María J. Giménez

Molecular and Immunological Characterization of Gluten Proteins Isolated from Oat Cultivars That Differ in Toxicity for Celiac Disease

A strict gluten-free diet (GFD) is the only currently available therapeutic treatment for patients with celiac disease (CD). Traditionally, treatment with a GFD has excluded wheat, barley and rye, while the presence of oats is a subject of debate. The most-recent research indicates that some cultivars of oats can be a safe part of a GFD. In order to elucidate the toxicity of the prolamins from oat varieties with low, medium, and high CD toxicity, the avenin genes of these varieties were cloned and sequenced, and their expression quantified throughout the grain development. At the protein level, we have accomplished an exhaustive characterization and quantification of avenins by RP-HPLC and an analysis of immunogenicity of peptides present in prolamins of different oat cultivars. Avenin sequences were classified into three different groups, which have homology with S-rich prolamins of Triticeae. Avenin proteins presented a lower proline content than that of wheat gliadin; this may contribute to the low toxicity shown by oat avenins. The expression of avenin genes throughout the development stages has shown a pattern similar to that of prolamins of wheat and barley. RP-HPLC chromatograms showed protein peaks in the alcohol-soluble and reduced-soluble fractions. Therefore, oat grains had both monomeric and polymeric avenins, termed in this paper gliadin- and glutenin-like avenins. We found a direct correlation between the immunogenicity of the different oat varieties and the presence of the specific peptides with a higher/lower potential immunotoxicity. The specific peptides from the oat variety with the highest toxicity have shown a higher potential immunotoxicity. These results suggest that there is wide range of variation of potential immunotoxicity of oat cultivars that could be due to differences in the degree of immunogenicity in their sequences.

Assessment of candidate reference genes for expression studies in Vicia faba L. by real-time quantitative PCR

Faba bean (Vicia faba L.) cultivation has declined in recent years due to several factors, including diseases and anti-nutritional compounds in the seeds. The introduction of disease resistance and the elimination of anti-nutritional factors in new varieties are important objectives in any breeding program for the species. Because of the faba bean’s huge genome, it is necessary to rely on synteny with related species in order to identify candidate genes responsible for the character under study. Quantification of expression level of candidate genes could help to validate them. Appropriate normalization is an essential prerequisite for obtaining accurate and reproducible quantification of gene expression level. Real-time quantitative PCR was used for evaluate the expression stability of 11 candidate reference genes. A wide set of samples, including different tissues, genotypes and several inoculations for the most important pathogens were employed. The expression stability of the candidate genes was analyzed using two different algorithms, geNorm and NormFinder, and results obtained from both algorithms were highly correlated for each experimental set. In all cases, either ACT1, CYP2 or ELF1A genes performed as the most stable genes in our experimental sets. They also represent part of the best combination of genes according to the geNorm and NormFinder algorithms. Our data showed the wide expression range of the selected genes, confirming that no single reference gene had a stable expression under these conditions in the faba bean. We recommend the use of ACT1, CYP2 and ELF1A as the most suitable reference genes to normalize gene expression for future studies in V. faba.

Diversification of the celiac disease α‐gliadin complex in wheat: a 33‐mer peptide with six overlapping epitopes, evolved following polyploidization

