The molecular diversity of α-gliadin genes in the tribe Triticeae

Many of the unique properties of wheat flour are derived from seed storage proteins such as the α-gliadins. In this study these α-gliadin genes from diploid Triticeae species were systemically characterized, and divided into 3 classes according to the distinct organization of their protein domains. Our analyses indicated that these α-gliadins varied in the number of cysteine residues they contained. Most of the α-gliadin genes were grouped according to their genomic origins within the phylogenetic tree. As expected, sequence alignments suggested that the repetitive domain and the two polyglutamine regions were responsible for length variations of α-gliadins as were the insertion/deletion of structural domains within the three different classes (I, II, and III) of α-gliadins. A screening of celiac disease toxic epitopes indicated that the α-gliadins of the class II, derived from the Ns genome, contain no epitope, and that some other genomes contain much fewer epitopes than the A, S(B) and D genomes of wheat. Our results suggest that the observed genetic differences in α-gliadins of Triticeae might indicate their use as a fertile ground for the breeding of less CD-toxic wheat varieties.     

Molecular characterization of α-gliadin gene sequences in Indian wheat cultivars vis-à-vis celiac disease eliciting epitopes

α-Gliadin proteins of the wheat gluten form a multigene family encoded by genomic loci Gli-A2, Gli-B2 and Gli-D2 located on the homoeologous wheat chromosomes 6AS, 6BS, and 6DS, respectively which upon partial digestion elicits celiac disease (CD) in the genetically susceptible individuals. The present investigation was planned to study the variations in the amino acid sequence of the α-gliadin proteins and CD eliciting epitopes in the Indian wheat cultivars. Representative wheat varieties released and cultivated in India during the period 1905–2011 were selected for studying the α-gliadin genes by cloning and sequencing followed by in silico analysis of the gene sequences. A lot of variation for α-gliadin gene sequences especially in T cell stimulatory epitopes glia-α9, glia-α20, glia-α2 and glia-α was observed in different wheat varieties. Modern varieties released during 1971–2011 had higher proportion of intact T-cell stimulatory epitopes. The old wheat varieties released in the period 1905–1970 on the other hand had large proportion of variant epitopes. We identified three wheat varieties namely C591, C273 and K78 having only variant epitopes at Gli-D2 and Gli-B2 and both intact and variant epitopes at Gli-A2. Identification of lower proportion of T-cell stimulatory epitopes in these three varieties is the first step towards developing a wheat variety less immunogenic for celiac disease patients. The gene sequences of the selected varieties have been submitted at NCBI with accession numbers GenBank KJ410473–KJ410488.     

A universal approach to eliminate antigenic properties of alpha-gliadin peptides in celiac disease

Celiac disease is caused by an uncontrolled immune response to gluten, a heterogeneous mixture of wheat storage proteins, including the α-gliadins. It has been shown that α-gliadins harbor several major epitopes involved in the disease pathogenesis. A major step towards elimination of gluten toxicity for celiac disease patients would thus be the elimination of such epitopes from α-gliadins. We have analyzed over 3,000 expressed α-gliadin sequences from 11 bread wheat cultivars to determine whether they encode for peptides potentially involved in celiac disease. All identified epitope variants were synthesized as peptides and tested for binding to the disease-associated HLA-DQ2 and HLA-DQ8 molecules and for recognition by patient-derived α-gliadin specific T cell clones. Several specific naturally occurring amino acid substitutions were identified for each of the α-gliadin derived peptides involved in celiac disease that eliminate the antigenic properties of the epitope variants. Finally, we provide proof of principle at the peptide level that through the systematic introduction of such naturally occurring variations α-gliadins genes can be generated that no longer encode antigenic peptides. This forms a crucial step in the development of strategies to modify gluten genes in wheat so that it becomes safe for celiac disease patients. It also provides the information to design and introduce safe gluten genes in other cereals, which would exhibit improved quality while remaining safe for consumption by celiac disease patients.     

