Marta Rodríguez-Quijano

Down-regulating γ-gliadins in bread wheat leads to non-specific increases in other gluten proteins and has no major effect on dough gluten strength.

Background Gliadins are a major component of gluten proteins but their role in the mixing of dough is not well understood because their contribution to wheat flour functional properties are not as clear as for the glutenin fraction. Methodology/Principal Findings Transgenic lines of bread wheat with γ-gliadins suppressed by RNAi are reported. The effects on the gluten protein composition and on technological properties of flour were analyzed by RP-HPLC, by sodium dodecyl sulfate sedimentation (SDSS) test and by Mixograph analysis. The silencing of γ-gliadins by RNAi in wheat lines results in an increase in content of all other gluten proteins. Despite the gluten proteins compensation, in silico analysis of amino acid content showed no difference in the γ-gliadins silenced lines. The SDSS test and Mixograph parameters were slightly affected by the suppression of γ-gliadins. Conclusions/Significance Therefore, it is concluded that γ-gliadins do not have an essential functional contribution to the bread-making quality of wheat dough, and their role can be replaced by other gluten proteins.

One Hundred Years of Grain Omics: Identifying the Glutens That Feed the World.

Glutens, the storage proteins in wheat grains, are a major source of protein in human nutrition. The protein composition of wheat has therefore been an important focus of cereal research. Proteomic tools have been used to describe the genetic diversity of wheat germplasms from different origins at the level of polymorphisms in alleles encoding glutenin and gliadin, the two main proteins of gluten. More recently, proteomics has been used to understand the impact of specific gluten proteins on wheat quality. Here we review the impact of proteomics on the study of gluten proteins as it has evolved from fractionation and electrophoretic techniques to advanced mass spectrometry. In the postgenome era, proteomics is proving to be essential in the effort to identify and understand the interactions between different gluten proteins. This is helping to fill in gaps in our knowledge of how the technological quality of wheat is determined by the interaction between genotype and environment. We also collate information on the various storage protein alleles identified and their prevalence, which makes it possible to infer the effects of wheat selection on grain protein content. We conclude by reviewing the more recent use of transgenesis aimed at improving the quality of gluten.

Validation of microsatellite markers for cytotype discrimination in the model grass Brachypodium distachyon

Brachypodium distachyon (L.) P. Beauv. (2n = 2x = 10) is a small annual grass species where the existence of three different cytotypes (10, 20, and 30 chromosomes) has long been regarded as a case of autopolyploid series with x = 5. However, it has been demonstrated that the cytotypes assumed to be polyploids represent two separate Brachypodium species recently named as Brachypodium stacei (2n = 2x = 20) and Brachypodium hybridum (2n = 4x = 30). The aim of this study was to find a PCR-based alternative approach that could replace standard cytotyping methods (i.e., chromosome counting and flow cytometry) to characterize each of the three Brachypodium species. We have analyzed with four microsatellite (SSR) markers 83 B. distachyon-type lines from varied locations in Spain, including the Balearic and Canary Islands. Within this set of lines, 64, 4, and 15 had 10, 20, and 30 chromosomes, respectively. The surveyed markers produced cytotype-specific SSR profiles. So, a single amplification product was generated in the diploid samples, with nonoverlapping allelic ranges between the 2n = 10 and 2n = 20 cytotypes, whereas two bands, one in the size range of each of the diploid cytotypes, were amplified in the 2n = 30 lines. Furthermore, the remarkable size difference obtained with the SSR ALB165 allowed the identification of the Brachypodium species by simple agarose gel electrophoresis.

Polymorphism, variation and genetic identity of Spanish common wheat germplasm based on gliadin alleles