The gluten proteins from wheat, barley and rye are responsible both for celiac disease (CD) and for non-celiac gluten sensitivity, two pathologies affecting up to 6-8% of the human population worldwide. The wheat α-gliadin proteins contain three major CD immunogenic peptides: p31-43, which induces the innate immune response; the 33-mer, formed by six overlapping copies of three highly stimulatory epitopes; and an additional DQ2.5-glia-α3 epitope which partially overlaps with the 33-mer. Next-generation sequencing (NGS) and Sanger sequencing of α-gliadin genes from diploid and polyploid wheat provided six types of α-gliadins (named 1-6) with strong differences in their frequencies in diploid and polyploid wheat, and in the presence and abundance of these CD immunogenic peptides. Immunogenic variants of the p31-43 peptide were found in most of the α-gliadins. Variants of the DQ2.5-glia-α3 epitope were associated with specific types of α-gliadins. Remarkably, only type 1 α-gliadins contained 33-mer epitopes. Moreover, the full immunodominant 33-mer fragment was only present in hexaploid wheat at low abundance, probably as the result of allohexaploidization events from subtype 1.2 α-gliadins found only in Aegilops tauschii, the D-genome donor of hexaploid wheat. Type 3 α-gliadins seem to be the ancestral type as they are found in most of the α-gliadin-expressing Triticeae species. These findings are important for reducing the incidence of CD by the breeding/selection of wheat varieties with low stimulatory capacity of T cells. Moreover, advanced genome-editing techniques (TALENs, CRISPR) will be easier to implement on the small group of α-gliadins containing only immunogenic peptides.

Targeting of prolamins by RNAi in bread wheat: effectiveness of seven silencing‐fragment combinations for obtaining lines devoid of coeliac disease epitopes from highly immunogenic gliadins

Gluten proteins are responsible for the viscoelastic properties of wheat flour but also for triggering pathologies in susceptible individuals, of which coeliac disease (CD) and noncoeliac gluten sensitivity may affect up to 8% of the population. The only effective treatment for affected persons is a strict gluten-free diet. Here, we report the effectiveness of seven plasmid combinations, encompassing RNAi fragments from α-, γ-, ω-gliadins, and LMW glutenin subunits, for silencing the expression of different prolamin fractions. Silencing patterns of transgenic lines were analysed by gel electrophoresis, RP-HPLC and mass spectrometry (LC-MS/MS), whereas gluten immunogenicity was assayed by an anti-gliadin 33-mer monoclonal antibody (moAb). Plasmid combinations 1 and 2 downregulated only γ- and α-gliadins, respectively. Four plasmid combinations were highly effective in the silencing of ω-gliadins and γ-gliadins, and three of these also silenced α-gliadins. HMW glutenins were upregulated in all but one plasmid combination, while LMW glutenins were downregulated in three plasmid combinations. Total protein and starch contents were unaffected regardless of the plasmid combination used. Six plasmid combinations provided strong reduction in the gluten content as measured by moAb and for two combinations, this reduction was higher than 90% in comparison with the wild type. CD epitope analysis in peptides identified in LC-MS/MS showed that lines from three plasmid combinations were totally devoid of CD epitopes from the highly immunogenic α- and ω-gliadins. Our findings raise the prospect of breeding wheat species with low levels of harmful gluten, and of achieving the important goal of developing nontoxic wheat cultivars.

Low-gluten, nontransgenic wheat engineered with CRISPR/Cas9

Summary 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.

Phenolic content variability and its chromosome location in tritordeum.

For humans, wheat is the most important source of calories, but it is also a source of antioxidant compounds that are involved in the prevention of chronic disease. Among the antioxidant compounds, phenolic acids have great potential to improve human health. In this paper we evaluate the effect of environmental and genetic factors on the phenolics content in the grain of a collection of tritordeums with different cytoplasm and chromosome substitutions. To this purpose, tritordeum flour was used for extraction of the free, conjugates and bound phenolic compounds. These phenolic compounds were identified and quantified by RP-HPLC and the results were analyzed by univariate and multivariate methods. This is the first study that describes the composition of phenolic acids of the amphiploid tritordeum. As in wheat, the predominant phenolic compound is ferulic acid. In tritordeum there is great variability for the content of phenolic compounds and the main factor which determines its content is the genotype followed by the environment, in this case included in the year factor. Phenolic acid content is associated with the substitution of chromosome DS1D(1Hch) and DS2D(2Hch), and the translocation 1RS/1BL in tritordeum. The results show that there is high potential for further improving the quality and quantity of phenolics in tritordeum because this amphiploid shows high variability for the content of phenolic compounds.