A New Class of Wheat Gliadin Genes and Proteins

The utility of mining DNA sequence data to understand the structure and expression of cereal prolamin genes is demonstrated by the identification of a new class of wheat prolamins. This previously unrecognized wheat prolamin class, given the name δ-gliadins, is the most direct ortholog of barley γ3-hordeins. Phylogenetic analysis shows that the orthologous δ-gliadins and γ3-hordeins form a distinct prolamin branch that existed separate from the γ-gliadins and γ-hordeins in an ancestral Triticeae prior to the branching of wheat and barley. The expressed δ-gliadins are encoded by a single gene in each of the hexaploid wheat genomes. This single δ-gliadin/γ3-hordein ortholog may be a general feature of the Triticeae tribe since examination of ESTs from three barley cultivars also confirms a single γ3-hordein gene. Analysis of ESTs and cDNAs shows that the genes are expressed in at least five hexaploid wheat cultivars in addition to diploids Triticum monococcum and Aegilops tauschii. The latter two sequences also allow assignment of the δ-gliadin genes to the A and D genomes, respectively, with the third sequence type assumed to be from the B genome. Two wheat cultivars for which there are sufficient ESTs show different patterns of expression, i.e., with cv Chinese Spring expressing the genes from the A and B genomes, while cv Recital has ESTs from the A and D genomes. Genomic sequences of Chinese Spring show that the D genome gene is inactivated by tandem premature stop codons. A fourth δ-gliadin sequence occurs in the D genome of both Chinese Spring and Ae. tauschii, but no ESTs match this sequence and limited genomic sequences indicates a pseudogene containing frame shifts and premature stop codons. Sequencing of BACs covering a 3 Mb region from Ae. tauschii locates the δ-gliadin gene to the complex Gli-1 plus Glu-3 region on chromosome 1.     

Detailed analysis of the expression of an alpha-gliadin promoter and the deposition of alpha-gliadin protein during wheat grain development

Background and Aims

Alpha-gliadin proteins are important for the industrial quality of bread wheat flour, but they also contain many epitopes that can trigger celiac (cœliac) disease (CD). The B-genome-encoded α-gliadin genes, however, contain very few epitopes. Controlling α-gliadin gene expression in wheat requires knowledge on the processes of expression and deposition of α-gliadin protein during wheat grain development.

Methods

A 592-bp fragment of the promotor of a B-genome-encoded α-gliadin gene driving the expression of a GUS reporter gene was transformed into wheat. A large number of transgenic lines were used for data collection. GUS staining was used to determine GUS expression during wheat kernel development, and immunogold labelling and tissue printing followed by staining with an α-gliadin-specific antibody was used to detect α-gliadin protein deposited in developing wheat kernels. The promoter sequence was screened for regulatory motifs and compared to other available α-gliadin promoter sequences.

Key Results

GUS expression was detected primarily in the cells of the starchy endosperm, notably in the subaleurone layer but also in the aleurone layer. The α-gliadin promoter was active from 11 days after anthesis (DAA) until maturity, with an expression similar to that of a 326-bp low molecular weight (LMW) subunit gene promoter reported previously. An α-gliadin-specific antibody detected α-gliadin protein in protein bodies in the starchy endosperm and in the subaleurone layer but, in contrast to the promoter activity, no α-gliadin was detected in the aleurone cell layer. Sequence comparison showed differences in regulatory elements between the promoters of α-gliadin genes originating from different genomes (A and B) of bread wheat both in the region used here and upstream.

