Polymorphism and inheritance of gliadin polypeptides in T. monococcum L.

Summary More than 80 different gliadin electrophoretic patterns (spectra) have been found in 109 accessions of the diploid wheat Triticum monococcum. Each pattern consists of 15–20 gliadin bands. Some patterns are clearly related and might arise from one another through single mutations in the gliadin-coding loci. From the analysis of 15 grains of each, only 61 accessions were found to be uniform; others consisted of two or more grain variants differing in their gliadin spectrum. An analysis of F₂ grains from three crosses between different accessions showed that groups (blocks) of components are jointly and codominantly inherited. Two independent major Gli loci were established. The close resemblance of the composition of some blocks of T. monococcum to some of those in polyploid wheats indicates that one locus in each T. monococcum genotype is located on chromosome 1A (Gli-A1) and the other on 6A (Gli-A2). However, the blocks of T. monococcum include more bands than corresponding (equivalent) blocks of polyploid wheats. Two out of 275 F₂ grains of the cross k-14244 x k-20409 were found to have gliadin spectra which can be explained as a result of intralocus recombination. Also, a second gliadin-coding locus on chromosome 1A was found in the cross k-46140 x k-46753. This locus recombines with the main Gli-A1 locus with a frequency of about 22% and was clearly analogous to the additional Gli locus found earlier on chromosome 1A of certain polyploid wheats.     

Use of selected enterococci and Rhizopus oryzae proteases to hydrolyse wheat proteins responsible for celiac disease

Aims:  This work aimed at using a pool of selected enterococci and fungal proteases to hydrolyse wheat gluten during long-time fermentation.
Methods and Results:  A liquid dough made with wheat flour (20% w/w) was fermented with three Enterococcus strains (dough A) or with the combination of enterococci and Rhizopus oryzae proteases (dough B). After 48 h of fermentation, dough A and B had a concentration of water-soluble peptides approximately threefold higher than the chemically acidified dough (CAD), used as the control. The same was found for the concentration of free amino acids, being higher in dough B with respect to dough A. SDS-PAGE analysis showed that albumin and glutenin fractions were partially hydrolysed, while gliadins almost disappeared in dough A and B, as confirmed by two-dimensional electrophoresis, RP-HPLC and R5-ELISA analyses.
Conclusions:  The combined use of enterococci and fungal proteases showed a decrease of the gluten concentration of more than 98% during long-time fermentation.
Significance and Impact of the Study:  The use of the mixture of selected enterococci and R. oryzae proteases should be considered as a potential tool to decrease gluten concentration in foods.     

Low‐gluten,nontransgenic wheat engineered with CRISPR/Cas9

Coeliac disease is an autoimmune disorder triggered in genetically predisposed individuals by the ingestion of gluten proteins from wheat, barley and rye. The α‐gliadin gene family of wheat contains four highly stimulatory peptides, of which the 33‐mer is the main immunodominant peptide in patients with coeliac. We designed two sgRNAs to target a conserved region adjacent to the coding sequence for the 33‐mer in the α‐gliadin genes. Twenty‐one mutant lines were generated, all showing strong reduction in α‐gliadins. Up to 35 different genes were mutated in one of the lines of the 45 different genes identified in the wild type, while immunoreactivity was reduced by 85%. Transgene‐free lines were identified, and no off‐target mutations have been detected in any of the potential targets. The low‐gluten, transgene‐free wheat lines described here could be used to produce low‐gluten foodstuff and serve as source material to introgress this trait into elite wheat varieties.     

Effects of an Agropyron chromosome on endosperm proteins in common wheat (Triticum aestivum L.)

An Agropyron chromosome having a gene conferring blue color on the aleurone layer of the kernel endosperm causes a 15% increase in total grain protein content when it is added to the common wheat (2n=42) complement. In contrast, there is no effect of this chromosome on total protein content if it replaced part of a wheat chromosome. Endosperm protein components of isolines having blue aleurone due to the Agropyron chromosome being added (2n=44) or translocated (2n=42) were compared to normal nonblue isoline counterparts. Gliadin proteins separated by aluminum lactate (pH 3.2) polyacrylamide gel electrophoresis (PAGE) in one or two dimensions showed greater staining intensity for the blue addition isolines (2n=44) than nonblue (2n=42) isolines. However, the 42-chromosome blue isoline did not show increased protein staining over the nonblue isoline, but at least five protein differences were detected between the lines. SDS-PAGE showed that blue and nonblue differences were expressed primarily in the gliadins, but also in the glutenin, globulin, and albumin proteins.This research was supported by a D. F. Jones Postdoctoral Fellowship to K. M. Soliman and by Western Regional Project W-132, Genotype-environment interactions related to end-product uses in small grains.     