Progress and perspectives for carotenoid accumulation in selected Triticeae species

Plant carotenoids are C40 isoprenoids with multiple biological roles. Breeding for carotenoid content in rice, maize and wheat is a relevant issue, both for their importance in human health and nutrition and for their influence in food colouration in products such as pasta from durum wheat. Regarding human health, vitamin A deficiency (VAD) is one of the major causes of malnutrition in the world. As many as 500 000 children become blind due to VAD each year with many of them dying from VAD-related illness within 1 year. This review presents the main results in the improvement of endosperm carotenoid levels in rice, maize and wheat considering the methodology used, either transgenic or non-transgenic; the breeding target, such as provitamin A or total carotenoid content; the identification of new carotenogenic genes/alleles related to the available variation for this trait; and the development of new functional markers for marker-assisted selection. A comparative overview among these species and key areas for further improvement are also identified. Carotenoid enhancement in grasses would benefit from comparative studies among Triticeae species since they allow the understanding of the diversity basis. Therefore, the comparative overview given in this work will be relevant not only to rice, maize and wheat but also to other Triticeae species.

Exploitation of nuclear and cytoplasm variability in Hordeum chilense for wheat breeding.

Our work in this area is supported by grants (to S. G. A.) AGL2008-03720, and P09-AGR-4817 from Spanish Ministry of Science and Innovation, Junta de Andalucia and FEDER. C. R.-S. acknowledges financial support from CSIC (JAE-Doc program).

First Resistance Mechanisms Characterization in Glyphosate-Resistant Leptochloa virgata

Leptochloa virgata (L.) P. Beauv. is an annual weed common in citrus groves in the states of Puebla and Veracruz, Mexico limiting their production. Since 2010, several L. virgata populations were identified as being resistant to glyphosate, but studies of their resistance mechanisms developed by this species have been conducted. In this work, three glyphosate-resistant populations (R8, R14, and R15) collected in citrus orchards from Mexico, were used to study their resistance mechanisms comparing them to one susceptible population (S). Dose-response and shikimic acid accumulation assays confirmed the glyphosate resistance of the three resistant populations. Higher doses of up to 720 g ae ha-1 (field dose) were needed to control by 50% plants of resistant populations. The S population absorbed between 7 and 13% more 14C-glyphosate than resistant ones, and translocated up to 32.2% of 14C-glyphosate to the roots at 96 h after treatment (HAT). The R8, R14, and R15 populations translocated only 24.5, 26.5, and 21.9%, respectively. The enzyme activity of 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) was not different in the S, R8 and R14 populations. The R15 Population exhibited 165.9 times greater EPSPS activity. Additionally, this population showed a higher EPSPS basal activity and a substitution in the codon 106 from Proline to Serine in the EPSPS protein sequence. EPSPS gene expression in the R15 population was similar to that of S population. In conclusion, the three resistant L. virgata populations show reduced absorption and translocation of 14C-glyphosate. Moreover, a mutation and an enhanced EPSPS basal activity at target-site level confers higher resistance to glyphosate. These results describe for the first time the glyphosate resistance mechanisms developed by resistant L. virgata populations of citrus orchards from Mexico.

Introgression of 1Dx5+1Dy10 into tritordeum.

Abstract.The uses of hexaploid tritordeum as a crop for human consumption require improvement of its bread-making quality. For this purpose chromosome 1D of bread wheat with the Glu-D1 allele encoding for high-molecular-weight glutenin subunits Dx5+Dy10 was introgressed into tritordeum. Different primary tritordeums were crossed with wheats carrying subunits Dx5+Dy10. The hybrids were backcrossed to tritordeum and seeds for the next backcross (or selfing) were selected for the presence of chromosome 1D using SDS-PAGE. Forty two chromosome plants carrying subunits Dx5+Dy10 were obtained after two backcrosses and selfing. Chromosome characterization of these plants using fluorescence in situ hybridisation (FISH) proved that either chromosome substitution 1Hch/1D or 1A/1D had been obtained. A homozygous plant with a translocation of the entire 1DL arm to 1HchS was also obtained. The complete chromosome substitution lines have better agronomic characteristics than the lines with translocations.