Conclusions

The results suggest that additional regulator elements upstream of the promoter region used may specifically repress expression in the aleurone cell layer. Observed differences in expression regulator motifs between the α-gliadin genes on the different genomes (A and B) of bread wheat leads to a better understanding how α-gliadin expression can be controlled.Key words: Alpha-gliadin, promoter, expression, deposition, wheat, Triticum aestivum, grain development     

Molecular cloning of novel α-gliadin genes from Crithopsis delileana and the evolution analysis with those from Triticeae

The α-gliadins from Crithopsis delileana (Schult) Roshev (2n=2x=14, KK) were investigated by Acid polyacrylamide gel electrophoresis (A-PAGE) analysis. It was indicated that the electrophoresis mobility of gliadins from C.delileana had obvious difference with those from common wheat in α, γ and ω region. Using primers designed from published sequences of α-gliadin genes, three α-gliadin genes were isolated from C. delileana, which were designated as gli-ka1, gli-ka2 and gli-ka3, respectively. Two in-frame stop codons were found in the coding sequences of gli-ka3, indicating that gli-ka3 could be a pseudogene. The gli-ka2 was a gliadin with an odd number of cysteines, resulting from a non-synonymous mutation. This change might lead to the interactive behavior of gli-ka2. Three α-gliadin genes of C. delileana had the similar but not identical primary structures to the corresponding gene sequences from other wheat related species. By the alignment of α-gliadin genes from Triticeae, phylogenetic analysis indicated that three α-gliadin genes of C. delileana clustered together with all α-gliadin genes from Ee genome of Lophopyrum elongatum by an interior paralleled branch.     

Characterization of two novel γ-gliadin genes encoded by K genome of Crithopsis delileana and evolution analysis with those from Triticeae

By acid polyacrylamide gel electrophoresis (A-PAGE) analysis, it was indicated that the electrophoresis mobility of gliadins from Crithopsis delileana (Schult) Roshev (2n=2x=14, KK) had obvious difference with those from common wheat in α, γ and ω region. Using homologous primers, two γ-gliadin genes (gli-Kr1 and gli-Kr2) were isolated from C. delileana, which had been deposited in the GenBank under accession numbers EU283818 and EU283821, respectively. Two γ -gliadin genes of C. delileana had the similar primary structures to the corresponding gene sequences from other wheat related species. The differences were mainly resulted from substitutions, insertions and deletions involving single amino acid residues or motifs of γ-gliadins. The repetitive domains of gli-Kr1 and gli-Kr2 from C. delileana are shorter than most of other sequences. By the alignment of γ-gliadin genes from A, B, D, A^m, A^u, S, S^l, S^sh, S^s and S^b genomes of Triticum and Aegilops, R genome of Secale (γ-secalin), Ee genome of Lophopyrum and K genome of Crithopsis in Triticeae, phylogenetic analysis indicated that two γ-gliadin genes of C. delileana could be clustered together with a γ-gliadin genefrom Ssh genome of Aegilops by an interior paralleled branch. It was the first time that the γ-gliadin genes encoded by K genome of C. delileana were characterized. These could offer precious information for better understanding the qualities associated with gliadins, the response in coeliac disease and studying the evolutionary relationship of gliadins in Triticeae.     

The γ-gliadin gene content of a derivative from a somatic hybrid between bread wheat and tall wheatgrass

A PCR-based strategy was applied to obtain the DNA sequence of γ-gliadin open reading frames present in line II-12, a derivative from a somatic hybrid between bread wheat (Triticum aestivum L.) cv. Jinan177 and tall wheatgrass (Lophopyrum ponticum, 10×). A total 50 analysable sequences were obtained, 18 from II-12 and 16 each from the parents. Amplicon length ranged from 720 to 936 bp, corresponding to a putative mature protein of 239–309 residues. The primary structure of these putative proteins comprised five domains, of which only two varied in length. Phylogenetic analyses showed that the mature γ-gliadin sequences fell into four major clades. Group 1 contained sequences shared between II-12 and L. ponticum, suggesting that some L. ponticum γ-gliadin genes are present in the introgression line. Group 3 has five Jinan177 and five II-12 sequences, indicating that II-12 also carries wheat versions of Gli-1. Group 2 and 4 comprised four and two II-12, three and one Jinan177 as well as one and four L. ponticum sequences, respectively. Fewer genes encoding coeliac disease epitopes were present in II-12 than in the wheat donor parent. Three II-12 γ-gliadins and one from the wheat parent contained an odd number of cysteine residues, and two of them had an additional cysteine residue at the amino end of domain V. The possible use of II-2 for improving quality of bread wheat is discussed.     