Gliadin analysis has shown that Spanish common wheat germplasm is highly polymorphic and rather unique. In total, 81 different alleles, 70 of them catalogued, were identified in the 52 Spanish landraces studied, and 25 new gliadin alleles, not present in germplasm from other countries, were found. Eleven alleles were identified at the Gli-A1 locus, 14 at the Gli-B1 ,9a t the Gli-D1, 17 at the Gli-A2, 14 at the Gli-B2 and 16 at the Gli-D2. Comparison of gliadin alleles among varieties released before and after 1966 revealed significant differences for 46 gliadin alleles. Forty-six alleles (36 of them catalogued) present in the material developed before 1966 were lost. In contrast, 25 alleles (23 of them catalogued) appear for the first time in the genepool bred after 1966. On the other hand, most of the Gli alleles frequent in recent Spanish cultivars were already present in Spanish landraces. In general, the results showed that Spanish varieties have suffered qualitative alterations of genetic variation during the last 35 years mainly due to changes in breeding goals and crosses with foreign germplasm. Seventy-five percent of the landraces analysed were shown to have maintained their genetic identity in the genebank although they have been regenerated several times during the last century. However, mistakes in three accessions modified 10 alleles (allele losses or presence of new alleles), and error in one suggests alteration of five alleles. These results demonstrate that great care during maintenance and handling of samples is an important requirement in conserving germplasm collections. Gliadin alleles allowed us to identify 48 different genotypes, some accession duplicates and one intermixing. Also, several mistakes could be resolved using gliadins as genetic markers. # 2002 Elsevier Science B.V. All rights reserved.

Assessment of storage protein variation in relation to some morphological characters in a sample of Spanish landraces of common wheat (Triticum aestivum L. ssp. aestivum)

Fifty nine Spanish landraces of common wheat belonging to differentagrotypes were evaluated with four morphological spike characters and 11 seedprotein loci in order to study the level of agreement between the agrotypeclassification and the morphological and biochemical variation of the wheats. Asa result of the work 22 new gliadin alleles were found and 12 of them werecatalogued. Multivariate and cluster analysis performed on the two sets of data(morphological plus biochemical and biochemical only) showed that there was aclear relationship between seed protein composition and the wheat classificationfor the agrotypes ‘Barbilla’, ‘Candeal’ and‘Negrete’. For the other groups, agrotype classification was notconsistent with the variation for biochemical characters although, some allelesin common were found except for the ‘Jeja’ type. Morphologicaltraits revealed that they could be useful as first step in discriminating amongagrotypes. In contrast, biochemical markers showed that genetic variation amongaccessions was higher than among agrotypes. The protein lociGli-B1,Gli-A2,Gli-D2 andGli-A1 were shown to be the mostpolymorphic and useful in discriminating between the accessions studied.

New insights into wheat toxicity: Breeding did not seem to contribute to a prevalence of potential celiac disease’s immunostimulatory epitopes

Gluten proteins, namely gliadins, are the primary trigger of the abnormal immune response in celiac disease. It has been hypothesised that modern wheat breeding practices may have contributed to the increase in celiac disease prevalence during the latter half of the 20th century. Our results do not support this hypothesis as Triticum aestivum spp. vulgare landraces, which were not subjected to breeding practices, presented higher amounts of potential celiac diseases immunostimulatory epitopes when compared to modern varieties. Furthermore, high variation between wheat varieties concerning the toxic epitopes amount was observed. We carried out quantitative analysis of gliadin types by RP-HPLC to verify its correlation with the amount of toxic epitopes: ω-type gliadins content explain about 40% of the variation of toxic epitopes in tetraploid wheat varieties. This research provides new insights regarding wheat toxicity and into the controversial idea that human practices may have conducted to an increased exposure to toxic epitopes.

Efficient chemo-enzymatic gluten detoxification: reducing toxic epitopes for celiac patients improving functional properties

Protein engineering of gluten, the exogenous effector in celiac disease, seeking its detoxification by selective chemical modification of toxic epitopes is a very attractive strategy and promising technology when compared to pharmacological treatment or genetic engineering of wheat. Here we present a simple and efficient chemo-enzymatic methodology that decreases celiac disease toxic epitopes of gluten proteins improving its technological value through microbial transglutaminase-mediated transamidation of glutamine with n-butylamine under reducing conditions. First, we found that using low concentrations of amine-nucleophile under non-reducing conditions, the decrease in toxic epitopes is mainly due to transglutaminase-mediated cross-linking. Second, using high amine nucleophile concentrations protein cross-linking is substantially reduced. Third, reducing conditions increase 7-fold the transamidation reaction further decreasing toxic epitopes amount. Fourth, using n-butylamine improves gluten hydrophobicity that strengthens the gluten network. These results open the possibility of tailoring gluten for producing hypoallergenic flours while still taking advantage of the unique viscoelastic properties of gluten.

