Dataset Information

Elimination of Omega-1,2 Gliadins From Bread Wheat (Triticum aestivum) Flour: Effects on Immunogenic Potential and End-Use Quality.

ABSTRACT: The omega-1,2 gliadins are a group of wheat gluten proteins that contain immunodominant epitopes for celiac disease (CD) and also have been associated with food allergies. To reduce the levels of these proteins in the flour, bread wheat (Triticum aestivum cv. Butte 86) was genetically transformed with an RNA interference plasmid that targeted a 141 bp region at the 5' end of an omega-1,2 gliadin gene. Flour proteins from two transgenic lines were analyzed in detail by quantitative two-dimensional gel electrophoresis and tandem mass spectrometry. In one line, the omega-1,2 gliadins were missing with few other changes in the proteome. In the other line, striking changes in the proteome were observed and nearly all gliadins and low molecular weight glutenin subunits (LMW-GS) were absent. High molecular weight glutenin subunits (HMW-GS) increased in this line and those that showed the largest increases had molecular weights slightly less than those in the non-transgenic, possibly due to post-translational processing. In addition, there were increases in non-gluten proteins such as triticins, purinins, globulins, serpins, and alpha-amylase/protease inhibitors. Reactivity of flour proteins with serum IgG and IgA antibodies from a cohort of CD patients was reduced significantly in both transgenic lines. Both mixing time and tolerance were improved in the line without omega-1,2 gliadins while mixing properties were diminished in the line missing most gluten proteins. The data suggest that biotechnology approaches may be used to create wheat lines with reduced immunogenic potential in the context of gluten sensitivity without compromising end-use quality.

SUBMITTER: Altenbach SB

PROVIDER: S-EPMC6521778 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Elimination of Omega-1,2 Gliadins From Bread Wheat (<i>Triticum aestivum</i>) Flour: Effects on Immunogenic Potential and End-Use Quality.

Altenbach Susan B SB Chang Han-Chang HC Yu Xuechen B XB Seabourn Bradford W BW Green Peter H PH Alaedini Armin A

Frontiers in plant science 20190509

The omega-1,2 gliadins are a group of wheat gluten proteins that contain immunodominant epitopes for celiac disease (CD) and also have been associated with food allergies. To reduce the levels of these proteins in the flour, bread wheat (<i>Triticum aestivum</i> cv. Butte 86) was genetically transformed with an RNA interference plasmid that targeted a 141 bp region at the 5' end of an omega-1,2 gliadin gene. Flour proteins from two transgenic lines were analyzed in detail by quantitative two-dim ...[more]

PMID: 31143195

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Project description:Background and aimsGluten proteins are the major storage protein fraction in the mature wheat grain. They are restricted to the starchy endosperm, which forms white flour on milling, and interact during grain development to form large polymers which form a continuous proteinaceous network when flour is mixed with water to give dough. This network confers viscosity and elasticity to the dough, enabling the production of leavened products. The starchy endosperm is not a homogeneous tissue and quantitative and qualitative gradients exist for the major components: protein, starch and cell wall polysaccharides. Gradients in protein content and composition are the most evident and are of particular interest because of the major role played by the gluten proteins in determining grain processing quality.MethodsProtein gradients in the starchy endosperm were investigated using antibodies for specific gluten protein types for immunolocalization in developing grains and for western blot analysis of protein extracts from flour fractions obtained by sequential abrasion (pearling) to prepare tissue layers.Key resultsDifferential patterns of distribution were found for the high-molecular-weight subunits of glutenin (HMW-GS) and γ-gliadins when compared with the low-molecular-weight subunits of glutenin (LMW-GS), ω- and α-gliadins. The first two types of gluten protein are more abundant in the inner endosperm layers and the latter more abundant in the subaleurone. Immunolocalization also showed that segregation of gluten proteins occurs both between and within protein bodies during protein deposition and may still be retained in the mature grain.ConclusionsQuantitative and qualitative gradients in gluten protein composition are established during grain development. These gradients may be due to the origin of subaleurone cells, which unlike other starchy endosperm cells derive from the re-differentiation of aleurone cells, but could also result from the action of specific regulatory signals produced by the maternal tissue on specific domains of the gluten protein gene promoters.