Identification and DNA sequence analysis of a γ-type gliadin cDNA plasmid from winter wheat

Summary Recombinant cDNA plasmids possessing the coding sequences for the -type gliadins were isolated from a cDNA library prepared from wheat seed poly (A⁺) RNA. One of these plasmids, pGliB48, specifically hybridizes to poly (A⁺) RNA molecules 1 400–1 500 bases in length that direct the synthesis of polypeptides at 38 Kd and 46 Kd, the latter size characteristic of the -type gliadins. The cDNA sequence of pGliB48 was determined and encompasses the 3 untranslated region as well as 245 amino acids from the C-terminus of the -type gliadin polypeptide. The 5-end of the DNA coding sequence consists of a tandem repeat unit composed of eight amino acids. Localized regions of homology are observed for the /-type and -type gliadin cDNA sequences.     

Wheat protein inhibitors of α-amylase

The location in the seed, molecular properties and biological role of protein α-amylase inhibitors from wheat are discussed. Inhibition specificity of albumin inhibitors and structural features essential for interaction with inhibited amylases are also examined. The possible significance of these naturally occurring inhibitors in relation to their presence in foods in active form is described. Finally, genetic aspects of the albumin inhibitor production and the possibility of improving nutritional value and insect re     

Biochemical and molecular characterization of gliadins

Gliadins account for about 40–50% of the total proteins in wheat seeds and play an important role in the nutritional and processing quality of flour. Usually, gliadins can be divided into α-(α/β), γ-, and ω-groups, whereas the low-molecular-weight (LMW) gliadins are novel seed storage proteins. The low-molecular-weight glutenin subunits (LMW-GSs) are also designated as gliadins in a few publications. The genes encoding gliadins are mainly located on the short arms of group 6 and group 1 chromosomes, and not evenly distributed. Repetitive sequences cover most of the uncoding regions, which attributed greatly to the evolution of wheat genome. The primary structure of each gliadin is divided into several domains, and the long repetitive domains consist of peptide motifs. Conserved cysteine residues mainly form intramolecular disulfide bonds. The rare potential intermolecular disulfide bonds and the long repetitive domains play an important role in the quality of wheat flour. There is a general idea that gliadin genes, even prolamin genes, have a common origin and subsequent divergence leads to gene polymorphism. The γ-gliadins are considered to be the most ancient of the wheat prolamin family. Several elements in the 5′-flanking (e.g., CAAT and TATA box) and the 3′-flanking sequences have been detected, which has been shown to be necessary for the proper expression of gliadins. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 5, pp. 796–807. The text was submitted by the authors in English.     

The wheat ω-gliadin genes: structure and EST analysis

A survey and analysis is made of all available ω-gliadin DNA sequences including ω-gliadin genes within a large genomic clone, previously reported gene sequences, and ESTs identified from the large wheat EST collection. A contiguous portion of the Gli-B3 locus is shown to contain two apparently active ω-gliadin genes, two pseudogenes, and four fragments of the 3′ portion of ω-gliadin sequences. Comparison of ω-gliadin sequences allows a phylogenetic picture of their relationships and genomes of origin. Results show three groupings of ω-gliadin active gene sequences assigned to each of the three hexaploid wheat genomes, and a fourth group thus far consisting of pseudogenes assigned to the A-genome. Analysis of ω-gliadin ESTs allows reconstruction of two full-length model sequences encoding the AREL- and ARQL-type proteins from the Gli-A3 and Gli-D3 loci, respectively. There is no DNA evidence of multiple active genes from these two loci. In contrast, ESTs allow identification of at least three to four distinct active genes at the Gli-B3 locus of some cultivars. Additional results include more information on the position of cysteines in some ω-gliadin genes and discussion of problems in studying the ω-gliadin gene family. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Screening of high-quality bread wheat dihaploid lines by the use of biochemical markers

This work was undertaken to evaluate the storage protein composition of 13 doubled haploid bread wheat (Triticum aestivum L.) lines in order to identify those carrying promising alleles. For this, 15–20 seeds per each doubled haploid line were used to determine alleles at the loci for high-molecular-weight glutenin subunits and gliadins. Acid polyacrylamide gel electrophoresis was applied to identify glianin alleles, whereas SDS electrophoresis was used in the case of high-molecular-weight glutenin subunits. The identification of doubled haploid lines (DHLs) with respect to their storage protein composition enabled the classification of the DHLs derived from the cross Acheloos × Vergina in four different classes. Furthermore, protein composition analysis revealed that DHLs derived from the F₁ Penios × Kavkaz were identical. In addition, these lines were found to carry 1BL/1RS translocation, which is associated with high yield potential and resistance to biotic and abiotic stress conditions. Finally, it was observed that, in two cases, one rare biotype of the parental varieties was involved in the crosses. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 3, pp. 444–448. The text was submitted by the authors in English.