Phylogenetic relationship of a new class of LMW-GS genes in the M genome of Aegilops comosa

A new class of low molecular weight glutenin subunit (LMW-GS) genes was isolated and characterized from Aegilops comosa (2n?=?2x?=?14, MM). Although their DNA structure displayed high similarity to LMW-i type genes, there are some key differences. The deduced amino acid sequences of their mature proteins showed that the first amino acid residue of each gene was leucine and therefore they were designated as LMW-l type subunits. An extra cysteine residue was present in the signal peptide and the first cysteine residue of mature proteins located at the end of repetitive domain. Additionally, a long insertion of 10?C22 residues (LGQQPQ_5?C17) occurred in the end of the C-terminal II. Comparative analysis demonstrated that LMW-l type glutenin genes possessed a great number of single-nucleotide polymorphisms and insertions/deletions. A new classification system was proposed according to the gene structure and phylogenetic analysis. In this new system, LMW-GS is classified into two major classes, LMW-M and LMW-I, with each including two subclasses. The former included LMW-m and LMW-s types while the latter contained LMW-l and LMW-i types. Analysis of their evolutionary origin showed that the LMW-l genes diverged from the group 2 of LMW-m type genes at about 12?C14?million years ago (MYA) while LMW-i type evolved from LMW-l type at approximately 8?C12 MYA. The LMW-s type was a variant form of group 1 of LMW-m type and their divergence occurred about 4?C6 MYA. In addition to homologous recombination, non-homologous illegitimate recombination could be an important molecular mechanism for the origin and evolution of LMW-GS gene family. The secondary structure prediction suggested that the novel LMW-l type subunits, such as AcLMW-L1 and AcLMW-L2, may have positive effects on dough properties.     

A novel chimeric low-molecular-weight glutenin subunit gene from the wild relatives of wheat Aegilops kotschyi and Ae. juvenalis: evolution at the Glu-3 loci

Four LMW-m and one novel chimeric (between LMW-i and LMW-m types) low-molecular-weight glutenin subunit (LMW-GS) genes from Aegilops neglecta (UUMM), Ae. kotschyi (UUSS), and Ae. juvenalis (DDMMUU) were isolated and characterized. Sequence structures showed that the 4 LMW-m-type genes, assigned to the M genome of Ae. neglecta, displayed a high homology with those from hexaploid common wheat. The novel chimeric gene, designed as AjkLMW-i, was isolated from both Ae. kotschyi and Ae. juvenalis and shown to be located on the U genome. Phylogentic analysis demonstrated that it had higher identity to the LMW-m-type than the LMW-i-type genes. A total of 20 single nucleotide polymorphisms (SNPs) were detected among the 4 LMW-m genes, with 13 of these being nonsynonymous SNPs that resulted in amino acid substitutions in the deduced mature proteins. Phylogenetic analysis demonstrated that it had higher identity to the LMW-m-type than the LMW-i-type genes. The divergence time estimation showed that the M and D genomes were closely related and diverged at 5.42 million years ago (MYA) while the differentiation between the U and A genomes was 6.82 MYA. We propose that, in addition to homologous recombination, an illegitimate recombination event on the U genome may have occurred 6.38 MYA and resulted in the generation of the chimeric gene AjkLMW-i, which may be an important genetic mechanism for the origin and evolution of LMW-GS Glu-3 alleles as well as other prolamin genes.     

Molecular characterization and genomic organization of low molecular weight glutenin subunit genes at the Glu-3 loci in hexaploid wheat (Triticum aestivum L.)