Project description:BACKGROUND:Omega-5 gliadins are a group of highly repetitive gluten proteins in wheat flour encoded on the 1B chromosome of hexaploid wheat. These proteins are the major sensitizing allergens in a severe form of food allergy called wheat-dependent exercise-induced anaphylaxis (WDEIA). The elimination of omega-5 gliadins from wheat flour through biotechnology or breeding approaches could reduce the immunogenic potential and adverse health effects of the flour. RESULTS:A mutant line missing low-molecular weight glutenin subunits encoded at the Glu-B3 locus was selected previously from a doubled haploid population generated from two Korean wheat cultivars. Analysis of flour from the mutant line by 2-dimensional gel electrophoresis coupled with tandem mass spectrometry revealed that the omega-5 gliadins and several gamma gliadins encoded by the closely linked Gli-B1 locus were also missing as a result of a deletion of at least 5.8 Mb of chromosome 1B. Two-dimensional immunoblot analysis of flour proteins using sera from WDEIA patients showed reduced IgE reactivity in the mutant relative to the parental lines due to the absence of the major omega-5 gliadins. However, two minor proteins showed strong reactivity to patient sera in both the parental and the mutant lines and also reacted with a monoclonal antibody against omega-5 gliadin. Analysis of the two minor reactive proteins by mass spectrometry revealed that both proteins correspond to omega-5 gliadin genes encoded on chromosome 1D that were thought previously to be pseudogenes. CONCLUSIONS:While breeding approaches can be used to reduce the levels of the highly immunogenic omega-5 gliadins in wheat flour, these approaches are complicated by the genetic linkage of different classes of gluten protein genes and the finding that omega-5 gliadins may be encoded on more than one chromosome. The work illustrates the importance of detailed knowledge about the genomic regions harboring the major gluten protein genes in individual wheat cultivars for future efforts aimed at reducing the immunogenic potential of wheat flour.

Project description:BackgroundThe analyses of protein synthesis, accumulation and regulation during grain development in wheat are more complex because of its larger genome size compared to model plants such as Arabidopsis and rice. In this study, grains from two wheat cultivars Jimai 20 and Zhoumai 16 with different gluten quality properties were harvested at five development stages, and were used to displayed variable expression patterns of grain proteins.ResultsProteome characterization during grain development in Chinese bread wheat cultivars Jimai 20 and Zhoumai 16 with different quality properties was investigated by 2-DE and tandem MALDI-TOF/TOF-MS. Identification of 117 differentially accumulated protein spots representing 82 unique proteins and five main expression patterns enabled a chronological description of wheat grain formation. Significant proteome expression differences between the two cultivars were found; these included 14 protein spots that accumulated in both cultivars but with different patterns and 27 cultivar-different spots. Among the cultivar-different protein spots, 14 accumulated in higher abundance in Jimai 20 than in Zhoumai 16, and included NAD-dependent isocitrate dehydrogenase, triticin precursor, LMW-s glutenin subunit and replication factor C-like protein. These proteins are likely to be associated with superior gluten quality. In addition, some proteins such as class II chitinase and peroxidase 1 with isoforms in developing grains were shown to be phosphorylated by Pro-Q Diamond staining and phosphorprotein site prediction. Phosphorylation could have important roles in wheat grain development. qRT-PCR analysis demonstrated that transcriptional and translational expression patterns of many genes were significantly different.ConclusionsWheat grain proteins displayed variable expression patterns at different developmental stages and a considerable number of protein spots showed differential accumulation between two cultivars. Differences in seed storage proteins were considered to be related to different quality performance of the flour from these wheat cultivars. Some proteins with isoforms were phosphorylated, and this may reflect their importance in grain development. Our results provide new insights into proteome characterization during grain development in different wheat genotypes.

