Variation and classification of B low-molecular-weight glutenin subunit alleles in durum wheat

 The B low-molecular-weight (LMW) glutenin subunit composition of a collection of 88 durum wheat cultivars was analyzed. Extensive variation has been found and 18 different patterns were detected. Each cultivar exhibited 4–8 subunits, and altogether 20 subunits of different mobility were identified. The genetic control of all these subunits was determined through the analysis of nine F₂ populations and one backcross. Five subunits were controlled at the Glu-A3 locus, 14 at Glu-B3 and 1 at Glu-B2. At the Glu-A3 locus each cultivar possessed from zero to three bands and eight alleles were identified. At the Glu-B3 locus each cultivar showed four or five bands and nine alleles were detected. Only one band was encoded by the Glu-B2 locus. A nomenclature for these alleles is proposed and the relationship between them and the commonly used LMW-model nomenclature is discussed. Received: 10 February 1997 / Accepted: 25 April 1997     

Identification of new low-molecular-weight glutenin subunit genes in wheat

To clarify the composition of low-molecular-weight glutenin subunits (LMW-GSs) in a soft wheat cultivar, we cloned and characterized LMW-GS genes from a cDNA library and genomic DNA in Norin 61. Based on alignment of the conserved N- and C- terminal domains of the deduced amino-acid sequences, these genes are classified into 12 groups. One of these groups (group 5), the corresponding gene of which has not been reported previously, contains two additional hydrophobic amino-acid clusters interrupting the N-terminal repetitive domain. Other groups (groups 11 and 12), which were not identified in other cultivars as a protein product, showed all eight cysteines in the C-terminal conserved domain. With specific primer sets for these groups it was revealed that Glu-D3 and Glu-A3 encoded the former and the latter, respectively. Both groups of genes were expressed in immature seeds. The presence of these groups of LMW-GSs may affect the dough strength of soft wheat. Received: 26 March 2001 / Accepted: 16 July 2001     

A 1B-coded low-molecular-weight glutenin subunit associated with quality in durum wheats shows strong similarity to a subunit present in some bread wheat cultivars

Received: 25 May 1999 / Accepted: 22 June 1999     

The low-molecular-weight glutenin subunit proteins of primitive wheats. IV. Functional properties of products from individual genes

 Three genes encoding the low-molecular-weight glutenin subunits (LMW-GSs), LMWG-E2 and LMWG-E4, from A-genome diploid wheat species, and LMW-16/10 from a D-genome diploid wheat, were expressed in bacteria. The respective proteins were produced on a relatively large scale and compared with respect to their effects on flour-processing properties such as dough mixing, extensibility and maximum resistance; these are important features in the end-use of wheat for producing food products. The LMWG-E2 and LMWG-E4 proteins caused significant increases in peak resistance and mixing time, compared to the control, when incorporated into dough preparations. The LMWG-16/10 protein was qualitatively less effective in producing these changes. All three proteins also conferred varying degrees of decrease in dough breakdown. LMWG-E2 and LMWG-E4 caused significant increases in dough extensibility, and decreases in maximum resistance, relative to the control. LMW-16/10 did not show a significant effect on extensibility but showed a significant decrease in maximum resistance. The refinement of relating specific features of the structure of the LMW-GS genes to the functional properties of their respective proteins is discussed. Received: 24 November 1997 / Accepted: 18 August 1998     

Molecular characterization of a low-molecular-weight glutenin cDNA clone from Triticum durum

Summary A full-length, low-molecular-weight (LMW) glutenin cDNA clone, pTdUCD1, has been isolated from a Triticum durum cv Mexicali wheat cDNA library. The complete sequence was determined and compared to the LMW glutenin genes that have been isolated from hexaploid wheat, Triticum aestivum. This cDNA codes for a protein of 295 amino acids (33,414 daltons) including a 20-amino acid signal peptide as deduced from the DNA sequence. Northern analysis showed that this cDNA hybridizes to a family of related sequences ranging in length from 1,200 to 1,000 nucleotides. This gene is similar but not identical to previously published LMW glutenin gene sequences. The most striking characteristic of all cloned LMW glutenin genes is the conservation of eight cysteine residues, which could be involved in potential secondary or tertiary structure, disulfide bond interactions. This paper presents a structural map defining distinct regions of the LMW glutenin gene family.     