Molecular characterization of the celiac disease epitope domains in α-gliadin genes in Aegilops tauschii and hexaploid wheats (Triticum aestivum L.)

Nineteen novel full-ORF α-gliadin genes and 32 pseudogenes containing at least one stop codon were cloned and sequenced from three Aegilops tauschii accessions (T15, T43 and T26) and two bread wheat cultivars (Gaocheng 8901 and Zhongyou 9507). Analysis of three typical α-gliadin genes (Gli-At4, Gli-G1 and Gli-Z4) revealed some InDels and a considerable number of SNPs among them. Most of the pseudogenes were resulted from C to T change, leading to the generation of TAG or TAA in-frame stop codon. The putative proteins of both Gli-At3 and Gli-Z7 genes contained an extra cysteine residue in the unique domain II. Analysis of toxic epitodes among 19 deduced α-gliadins demonstrated that 14 of these contained 1–5 T cell stimulatory toxic epitopes while the other 5 did not contain any toxic epitopes. The glutamine residues in two specific ployglutamine domains ranged from 7 to 27, indicating a high variation in length. According to the numbers of 4 T cell stimulatory toxic epitopes and glutamine residues in the two ployglutamine domains among the 19 α-gliadin genes, 2 were assigned to chromosome 6A, 5 to chromosome 6B and 12 to chromosome 6D. These results were consistent with those from wheat cv. Chinese Spring nulli-tetrasomic and phylogenetic analysis. Secondary structure prediction showed that all α-gliadins had high content of β-strands and most of the α-helixes and β-strands were present in two unique domains. Phylogenetic analysis demonstrated that α-gliadin genes had a high homology with γ-gliadin, B-hordein, and LMW-GS genes and they diverged at approximate 39 MYA. Finally, the five α-gliadin genes were successfully expressed in E. coli, and their expression amount reached to the maximum after 4 h induced by IPTG, indicating that the α-gliadin genes can express in a high level under the control of T₇ promoter.     

Development of a wheat single gene FISH map for analyzing homoeologous relationship and chromosomal rearrangements within the Triticeae

Key message

A cytogenetic map of wheat was constructed using FISH with cDNA probes. FISH markers detected homoeology and chromosomal rearrangements of wild relatives, an important source of genes for wheat improvement.

Abstract

To transfer agronomically important genes from wild relatives to bread wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) by induced homoeologous recombination, it is important to know the chromosomal relationships of the species involved. Fluorescence in situ hybridization (FISH) can be used to study chromosome structure. The genomes of allohexaploid bread wheat and other species from the Triticeae tribe are colinear to some extent, i.e., composed of homoeoloci at similar positions along the chromosomes, and with genic regions being highly conserved. To develop cytogenetic markers specific for genic regions of wheat homoeologs, we selected more than 60 full-length wheat cDNAs using BLAST against mapped expressed sequence tags and used them as FISH probes. Most probes produced signals on all three homoeologous chromosomes at the expected positions. We developed a wheat physical map with several cDNA markers located on each of the 14 homoeologous chromosome arms. The FISH markers confirmed chromosome rearrangements within wheat genomes and were successfully used to study chromosome structure and homoeology in wild Triticeae species. FISH analysis detected 1U-6U chromosome translocation in the genome of Aegilops umbellulata, showed colinearity between chromosome A of Ae. caudata and group-1 wheat chromosomes, and between chromosome arm 7S#3L of Thinopyrum intermedium and the long arm of the group-7 wheat chromosomes.     