In this study, we report on the molecular characterization and genomic organization of the low molecular weight glutenin subunit (LMW-GS) gene family in hexaploid wheat (Triticum aestivum L.). Eighty-two positive BAC clones were identified to contain LMW-GS genes from the hexaploid wheat ‘Glenlea’ BAC library via filter hybridization and PCR validation. Twelve unique LMW glutenin genes and seven pseudogenes were isolated from these positive BAC clones by primer-template mismatch PCR and subsequent primer walking using hemi-nested touchdown PCR. These genes were sequenced and each consisted of a single-open reading frame (ORF) and untranslated 5′ and 3′ flanking regions. All 12 LMW glutenin subunits contained eight cysteine residues. The LMW-m-type subunits are the most abundant in hexaploid wheat. Of the 12 LMW-GS, 1, 2 and 9 are i-type, s-type and m-type, respectively. The phylogenetic analysis suggested that the LMW-i type gene showed greater differences to LMW-s and LMW-m-type genes, which, in turn, were more closely related to one another. On the basis of their N-terminal sequences, they were classified into nine groups. Fingerprinting of the 82 BAC clones indicated 30 BAC clones assembled into eight contigs, while the remaining clones were singletons. BAC end sequencing of the 82 clones revealed that long terminal repeat (LTR) retrotransposons were abundant in the Glu-3 regions. The average physical distance between two adjacent LMW-GS genes was estimated to be 81 kb. Most of LMW-GS genes are located in the d-genome, suggesting that the Glu-D3 locus is much larger than the Glu-B3 locus and Glu-A3 locus. Alignments of sequences indicated that the same type (starting with the same N-terminal sequence) LMW-GS genes were highly conserved in the homologous genomes between hexaploid wheat and its donors such as durum wheat and T. tauschii. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of high-molecular-weight glutenin subunits from <Emphasis Type="Italic">Eremopyrum bonaepartis</Emphasis> and identification of a novel variant with unusual high molecular weight and altered cysteine residues

We characterized two high-molecular-weight glutenin subunit (HMW-GS) variants from Eremopyrum bonaepartis, determined their complete open reading frames, and further expressed them in a bacterial system. The variants have many novel structural features compared with typical subunits encoded by Glu-1 loci: 1Fx3.7 and 1Fy1.5 exhibit hybrid properties of x- and y-type subunits. In addition, unusual molecular mass and altered number and distribution of cysteine residues were unique features of HMW-GSs encoded by Glu-F1 from E. bonaepartis. The mature 1Fx3.7 subunit has a full length of 1,223 amino acid residues, making it the largest subunit found thus far, while 1Fy1.5 is just 496 residues. In addition, the mutated PGQQ repeat motif was found in the repetitive region of 1Fx3.7. Although it has a similar molecular mass to that previously reported for 1Dx2.2, 1Dx2.2* and 1S^shx2.9 subunits, 1Fx3.7 appears to have had a different evolutionary history. The N-terminal and repetitive regions have a total of four additional cysteine residues, giving 1Fx3.7 a total of eight cysteines, while 1Fy1.5 has only six cysteines because the GHCPTSPQQ nonapeptide at the end of the repetitive region is deleted. With its extra cysteine residues and the longest repetitive region, features that are relevant to good wheat quality, the 1Fx3.7 subunit gene could be an excellent candidate for applications in wheat quality improvement.     

A second ‘overexpression’ allele at the <Emphasis Type="Italic">Glu-B1</Emphasis> high-molecular-weight glutenin locus of wheat: sequence characterisation and functional effects

Bread is one of the major constituents of the human diet and wheat (Triticum aestivum L.) is the most important cereal for bread making. The gluten proteins (glutenins and gliadins) are recognised as important components affecting the processing quality of wheat flour. In this research, we investigated a particular glutenin subunit allele in an Australian cultivar, H45. Based on protein and DNA assays, the Glu-B1 allele of H45 seems to be Glu-B1al, an allele that includes a functional duplication of a gene encoding an x-type high-molecular-weight glutenin subunit, and is thought to increase dough strength through overexpression of that subunit. Yet H45 does not have the dough properties that would be expected if it carries the Glu-B1al allele. After confirming that H45 overexpresses Bx subunits and that it has relatively low un-extractable polymeric protein (an indicator of weak dough), we cloned and sequenced two Bx genes from H45. The sequences of the two genes differ from each other, and they each differ by four single-nucleotide polymorphisms (SNPs) from the sequence that has been reported for the Glu-B1al x-type glutenin genes of the Canadian wheat cultivar Glenlea. One of the SNPs leads to an extra cysteine residue in one of the subunits. The presence of this additional cysteine may explain the dough properties of H45 through effects on cross-linkage within or between glutenin subunits. We propose that the Glu-B1 allele of H45 be designated Glu-B1br, and we present evidence that Glu-B1br is co-inherited with low un-extractable polymeric protein.     