四川小麦地方品种AS1643中低分子量谷蛋白基因的克隆及其蛋白质的二级结构预测

根据小麦低分子量谷蛋白基因保守区序列设计引物P1/P2, 采用PCR法对四川小麦地方品种AS1643的基因组DNA进行扩增, 获得1条约900 bp的片段, 分离、纯化后连接到载体pMD18-T上, 对筛选阳性克隆测序, 获得1个低分子量谷蛋白基因LMW-AS1643(GenBank登录号: EF190322), 其编码区长度为909 bp, 可编码302个氨基酸残基组成的成熟蛋白。序列分析结果表明, LMW-AS1643具有典型的低分子量谷蛋白基因的基本结构, 其推导氨基酸序列与其它已知的LMW-GS相比, 最高相似性为93.40%。生物信息学分析表明, 在LMW-AS1643低分子量谷蛋白中, 无规则卷曲含量最高, 为67.90 %, 其次是a-螺旋, 占30.46 %, b-折叠含量最少, 为1.64 %。     

Atomic force microscopy of a hybrid high-molecular-weight glutenin subunit from a transgenic hexaploid wheat

The high-molecular-weight glutenin subunits (HMW-GS) of wheat gluten in their native form are incorporated into an intermolecularly disulfide-linked, polymeric system that gives rise to the elasticity of wheat flour doughs. These protein subunits range in molecular weight from about 70 K-90 K and are made up of small N-terminal and C-terminal domains and a large central domain that consists of repeating sequences rich in glutamine, proline, and glycine. The cysteines involved in forming intra- and intermolecular disulfide bonds are found in, or close to, the N- and C-terminal domains. A model has been proposed in which the repeating sequence domain of the HMW-GS forms a rod-like beta-spiral with length near 50 nm and diameter near 2 nm. We have sought to examine this model by using noncontact atomic force microscopy (NCAFM) to image a hybrid HMW-GS in which the N-terminal domain of subunit Dy10 has replaced the N-terminal domain of subunit Dx5. This hybrid subunit, coded by a transgene overexpressed in transgenic wheat, has the unusual characteristic of forming, in vivo, not only polymeric forms, but also a monomer in which a single disulfide bond links the C-terminal domain to the N-terminal domain, replacing the two intermolecular disulfide bonds normally formed by the corresponding cysteine side chains. No such monomeric subunits have been observed in normal wheat lines, only polymeric forms. NCAFM of the native, unreduced 93 K monomer showed fibrils of varying lengths but a length of about 110 nm was particularly noticeable whereas the reduced form showed rod-like structures with a length of about 300 nm or greater. The 110 nm fibrils may represent the length of the disulfide-linked monomer, in which case they would not be in accord with the beta-spiral model, but would favor a more extended conformation for the polypeptide chain, possibly polyproline II.     

The low-molecular-weight glutenin subunit proteins of primitive wheats. I. Variation in A-genome species

 A Tris-Tricine gel-electrophoresis system (Schaegger and von Jagow 1987), combined with a gradient gel, has been employed to provide an improved resolution of the B and C low-molecular-weight glutenin subunits (LMW-GSs) found in the endosperm of wheat grain. The gel system was used to document the variation in the gluten subunit proteins present in A-genome diploid wheats. The majority of LMW-GSs found in the A-genome diploid wheats were not present in normal bread wheats; the data suggest that they represent a rich source of new variation for the LMW-GSs which are considered to be very important in modulating wheat flour-processing properties. The analysis of variation in the nature of the LMW-GS genes, using PCR, demonstrated that the subclass of C-subunits assayed by primers from a previously published sequence did not show as much variation as the proteins. However, the data collected suggest that sufficient variation may exist in the LMW-GS genes of A-genome diploid wheats to use them as a source of genes for altering the flour-processing properties of hexaploid wheat. Received: 24 November 1997 / Accepted: 18 August 1998     

The low-molecular-weight glutenin subunit proteins of primitive wheats. II. The genes from A-genome species

 Three accessions of T. boeoticum were selected for the cloning and sequencing of novel low-molecular-weight glutenin subunit (LMW-GS) genes, based on the results of SDS-PAGE and PCR analyses of the LMW-GS diversity in A-genome wheat (Lee et al. 1998 a). A comparison of the nucleotide and deduced amino-acid sequences of three cloned genes, LMWG-E2, LMWG-E4 and LMWG-AQ1, both to each other and to other known LMW-GS genes was carried out. The N-terminal domains showed one variable position; GAG (coding for a glutamic acid) for the E-type, and GAT (coding for an aspartic acid) for the Q-type. The comparisons of the LMW-GSs in the literature and this paper define three different types of N-terminal sequences; METSCIPGLERPW and MDTSCIPGLERPW from the durum and A-genome wheats, and METRCIPGLERPW from the hexaploid and D-genome wheats. The repetitive domains were AC-rich at the nucleotide level and coded for a large number of glutamine residues; this region showed 16 variable positions changing 12 amino-acid residues, three triple nucleotide deletions/additions, a large deletion of 18 nucleotides in LMWG-E4 and a deletion of 12 nucleotides in LMWG-E2. In the C-terminal domains 26 variable positions were found and 12 of these mutations changed amino-acid residues; no deletions/ additions were present in this region. It was shown that the LMWG-E2 and LMWG-E4 genes could be expressed in bacteria and this allowed the respective protein products to be related back to the proteins defined as LMW-GSs in vivo. Received: 24 November 1997 / Accepted: 18 August 1998     

