energy values associated to unfolding process were calculated for most of the y-type subunits presenting a two state transition. In particular, different values were found for subunit Dy10 and Dy12, with the former being larger than the latter, suggesting a different stability of the two subunits. Possible implications for qualitative differences associated to allelic subunits are also discussed.

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.

Depolymerisation and Re-polymerisation of Wheat Glutenin During Dough Processing. II. Changes in Composition

tbIn view of the importance of the glutenin macropolymer (GMP) for dough properties and bread-making quality, the changes of composition of glutenin subunits in GMP were investigated for flours and doughs from three cultivars (Rektor, Camp Remy, Obelisk) and from a blend of two of those cultivars (Camp Remy/Obelisk). After mixing, the content of GMP, and the contents of highMrand lowMrglutenin subunits in GMP, decreased. The two subunit classes, and the individual subunits, decreased to different extents, however. This resulted in large changes in their proportions in GMP. After mixing, highMrglutenin subunits accounted for 8–17% of the GMP of dough, whereas in flour they accounted for 29–31%. During dough resting, the amounts of all glutenin subunit classes increased as the amount of GMP increased, but in most cases to lower final levels than those present in the flour. The proportions of highMrglutenin subunits in the GMP of dough increased to levels similar to those present in the GMP of flour (22–31%). The x-type/y-type highMrglutenin subunit molar ratio decreased significantly from 2·42 in the GMP from flour or 2·38 in the GMP from dough directly after mixing, to 1·78–1·83 after 90 min dough resting or longer. This indicates that the GMP became more enriched in y-type subunits during re-polymerisation. In contrast, the highMrglutenin subunit 12/subunit 9 molar ratio in GMP fell significantly from 1·79 for GMP from flour to 1·13 for dough directly after mixing, and increased upon resting to a level similar to that in the GMP from flour. The changes in composition during dough mixing and resting indicate that specific reactions involving glutenin subunits occur during depolymerisation of GMP and during re-polymerisation of the SDS-extractable glutenin polymers.

Detection of y-type Subunit at theGlu-A1Locus in Some Swedish Bread Wheat Lines

Abstract Electrophoretic and reversed phase high performance liquid chromatographic (RP–HPLC) analyses were performed on gluten proteins extracted from flours milled from two different Swedish bread wheat lines; these lines have been reported to possess a novel highMrglutenin subunit controlled by a gene at theGlu-A1locus, referred to as 21*. Although RP–HPLC indicated that subunit 21* has a surface hydrophobocity similar to that of the commonly occurring allelic subunits 1 or 2*, it differs from them in isoelectric point, being more basic when analysed by two dimensional gel electrophoresis (IEF/SDS–PAGE). RP–HPLC separations of highMrglutenin subunits showed the presence of an additional peak, the behaviour of which was similar to that of y-type subunits encoded by genes at theGlu-A1ylocus and present only in wild wheatsT. urartu(AA) orT. dicoccoides(AABB). Based on chromatographic results and on the tight linkage observed with subunit 21*, it is suggested that the additional component (indicated as 21*y), present in the breeding lines analysed, corresponds to the y-type subunit encoded at theGlu-A1locus. Genes encoding the subunits 21* and 21*y were also analysed by polymerase chain reaction (PCR). Contrary to what was observed for the polypeptide itself, the gene corresponding to subunit 21* was similar in size to that encoding subunit 2* and shorter than that corresponding to subunit 1. Moreover, the amplification product corresponding to the active 21*y gene was shorter than that of the allelic inactive gene present in the bread wheat cultivar Cheyenne. As reported for other highMrglutenin subunits, gene size differences observed were due to a different length of the repetitive region. Because cultivated polyploid wheats have been shown to have only the x-type subunit at theGlu-A1locus, it is speculated that the new combination, with both x- and y-type subunits expressed, might have been introgressed during breeding processes from the wild wheat progenitorsT. urartuorT. dicoccoides, which have genotypes expressing both types of subunits.

Purification and Characterisation of HighMrGlutenin Subunit 20 and its Linked y-type Subunit from Durum Wheat

HighMrglutenin subunit 20 and its linked y-type subunit, present in the durum wheat cultivar Lira, were purified by preparative reversed-phase high-performance liquid chromatography (RP–HPLC). Amino acid and N-terminal sequence analysis of subunit 20y confirmed that it corresponded to a y-type subunit. Moreover, the number and position of the cysteine residues in subunit 20 were determined by alkylation with the fluorogenic reagent 7-fluoro-4-sulfamoyl-2,1,3,-benzoxadiazole (ABD-F) and subsequent enzymic digestion with trypsin. N-terminal amino acid sequence analysis of the fluorescent peptides showed that subunit 20 had only two cysteine residues, one in the N-terminal region and the other in the C-terminal domain.

Molecular and Biochemical Characterisation of HMW Glutenin Subunits fromT. tauschiiand the D Genome of Hexaploid Wheat

Abstract Electrophoretic (urea SDS–PAGE) and chromatographic (RP–HPLC) analysis was performed on 8 allelic variants of HMW glutenin subunits derived from Triticum tauschii and from the D genome of a hexaploid wheat species ( Triticum macha ) and hexaploid landraces. These subunits had been previously identified using SDS–PAGE. The characterisation revealed that subunits Dy10 t and Dy12 t from T. tauschii could be differentiated from their bread wheat counterparts using both urea SDS–PAGE and RP–HPLC. In the latter case, the T. tauschii y-type subunits were clearly more hydrophobic than the Dy type subunits of bread wheat. The characterisation also suggested that subunit Dx5 t , derived from two separate T. tauschii accessions, did not contain the extra cysteine residue characteristic of Dx5 from bread wheat. RFLP analysis of the genes encoding the HMW glutenin subunits of interest suggested that the absence of Dx-type HMW glutenins in two hexaploid landraces was due to lack of expression of their encoding genes. The relationship between Hin dIII DNA fragment size and protein subunit size, as measured by electrophoretic mobility, is examined and discussed. Finally, the solubility properties of a HMW protein designated T1 (derived from T. tauschii accession AUS 18913) suggested that it was not a HMW glutenin subunit as was previously thought. Further studies are needed to clarify the identity of this subunit.