Characterizing HMW-GS alleles of decaploid <Emphasis Type="Italic">Agropyron elongatum</Emphasis> in relation to evolution and wheat breeding

Bread wheat quality is mainly correlated with high molecular weight glutenin subunits (HMW-GS) of endosperm. The number of HMW-GS alleles with good processing quality is limited in bread wheat cultivars, while there are plenty of HMW-GS alleles in wheat-related grasses to exploit. We report here on the cloning and characterization of HMW-GS alleles from the decaploid Agropyron elongatum. Eleven novel HMW-GS alleles were cloned from the grass. Of them, five are x-type and six y-type glutenin subunit genes. Three alleles Aex4, Aey7, and Aey9 showed high similarity with another three alleles from the diploid Lophopyrum elongatum, which provided direct evidence for the E^e genome origination of A. elongatum. It was noted that C-terminal regions of three alleles of the y-type genes Aey8, Aey9, and Aey10 showed more similarity with x-type genes than with other y-type genes. This demonstrates that there is a kind of intermediate state that appeared in the divergence between x- and y-type genes in the HMW-GS evolution. One x-type subunit, Aex4, with an additional cysteine residue, was speculated to be correlated with the good processing quality of wheat introgression lines. Aey4 was deduced to be a chimeric gene from the recombination between another two genes. How the HMW-GS genes of A. elongatum may contribute to the improvement of wheat processing quality are discussed.     

Phylogenetic analysis of C, M, N, and U genomes and their relationships with Triticum and other related genomes as revealed by LMW-GS genes at Glu-3 loci

Phylogenetic relationships between the C, U, N, and M genomes of Aegilops species and the genomes of common wheat and other related species were investigated by using three types of low-molecular-weight glutenin subunit (LMW-GS) genes at Glu-3 loci. A total of 20 LMW-GS genes from Aegilops and Triticum species were isolated, including 11 LMW-m type and 9 LMW-i type genes. Particularly, four LMW-m type and three LMW-i type subunits encoded by the genes on the C, N, and U genomes possessed an extra cysteine residue at conserved positions, which could provide useful information for understanding phylogenetic relationships among Aegilops and Triticum genomes. Phylogenetic trees constructed by using either LMW-i or the combination of LMW-m and LMW-s, as well as analysis of all the three types of LMW-GS genes together, demonstrated that the C and U genomes were closely related to the A genome, whereas the N and M genomes were closely related to the D genome. Our results support previous findings that the A genome was derived from Triticum uratu, the B genome was from Aegilops speltoides, and the D genome was from Aegilops tauschii. In addition, phylogenetic relationships among different genomes analysed in this study support the concept that Aegilops is not monophyletic.     

Cloning and molecular characterization of a novel x-type glutenin gene Glu-D(t)1 from Aegilops tauschii

A novel gene encoding an x-type high molecular weight glutenin subunit (HMW-GS), designated 1Dx1.1 ^t , was isolated from Aegilops tauschii. It is the largest HMW-GS gene reported so far in this species and its product has a slower mobility than that of subunit 1Ax1 in SDS-PAGE. The open reading frame (ORF) of the gene was 2,628 bp, encoding a protein of 874 amino acid residues. Comparisons of amino acid sequences showed that subunit 1Dx1.1^t had high similarity with other 1Dx subunits but also had two unique characteristics. Firstly, a tripeptide of consensus LQE present in the N-terminal domains of other 1Dx subunits was absent from subunit Dx1.1^t. Secondly, three copies of tandem duplications of the tripeptide motif GQQ and a novel tripeptide sequence (GQL) were present in its central repetitive domain. Phylogenetic analysis showed that subunit 1Dx1.1^t clustered with other known 1Dx subunits.     