Production and characterization of a transgenic bread wheat line over-expressing a low-molecular-weight glutenin subunit gene

The end-use properties, and thus the value, of wheat flours are determined to a large extent by the proteins that make up the polymeric network called gluten. Low molecular weight glutenin subunits (LMW-GS) are important components of gluten structure. Their relative amounts and/or the presence of specific components can influence dough visco-elasticity, a property that is correlated with the end-use properties of wheat flour. For these reasons, manipulation of gluten dough strength and elasticity is important. We are pursuing this goal by transforming the bread wheat cultivar Bobwhite with a LMW-GS gene driven by its own promoter. Particle bombardment of immature embryos produced several transgenic lines, one of which over-expressed the LMW-GS transgene. Southern blots confirmed that the transgene was integrated into the wheat genome, although segregation analyses showed that its expression was sometimes poorly transmitted to progeny. We have determined that the transgene-encoded LMW-GS accumulates to very high levels in seeds of this line, and that it is incorporated into the glutenin polymer, nearly doubling its overall amount. However, SDS sedimentation test values were lower from the transgenic material compared to a non transgenic flour. These results suggest that the widely accepted correlation between the amount of the glutenin polymers and flour technological properties might not be valid, depending on the components of the polymer.     

部分小麦高分子量谷蛋白亚基组成分析

利用十二烷基硫酸钠聚丙烯胺凝胶电泳（SDS－PAGE）分析了85个小麦材料的高分子量谷蛋白亚基的构成,其结果表明：（1）目前生产中应用的优质小麦品种,大部分具有1A上的优质亚基1,1B上的14＋15／17＋18或1D上的5＋10,个别品种还同时聚合有1A,1B,1D上的优质亚基;（2）在所分析的28个八倍体小偃麦中,多数材料含有1,2^*和5＋10等优质亚基;（3）在本实验室创造的材料中,来源于中间偃麦草和普通小麦杂交的后代材料中大部分具有14＋15亚基。此外,个别种质材料还含有Payne亚基命名系统中未命名的一些稀有的高分子量谷蛋白亚基。     

Altered functional properties of tritordeum by transformation with HMW glutenin subunit genes

The high-molecular-weight (HMW) subunits of wheat glutenin are the major determinants of the gluten visco-elasticity that allows wheat doughs to be used to make bread, pasta and other food products. In order to increase the proportions of the HMW subunits, and hence improve breadmaking performance, particle bombardment was used to transform tritordeum, a fertile amphiploid between wild barley and pasta wheat, with genes encoding two HMW glutenin subunits (1Ax1 and 1Dx5). Of the 13 independent transgenic lines recovered (a transformation frequency of 1.4%) six express the novel HMW subunits at levels similar to, or higher than, those of the endogenous subunits encoded on chromosome 1B. Small-scale mixograph analysis of T₂ seeds from a line expressing the transgene for 1Dx5 indicated that the addition of novel HMW subunits can result in significant improvements in dough strength and stability, thus demonstrating that transformation can be used to modify the functional properties of tritordeum for improved breadmaking. Received: 15 January 1999 / Accepted: 5 February 1999     

Genetic polymorphism in low-molecular-weight glutenin genes from Triticum aestivum, variety Chinese Spring

Low-molecular-weight (LMW) glutenin subunits consist mainly of two domains, one at the N- terminus which contains repeats of short amino-acid motifs, and a non-repetitive one rich in cysteine, at the C- terminal region. In previous reports, polyacrylamide-gel electrophoresis has been used to show that large size variation exists among LMW and HMW glutenin subunits, and it has been suggested that deletions and insertions within the repetitive region are responsible for these variations in length. In this study, PCR-amplification of genomic DNA (Triticum aestivum variety Chinese Spring) was used to isolate three full-length LMW glutenin genes: LMWG-MB1, LMWG-MB2 and LMWG-MB3. The deduced amino-acid sequences show a high similarity between these ORFs, and with those of other LMW glutenin genes. Comparisons indicate that LMWG-MB1 has probably lost a 12-bp fragment through deletion and that LMWG-MB1 and LMWG-MB2 have an insertion of 81 bp within the repetitive domain. The current study has shown direct evidence that insertions and/or deletions provide a mechanistic explanation for the allelic variation, and the resultant evolution, of prolamin genes. Single-base substitutions at identical sites generate stop codons in both LMWG-MB2 and LMWG-MB3 indicating that these clones are pseudogenes. Received: 7 May 1999 / Accepted: 17 June 1999     