节节麦-黑麦双二倍体α醇溶蛋白基因的克隆与序列分析

以人工合成节节麦-黑麦双二倍体基因组DNA为模板,用小麦种子醇溶蛋白保守引物进行PCR扩增,经克隆测序获得了843 ～ 897 bp共15个新的DNA序列(GenBank登录号为:JQ029719,JQ046392～JQ046405),分别编码280 ～298个氨基酸.序列比对结果表明,它们具有α-醇溶蛋白基因的典型结构特点,是α-醇溶蛋白基因家系成员,其中有两个序列为同义突变.利用14个新氨基酸序列与乳糜泻( celiac disease)病人毒性抗原相关序列的比对,发现有8个序列的Glia-α-2和Glia-α-9型抗原序列产生缺失和替换.与来自粗山羊草属和黑麦属的α-醇溶蛋白基因的编码氨基酸建立系统树,结果表明,14个DNA序列编码的氨基酸序列与粗山羊草属的相关序列聚在一起.     

Comparison of Alleles at <Emphasis Type="Italic">Gli-2</Emphasis> Loci of Common Wheat by Means of Two-Dimensional Electrophoresis of Gliadin

Electrophoretic mobility (EM) and molecular weight (MW) of some allelic variants of α- and β-gliadins contrlled by Gli-2 loci were compared by means of two-dimensional (APAGE × SDS) electrophoresis. Comparison of α-gliadins of the alleles Gli-A2b and Gli-A2p, of β-gliadins of the Gli-B2b and Gli-B2c, and of β-gliadins of the Gli-D2b, Gli-D2c, Gli-D2j, and Gli-D2r indicated that a gliadin with lower EM had, as a rule, bigger MW which is known to depend on the length of the polyglutamine domain of gliadin of α-type. However, allelic variants of the α-gliadin encoded by Gli-D2b and Gli-D2e differ in EM but not in apparent MW. It might be caused by a substitution of some charged/uncharged aminoacids in the polypeptide of gliadin. Allele Gli-B2o which is very frequent in up-to-date common wheat germplasm originated probably by means of unequal crossingover. Some alleles at Gli-A2 is found to control completely different blocks of gliadins and therefore might come to common wheat from different genotypes of the polymorphic diploid donor of the A genome. The results indicate that the reason of the known more vast polymorphism of gliadins controlled by Gli-2 loci as compared with Gli-1 loci is the considerable difference of the structure, first, of Gli-1 and Gli-2 loci (Gli-2 loci have more expressed genes per locus) and, second, of genes encoding gliadins of α- and γ-types (α-gliadins are shown to contain a long polyglutamine sequences highly variable in their length).     

Gamma-gliadin specific celiac disease antibodies recognize p31-43 and p57-68 alpha gliadin peptides in deamidation related manner as a result of cross-reaction

Celiac disease (CeD) is a T-cell-dependent enteropathy with autoimmune features where tissue transglutaminase (TG2)-mediated posttranslational modification of gliadin peptides has a decisive role in the pathomechanism. The humoral immune response is reported to target mainly TG2-deamidated γ-gliadin peptides. However, α-gliadin peptides, like p57-68, playing a crucial role in the T-cell response, and p31-43, a major trigger of innate responses, also contain B-cell gliadin epitopes and γ-gliadin like motifs. We aimed to identify if there are anti-gliadin-specific antibodies in CeD patients targeting the p31-43 and p57-68 peptides and to examine whether deamidation of these peptides could increase their antigenicity. We explored TG2-mediated deamidation of the p31-43 and p57-68 peptides, and investigated serum antibody reactivity toward the native and deamidated α and γ-gliadin peptides in children with confirmed CeD and in prospectively followed infants at increased risk for developing CeD. We affinity-purified antibody populations utilizing different single peptide gliadin antigens and tested their binding preferences for cross-reactivity in real-time interaction assays based on bio-layer interferometry. Our results demonstrate that there is serum reactivity toward p31-43 and p57-68 peptides, which is due to cross-reactive γ-gliadin specific antibodies. These γ-gliadin specific antibodies represent the first appearing antibody population in infancy and they dominate the serum reactivity of CeD patients even later on and without preference for deamidation. However, for the homologous epitope sequences in α-gliadins shorter than the core QPEQPFP heptapeptide, deamidation facilitates antibody recognition. These findings reveal the presence of cross-reactive antibodies in CeD patients recognizing the disease-relevant α-gliadins.