A novel high molecular weight glutenin subunit from Australopyrum retrofractum

We describe the sequence of a gene encoding a high molecular weight glutenin subunit (HMW-GS) expressed in the endosperm of the wheat relative Australopyrum retrofractum. Although the subunit has a similar primary structure to that HMW-GS genes present in other Triticeae species, its N-terminal domain is shorter, its central repetitive domain includes a unique dodecameric motif, and its C-terminal domain contain an extra cysteine residue. A phylogenetic analysis showed that the Glu-W1 gene is neither a true x- nor a true y-type subunit, although it is more closely related to the y-type genes present in the K and E genomes than to any other published HMW-GS gene. All these results indicated that this novel subunit may undergo a special evolutionary process different from other Triticeae species. A flour supplementation experiment showed that the Glu-W1 subunit has a negative effect on dough quality, which might be the result of interaction between the two closely placed cysteine residues in the C-terminal region.     

Biosynthesis of Grayanotoxins in Leucothoe grayana Max.Incorporation of Mevalonic Acid and (—)-Kaurene into Grayanotoxin-III

The main subunits of glutenin were separated by preparative SDS-PAGE with a Laemmli system (U. K. Laemmli, Nature, 227, 680 (1970)) and their cysteine (Cys) contents were determined by amino acid analysis. Amino acid compositions of glutenin subunits, determined in the present study, were different from those determined by Danno et al. [G. Danno, K. Kanazawa and M. Natake, Agric. Biol. Chem., 40, 739 (1976)]. We found that these differences were due to the different methods of hydrolysis of subunit polypeptides. That is, hydrolysis of subunit polypeptides extracted from gel and hydrolysis of polypeptides in gel without extraction. Cys contents of glutenin subunits were determined as S-pyridylethyl cysteine (PE-Cys). Although no PE-Cys was detected in B-4 or B-4′, all other subunits were shown to have 4mol Cys per mol protein, respectively.     

Characterization of the HMW glutenin subunits from Aegilops searsii and identification of a novel variant HMW glutenin subunit

High molecular weight (HMW) glutenin subunits are conserved seed storage proteins in wheat and related species. Here we describe a more detailed characterization of the HMW glutenin subunits from Aegilops searsii, which is diploid and contains the S^s genome related to the S genome of Aegilops speltoides and the A, B and D genomes of hexaploid wheat. SDS-PAGE experiments revealed two subunits (one x and one y) for each of the nine Ae. searsii accessions analyzed, indicating that the HMW glutenin subunit gene locus of Ae. searsii is similar to the Glu-1 locus found in wheat in containing both x and y genes. The primary structure of the four molecularly cloned subunits (from two Ae. searsii accessions) was highly similar to that of the previously reported x and y subunits. However, in one accession (IG49077), the last 159 residues of the x subunit (1S^sx49077), which contained the sequence element GHCPTSPQQ, were identical to those of the y subunit (1S^sy49077) from the same accession. Consequently, 1S^sx49077 contains an extra cysteine residue located at the C-terminal part of its repetitive domain, which is novel compared to the x-type subunits reported so far. Based on this and previous studies, the structure and expression of the Glu-1 locus in Ae. searsii is discussed. A hypothesis on the genetic mechanism generating the coding sequence for the novel 1S^sx49077 subunit is presented.     

Expression of epitope-tagged LMW glutenin subunits in the starchy endosperm of transgenic wheat and their incorporation into glutenin polymers

The low-molecular-weight (LMW) glutenin subunits are components of the highly cross-linked glutenin polymers that confer viscoelastic properties to gluten and dough. They have both quantitative and qualitative effects on dough quality that may relate to differences in their ability to form the inter-chain disulphide bonds that stabilise the polymers. In order to determine the relationship between dough quality and the amounts and properties of the LMW subunits, we have transformed the pasta wheat cultivars Svevo and Ofanto with three genes encoding proteins, which differ in their numbers or positions of cysteine residues. The transgenes were delivered under control of the high-molecular-weight (HMW) subunit 1Dx5 gene promoter and terminator regions, and the encoded proteins were C-terminally tagged by the introduction of the c-myc epitope. Stable transformants were obtained with both cultivars, and the use of a specific antibody to the c-myc epitope tag allowed the transgene products to be readily detected in the complex mixture of LMW subunits. A range of transgene expression levels was observed. The addition of the epitope tag did not compromise the correct folding of the trangenic subunits and their incorporation into the glutenin polymers. Our results demonstrate that the ability to specifically epitope-tag LMW glutenin transgenes can greatly assist in the elucidation of their individual contributions to the functionality of the complex gluten system.Communicated by J. W. Snape     

Role of individual disulfide bonds in the structural maturation of a low molecular weight glutenin subunit.