The low-molecular-weight glutenin subunit proteins of primitive wheats. III. The genes from D-genome species

 The isolation and characterisation by DNA sequencing of two different low molecular weight glutenin subunit (LMW-GS) genes from a genomic library derived from Triticum tauschii is described. These genes are similar (more than 90% similarity) but not identical to previously published LMW-GS gene sequences from cultivated wheats. A comparison of nucleotide sequence of the coding regions revealed the presence of insertions and deletions preferentially located in the region encoding the domains in the LMW-GS proteins rich in proline and glutamine and the middle part of the C-domain. The signal sequences, the amino-terminus and the remaining parts of the C-domain were conserved between all the LMW-GSs compared. The differences detected between the deduced amino-acid sequences in these three regions are only due to single nucleotide substitutions. The most important characteristic of all compared LMW-GS genes is the conservation of eight cysteine residues that could be involved in potential secondary or tertiary structure and disulphide-bond interactions. Comparisons between the 5′ and 3′ non-coding sequences of one of the isolated clones (LMW-16/10) with those of different prolamin genes from wheat, barley and rye led to the distinction of five different gene families, and confirmed the evolutionary relationships determined previously for these genes mainly on the basis of the coding region. In particular, the LMW-GS sequences are more closely related to the B-hordein sequences than to any other prolamin genes from wheat, barley and rye. Formal proof that the isolated genes coded for LMW-GSs, as defined by gel electrophoresis, was obtained by moving one of these genes (LMW-16/10) into a bacterial expression vector based on bacteriophage T7 RNA polymerase. The resulting plasmid directed the synthesis of large amounts of the mature form of the subunit in Escherichia coli. This protein exhibited solubility characteristics identical to those of the LMW-GSs and cross-reacted with antibodies reactive with these proteins. Received: 24 November 1997 / Accepted: 18 August 1998     

Study of high molecular weight wheat glutenin subunit 1Dx5 by 13C and 1H solid-state NMR spectroscopy. I. Role of covalent crosslinking

This work describes a carbon and proton solid-state NMR study of the hydration of a high molecular weight wheat glutenin subunit, 1Dx5. The effect of the presence of disulfide bonds on the hydration behavior of the subunit is investigated by a comparison of the unalkylated and alkylated forms of the protein. Hydration induces partial plasticization of the protein so that some segments become more mobile than others. The 13C cross-polarization and magic-angle spinning (MAS) spectra of the samples in the dry state and at two hydration levels (approximately 40 and approximately 65% D2O) were used to monitor the protein fraction resisting plasticization (trains). Conversely, 13C single pulse excitation and 1H-MAS experiments were used to gain information on the more plasticized segments (loops). The molecular motion of the two protein dynamic populations was further characterized by 13C T1 and 1H T(1rho), T2, and T1 relaxation times. The results suggest that hydration leads to the formation of a network held by a cooperative action of hydrogen bonded glutamines and some hydrophobic interactions. The looser protein segments are suggested to be glycine- and glutamine-rich segments. The primary structure is therefore expected to significantly determine the proportion of trains and loops in the network. The presence of disulfide bonds was observed to promote easier plasticization of the protein and the formation of a more mobile network, probably involving a higher number of loops and/or larger loops.     

小麦新品种(系)Glu-1位点等位基因变异研究

应用SDS-PAGE技术分析了40份小麦新品种(系)的高分子量麦谷蛋白亚基等位基因变异。在Glu-1位点共检测到10种变异类型,其中Glu-Al位点有3种类型：Null、1、26 ,Glu-B1位点有5种类型：7 8、7 9、14 15、7、17 18,Glu-D1位点有2种类型：2 12、5 10;Null(54．3％)、7 8(51．4％)和2 12(62．9％)分别是Glu-Al、Glu-B1和Glu-D1位点上的主要亚基变异类型。另外,在2份材料的Glu-B1和Glu-D1位点各检测到1个新的亚基,分别命名为1By8．1和1Dx5^ 。Glu-1位点的Nei‘s遗传变异指数平均为0,5648,Glu-B1的遗传多样性最高,Glu-D1最低。供试小麦材料Glu-1位点的HMW-GS组合共有17种类型,以(Null,7 8,2 12)组合为主要类型,占31．4％;有9种亚基组合类型分别只在1份材料中出现,占26.1％。结果表明,这些小麦新品种(系)存在着丰富的亚基组合类型。     