Genetic variability for waxy genes in Argentinean bread wheat germplasm

Amylose and amylopectin are the two polysaccharides that constitute starch in bread wheat and the enzyme GBSSI ( Granule-bound starch synthase I), also known as waxy protein, is responsible for amylose synthesis in storage tissues. Decrease of the amylose content in starch has been associated with the lack of waxy protein(s). In this work, different sets of PCR markers were used to characterize the genetic variability of waxy loci from 103 Argentinean bread wheat cultivars. For the Wx-A1 locus, Wx-A1a and a novel molecular allele designed Wx-A1g were detected. Wx-B1 locus showed three alleles ( Wx-B1a , Wx-B1b, Wx-B1e ), and Wx-D1 locus showed only the Wx-D1a allele. Novel single-locus allele specific markers for Wx-A1b, Wx-B1b and Wx-D1b null alleles were also described. To our best knowledge this is the first study focused to characterize the genetic variability for waxy genes in bread wheat cultivars from South America.

Comparison of alleles at the Gli-1 loci of common wheat by means of two-dimensional electrophoresis of gliadin and RFLP analysis

Allelic variants of the Gli-1 locus is known to control groups (blocks) of gliadin polypeptides (gliadins). Some allelic variants of blocks that differ in the electrophoretic (acid gel) mobility (EM) of only one gliadin of the block were compared using two-dimensional electrophoresis (SDS-PAGE) and the RFLP procedure. It was found that, in these pairs of similar alleles (Gli-B1f, Gli-B1s, and Gli-D1a as compared with Gli-B1e, Gli-B1n, and Gli-D1c, respectively), faster γ-gliadin had smaller molecular weight (MW). Alleles at the Gli-A1 locus (Gli-A1j, Gli-A1i, Gli-A1a, Gli-A1k, and Gli-A1f) differ in the EM of the γ-gliadin so that Gli-A1j controls the slowest γ-gliadin and Gli-A1f controls the fastest one. We found that, in this order of alleles, faster γ-gliadin always had smaller MW. It was suggested that similar alleles might arise from one another by spontaneous mutations changing the number of repeating sequences or length of the polyglutamine domain present in the γ-gliadin gene thereby influencing MW and EM of encoding polypeptide. Other mechanisms of the mutational appearance of new alleles were found earlier by comparison of allele pairs: Gli-D1a and Gli-D1k (gene silencing) and Gli-D1b and Gli-D1d (gene amplification). We discovered contrasting families of alleles at the Gli-B1 and at the Gli-D1 loci and also two variants of apparently the same allele Gli-D1a that differed in the number of encoded ω-gliadins. Families of alleles at one locus of T. aestivum might inherit from different genotypes of corresponding diploid donor, as we suggested earlier.

Effect of allelic variation at glutenin and puroindoline loci on bread-making quality: favorable combinations occur in less toxic varieties of wheat for celiac patients

Genetically diverse wheat samples, twenty-seven Triticum aestivum L. varieties, grown in two environments (Portugal and Spain) were analyzed for their allelic composition in high-molecular-weight glutenin subunits (HMW-GS), low-molecular-weight glutenin subunits (LMW-GS) and puroindolines, as well as their protein content, hardness, sodium dodecyl sulfate-sedimentation (SDS-S), mixograph mixing time and breakdown resistance (MT and BDR, respectively) parameters, and R5 reactivity. The environment showed significant effect on protein content, SDS-S and BDR parameters. In relation to HMW-GS quality effect, the allelic composition Glu-A1d, Glu-B1al, Glu-D1d presented the best results. From the complex Glu-3 loci (LMW-GS), only Glu-B3 locus showed a significant effect on the quality parameters. The Glu-B3ab allele is desirable considering the higher mean values for SDS-S, MT and hardness, and the lower mean values for BDR. Regarding puroindolines, Pina-D1a, Pinb-D1c allelic composition (Leu to Pro at position +60) showed the best quality potential. In addition, we found negative (SDS-S and MT) and positive (BDR) significant correlations between quality parameters and the amount of potential celiac disease toxic epitopes, suggesting that wheat breeding aiming at quality does not have a negative impact on wheat toxicity and on the other hand, emphasizes the need for a more comprehensive wheat breeding programs that encompass celiac disease problematic.