Gliadins and glutenins are the major storage proteins that accumulate in wheat endosperm cells during seed development. Although gliadins are mainly monomeric, glutenins consist of very large disulfide-linked polymers made up of high molecular weight and low molecular weight subunits. These polymers are among the largest protein molecules known in nature and are the most important determinants of the viscoelastic properties of gluten. As a first step toward the elucidation of the folding and assembly pathways that lead to glutenin polymer formation, we have exploited an in vitro system composed of wheat germ extract and bean microsomes to examine the role of disulfide bonds in the structural maturation of a low molecular weight glutenin subunit. When conditions allowing the formation of disulfide bonds were established, the in vitro synthesized low molecular weight glutenin subunit was recovered in monomeric form containing intrachain disulfide bonds. Conversely, synthesis under conditions that did not favor the formation of disulfide bonds led to the production of large aggregates from which the polypeptides could not be rescued by the post-translational generation of a more oxidizing environment. These results indicate that disulfide bond formation is essential for the conformational maturation of the low molecular weight glutenin subunit and suggest that early folding steps may play an important role in this process, allowing the timely pairing of critical cysteine residues. To determine which cysteines were important to maintain the protein in monomeric form, we prepared a set of mutants containing selected cysteine to serine substitutions. Our results show that two conserved cysteine residues form a critical disulfide bond that is essential in preventing the exposure of adhesive domains and the consequent formation of aberrant aggregates.     

Cloning and Expression of Low Molecular Weight Glutenin Genes from the Chinese Elite Wheat Cultivar ＂Xiaoyan 54＂

The low molecular weight （LMW） glutenln subunlts account for 40% of wheat gluten protein content by mass and these proteins are considered to significantly affect dough quality characteristics. Five new full-length LMW glutenln genes （designated LMW-5, LMW-7, LMW-42, LMW-58, and LMW-34） were isolated from the Chinese elite wheat cultivar ＂Xlaoyan 54＂ by PCR amplification of genomlc DNA using a pair of degenerate primers designed from the conserved sequences of the N- and C-terminal regions of published LMW glutenln genes. Deduced amino acid sequence analysis showed that LMW-5 belongs to the LMW-i type genes and that the other four belong to LMW-m type genes. Sequence comparisons revealed that point mutations occasionally occurred in signal peptide and N-terminus domains and often existed in domain III and domain V. Small insertions and deletions are represented in the repetitive domain. There is a stop codon after amino acid position 110 In the repetitive domain of LMW.34, indicating that It is a pseudogene. The other four genes have complete open reading frames and the putative mature regions of these genes were subcloned Into pET-30a expression vector and successfully expressed in Escherlchla coll. Protein sodium dodecyl sulfate-polyacrylamlde gel electro- phoresls analysis showed that all proteins expressed in E. coil by the four genes could be related to B-group LMW glutenln subunits of wheat.     

Characterization of Low Molecular Weight Glutenin Subunit Gene Representing Glu-B3 Locus of Indian Wheat Variety NP4

Low molecular weight (LMW) glutenin subunits represent major part (30%) of storage proteins in wheat endosperm and determine the quality of dough. Despite their importance few LMW glutenin genes have been characterized so far and none from Indian wheat variety. In the present investigation PCR technique was employed to characterize LMW-GS gene representing Glu-B3 locus from Indian bread wheat cultivar NP4. The deduced protein sequence coded by Glu-B3 locus of LMW-GS gene from NP4 showed the presence of regular structure of the repetitive domain with varying numbers of glutamine (Q) residues and the presence of 1^st cysteine residue within the repetitive domain at 40^th position in mature polypeptide. Such structure might increase and stabilize the gluten polymer through intermolecular interactions of the large numbers of glutamine side chains and cysteine residues for intermolecular disulphide bond formation leading to stronger dough quality of NP4. Moreover, Glu-B3 specific primers could also be used for identifying 1BL/1RS translocation in addition to amplifying LMW glutenin genes. There was no amplification in 1B/1R translocation lines as short arm of wheat was replaced by short arm of rye chromosome in these lines. Such information can be useful in wheat improvement for dough properties for better chapati and bread quality.     