     

Identification of novel α-gliadin genes

Ten novel α-gliadin genes (Gli-ta, Gli-turg1, Gli-turg2, Gli-turg3, Gli-turg4, Gli-turg5, Gli-turg6, Gli-cs1, Gli-cs2, and Gli-cs3) with unique characteristics were isolated from wheat (Triticum aestivumL.), among which Gli-cs1, Gli-cs2, Gli-cs3, and Gli-turg6 were pseudogenes. Gli-cs3 and nine other sequences were much larger and smaller, respectively, than the typical α-gliadins. This variation was caused by insertion or deletion of the unique domain I and a polyglutamine region, possibly the result of illegitimate recombination. Consequently, Gli-cs3 contained 10 cysteine residues, whereas there were 2 cysteine residues only in the other nine sequences. Gli-ta/Gli-ta-like α-gliadin genes are normally expressed during the development of seeds. SDS-PAGE analysis showed that in-vitro-expressed Gli-ta could form intermolecular disulphide bonds and could be chain extenders. A protein band similar in size to Gli-ta has been observed in seed extracts, and mass spectrometry results confirm that the band contains small molecular mass α-gliadins, which is a characteristic of the novel α-gliadins. Mass spectrometry results also indicated that the two cysteine residues of Gli-ta/Gli-ta-like proteins participated in the formation of intermolecular disulphide bonds in vivo.     

Development of chromosome 6D-specific markers for α-gliadin genes and their use in assessing dynamic changes at the Gli-2 loci

To develop chromosome 6D-specific point mutation (PM) markers for α-gliadin genes, 79 α-gliadin sequences cloned from Aegilops tauschii and another 40 α-gliadin genes with known chromosome locations were used in multi-sequence alignment and phylogenic analysis. Additional multiple alignment adjustments were performed manually to facilitate discovery of putative chromosome 6D-specific point mutations. A total of 85 PM primers were designed to detect 68 candidate chromosome 6D-specific point mutations. Experimental tests revealed 11 chromosome 6D-specific PM markers by using genomic DNA from homoeologous group 6 nullisomic–tetrasomic lines of Chinese Spring and putative diploid and tetraploid ancestors of hexaploid wheat as PCR templates. Detection of PM markers in one synthetic hexaploid wheat and its parental lines indicated that some α-gliadin genes were lost from Gli-2 loci during the formation of hexaploid wheat by amphidiploidization of the genomes of Triticum turgidum and Ae. tauschii. Detection of these PM markers in Ae. tauschii, T. aestivum and its four subspecies indicated that at least two genetically distinct sources of Ae. tauschii contributed germplasm to the D genome of T. aestivum.     

The Genome of Bread Wheat <Emphasis Type="Italic">Triticum aestivum</Emphasis> L.: Unique Structural and Functional Properties

Polyploidy is the major mechanism of speciation in flowering plants. All genomes of ancient species that are the progenitors of extant plant species experienced polyploidization. Three consecutive stages of polyploidization, i.e., ancient polyploidization, tetra-, and hexaploidization, resulted in the emergence of modern allohexaploid bread wheat Triticum aestivum L. with the BBAADD genome. Polyploidization and subsequent stabilization of the polyploid genome of T. aestivum led, on one hand, to cytological diploidization and, on the other hand, to structural and functional asymmetry of its three subgenomes. In recent years, there has been a sharp increase in the data accumulation on the origin and structure of the bread wheat genomes a result of analysis of genomes and transcripomes of natural and synthetic wheats using modern mapping and sequencing methods. This review provides up-to-date information on the peculiarities of the T. aestivum genome reorganization, which affected its structure and functioning.