Chromosomal control of glutenin subunits in aneuploid lines of wheat: analysis by reversed-phase high-performance liquid chromatography

Summary Glutenin subunits from nullisomic-tetrasomic and ditelocentric lines of the hexaploid wheat variety ‘Chinese Spring’ (CS) and from substitution lines of the durum wheat variety ‘Langdon’ were fractionated by reversed-phase high-performance liquid chromatography (RP-HPLC) at 70 °C using a gradient of acetonitrile in the presence of 0.1% trifluoroacetic acid. Nineteen subunits were detected in CS. The presence and amounts of four early-eluted subunits were found, through aneuploid analysis, to be controlled by the long arms of chromosomes 1D (1DL) (peaks 1–2) and 1B (1BL) (peaks 3–4). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that these four subunits are the high molecular weight subunits of glutenin, which elute in the order 1Dy, 1Dx, 1By, and 1Bx. Similar amounts of 1DL subunits were present (6.3 and 8.8% of total glutenin), but 1BL subunits differed more in abundance (5.4 and 9.5%, respectively). Results indicate that most late-eluting CS glutenin subunits were coded by structural genes on the short arms of homoeologous group 1 chromosomes: 6 by 1DS, 5 by 1AS, and 4 by 1BS. Glutenin of tetraploid ‘Langdon’ durum wheat separated into nine major subunits: 6 were coded by genes on 1B chromosomes, and 3 on 1A chromosomes. Gene locations for glutenin subunits in the tetraploid durum varieties ‘Edmore’ and ‘Kharkovskaya-5’ are also given. These results should make RP-HPLC a powerful tool for qualitative and quantitative genetic studies of wheat glutenin. The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned Stationed at the Northern Regional Research Center, Peoria.     

Effect of gliadins and HMW and LMW subunits of glutenin on dough properties in the F6 recombinant inbred lines from a bread wheat cross

The storage proteins of 64 F₂-derived F₆ recombinant inbred lines (RILs) from the bread wheat cross Prinqual/Marengo were analyzed. Parents differed at four loci: Gli-B1 (coding for gliadins), Glu-B1 (coding for HMW glutenin subunits), Glu-A3/Gli-A1 (coding for LMW glutenin subunits/gliadins) and Glu-D3 (coding for LMW glutenin subunits). The effect of allelic variation at these loci on tenacity, extensibility and dough strength as measured by the Chopin alveograph was determined. Allelic differences at the Glu-B1 locus had a significant effect on only tenacity. None of the allelic differences at either the Glu-A3/Gli-A1 or Glu-D3 loci had a significant effect on quality criteria. Allelic variation at the Gli-B1 locus significantly affected all of the dough properties. Epistatic effects between some of the loci considered contributed significantly to the variation in dough quality. Additive and epistatic effects each accounted for 15% of the variation in tenacity. Epistasis accounted for 15% of the variation in extensibility, whereas additive effects accounted for 4%. Epistasis accounted for 14% of the variation in dough strength, and additivity for 9%. The relative importance of epistatic effects suggest that they should be included in predictive models when breeding for breadmaking quality.     

Molecular characterization of a LMW-GS gene located on chromosome 1B and the development of primers specific for the Glu-B3 complex locus in durum wheat

 Low-molecular-weight glutenin subunits (LMW-GS) represent a specific class of wheat storage proteins encoded at the Glu-3 loci. Particularly interesting are the LMW-GS encoded at the Glu-B3 locus because they have been shown to play an important role in determining the pasta-making properties of durum wheat. Genes encoding LMW-GS have been characterized but only a few of them have been assigned to specific loci. Notably, no complete LMW-GS gene encoded at the Glu-B3 locus has yet been described. The present paper reports the isolation and characterization of a lmw-gs gene located at the Glu-B3 locus. The clone involved, designated pLDNLMW1B, contains the entire coding region and 524 bp of the 5′ upstream region. A nucleotide comparison between the pLDNLMW1B clone and other LMW-GS genes showed the presence of some peculiar structural characteristics, such as short insertions in the promoter region, the presence of a cysteine codon in the repetitive domain, and a more regular structure of this region, which could be important for its tissue-specific expression and for the functional properties of the encoded protein, respectively. Received : 30 May 1997 / Accepted : 29 July 1997