Generation of high frequency of novel alleles of the high molecular weight glutenin in somatic hybridization between bread wheat and tall wheatgrass

Somatic hybridization between bread wheat and tall wheatgrass (Agropyron elongatum) has generated fertile introgression progenies with novel combinations of high molecular weight glutenin subunits (HMW-GS). Most of these novel HMW-GS alleles were stably inherited. Sixteen HMW-GS sequences were PCR amplified from three introgression progeny lines and sequenced. The alignment of these sequences indicated that five, probably derived from point mutations of the parental genes, whereas eight likely represent the product of replication slippage. Three Glu-1Ay sequences appear to have lost the transposon presented in the parental gene. Two subunits carry an additional cysteine residue, which may be favorable to the quality of end-use product. We demonstrate that novel HMW-GS alleles can be rapidly generated via asymmetric somatic hybridization. Heng Liu and Shuwei Liu have contributed equally to the work.     

Role of the amino- and carboxy-terminal regions in the folding and oligomerization of wheat high molecular weight glutenin subunits

The high molecular weight glutenin subunits are considered one of the most important components of wheat (Triticum aestivum) gluten, but their structure and interactions with other gluten proteins are still unknown. Understanding the role of these proteins in gluten formation may be aided by analyses of the conformation and interactions of individual wild-type and modified subunits expressed in heterologous systems. In the present report, the bacterium Escherichia coli was used to synthesize four naturally occurring X- and Y-type wheat high molecular weight glutenin subunits of the Glu-1D locus, as well as four bipartite chimeras of these proteins. Naturally occurring subunits synthesized in the bacteria exhibited sodium dodecyl sulfate-polyacrylamide gel electrophoresis migration properties identical to those of high molecular weight glutenin subunits extracted from wheat grains. Wild-type and chimeric subunits migrated in sodium dodecyl sulfate gels differently than expected based on their molecular weights due to conformational properties of their N- and C-terminal regions. Results from cycles of reductive cleavage and oxidative reformation were consistent with the formation of both inter- and intramolecular disulfide bonds in patterns and proportions that differed among specific high molecular weight glutenin species. Comparison of the chimeric and wild-type proteins indicated that the two C-terminal cysteines of the Y-type subunits are linked by intramolecular disulfide bonds, suggesting that the role of these cysteines in glutenin polymerization may be limited.     

ChAy/Bx, a novel chimeric high-molecular-weight glutenin subunit gene apparently created by homoeologous recombination in Triticum turgidum ssp. dicoccoides

High-molecular-weight glutenin subunits (HMW-GSs) are of considerable interest, because they play a crucial role in determining dough viscoelastic properties and end-use quality of wheat flour. In this paper, ChAy/Bx, a novel chimeric HMW-GS gene from Triticum turgidum ssp. dicoccoides (AABB, 2n = 4x = 28) accession D129, was isolated and characterized. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that the electrophoretic mobility of the glutenin subunit encoded by ChAy/Bx was slightly faster than that of 1Dy12. The complete ORF of ChAy/Bx contained 1671 bp encoding a deduced polypeptide of 555 amino acid residues (or 534 amino acid residues for the mature protein), making it the smallest HMW-GS gene known from Triticum species. Sequence analysis showed that ChAy/Bx was neither a conventional x-type nor a conventional y-type subunit gene, but a novel chimeric gene. Its first 1305 nt sequence was highly homologous with the corresponding sequence of 1Ay type genes, while its final 366 nt sequence was highly homologous with the corresponding sequence of 1Bx type genes. The mature ChAy/Bx protein consisted of the N-terminus of 1Ay type subunit (the first 414 amino acid residues) and the C-terminus of 1Bx type subunit (the final 120 amino acid residues). Secondary structure prediction showed that ChAy/Bx contained some domains of 1Ay subunit and some domains of 1Bx subunit. The special structure of this HMW glutenin chimera ChAy/Bx subunit might have unique effects on the end-use quality of wheat flour. Here we propose that homoeologous recombination might be a novel pathway for allelic variation or molecular evolution of HMW-GSs.