Project description:Vitamin B9, particularly folic acid, is an essential molecule for human health. Wheat flour is one of the major sources of calorie intake by humans. The selection of folate-rich genotypes in wheat breeding can enhance the natural folate value in the daily diet. This study used a precise, high-performance liquid chromatography (HPLC) assay to analyze folate content in a 262-accession Chinese wheat mini-core collection (MCC) grown under three environments. Four folate derivatives in grains including tetrahydrofolate (THF), 5-methyltetrahydrofolate (5-CH3-THF), 5-formyltetrahydrofolate (5-CHO-THF), and 5,10-methenyltetrahydrofolate (5,10-CH+THF) were considered. An association analysis of water regimes, accession types, released years, geographical origin, and agronomic traits with folate content was conducted for the first time. There was a large amount of variation in folate content in the analyzed accessions, with genotype identified as the main influencing factor. Total folate content was significantly correlated with the content of the four MCC derivatives under the three environments. 5-CH3-THF and 5-CHO-THF were the most abundant among the four folate derivatives and were positively correlated with high folate content. The 12 accessions with the highest folate content showed an average of more than 80 μg/100 g. The analysis demonstrated that this Chinese wheat had not undergone extensive selection for folate content during breeding, which is unrelated to the geographical origin, accession types, winter/spring types, and grain colors of wheat. The content of THF, 5-CH3-THF, and 5,10-CH+THF was significantly negatively correlated with grain width, grain thickness, and thousand kernel weight. A relatively weak negative relationship manifested between folate contents and flowering date, whereas no significant correlation with tiller number, grain number per spike, maturity date, height, and spike length was detected. The investigation benefits wheat breeders for folate enhancement.

Project description:BACKGROUND:Celiac disease (CD) is caused by specific sequences of gluten proteins found in cereals such as bread wheat (Triticum aestivum ssp. aestivum) and spelt (T. aestivum ssp. spelta). Among them, the ?-gliadins display the highest immunogenicity, with four T-cell stimulatory epitopes. The toxicity of each epitope sequence can be reduced or even suppressed according to the allelic form of each sequence. One way to address the CD problem would be to make use of this allelic variability in breeding programs to develop safe varieties, but tools to track the presence of toxic epitopes are required. The objective of this study was to develop a tool to accurately detect and quantify the immunogenic content of expressed ?-gliadins of spelt and bread wheat. RESULTS:Four TaqMan probes that only hybridize to the canonical-i.e. toxic-form of each of the four epitopes were developed and their specificity was demonstrated. Six TaqMan probes targeting stable reference genes were also developed and constitute a tool to normalize qPCR data. The probes were used to measure the epitope expression levels of 11 contrasted spelt accessions and three ancestral diploid accessions of bread wheat and spelt. A high expression variability was highlighted among epitopes and among accessions, especially in Asian spelts, which showed lower epitope expression levels than the other spelts. Some discrepancies were identified between the canonical epitope expression level and the global amount of expressed ?-gliadins, which makes the designed TaqMan probes a useful tool to quantify the immunogenic potential independently of the global amount of expressed ?-gliadins. CONCLUSIONS:The results obtained in this study provide useful tools to study the immunogenic potential of expressed ?-gliadin sequences from Triticeae accessions such as spelt and bread wheat. The application of the designed probes to contrasted spelt accessions revealed a high variability and interesting low canonical epitope expression levels in the Asian spelt accessions studied.

Dataset Information

Elimination of Omega-1,2 Gliadins From Bread Wheat (Triticum aestivum) Flour: Effects on Immunogenic Potential and End-Use Quality.

Publications

Elimination of Omega-1,2 Gliadins From Bread Wheat (<i>Triticum aestivum</i>) Flour: Effects on Immunogenic Potential and End-Use Quality.

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