Characterization of Changes in Gluten Proteins in Low-Gliadin Transgenic Wheat Lines in Response to Application of Different Nitrogen Regimes

Gluten proteins are major determinants of the bread making quality of wheat but also of important gluten-related disorders. The gluten protein accumulation during grain filling is strongly influenced by nitrogen fertilization. We have characterized the gluten proteins in low-gliadin wheat lines as influenced by nitrogen treatments in two experiments. These transgenic lines, D783, D793, C655, D577, and E82 were obtained by using two different RNAi silencing fragments and two endosperm-specific promoters to drive the silencing fragments (d-hordein and γ-gliadin). In Experiment 1, we used three nitrogen fertilizer rates (120, 360, and 1080 mg N) added at sowing stage and combined with two sulfur rates (8 and 30 mg S); Experiment 2 included two nitrogen levels (120 and 1080 mg N), which were added according to the greatest demand per plant using split applications. The protein quantification was accomplished by Reverse-Phase High-Performance Liquid Chromatography and gluten content (ppm) determined using monoclonal antibody R5 (Competitive R5 ELISA). The results showed differences in protein accumulation between the two transgenic lines with the same silencing fragment but different promoter. Lines D793 and E82 showed low gliadin and an increment in glutenin content with increasing nitrogen. Competitive ELISA R5 showed a significant decrease in gluten content using split applications of nitrogen (Experiment 2) with 120 mg N compared to Experiment 1. In addition, line E82 ensures that variations in N fertilization will not result in increased gluten content.The Spanish Ministry of Economy and Competitiveness (Projects AGL2013-48946-C3-1-R and AGL2016-80566-P), the European Regional Development Fund (FEDER) and Junta de Andalucía (Project P11-AGR-7920) supported this work.Peer Reviewe

Polinucleótido que comprende secuencias de gliadinas de trigo y su uso para silenciamiento mediante iARN

Peer reviewedConsejo Superior de Investigaciones CientíficasT3 Traducción de patente europe

Polinucleótido que comprende secuencias de gliadinas de trigo y su uso para silenciamiento mediante RNAi

Polinucleótido que comprende secuencias de gliadinas de trigo y su uso para silenciamiento mediante RNAi. La presente invención se refiere al silenciamiento especifico de las (alfa), (beta), (gamma) y ! (omega)- gliadinas de trigo duro y harinero mediante RNA de interferencia (ARNi) por medio del empleo de un polinucleótido que se transcribe a un hpRNA (hairpin RNA). Además, la presente invención también se refiere a un vector, célula, planta o semilla que comprenden el polinucleótido, cuya expresión se dirige de forma específica en tejidos concretos de las semillas de trigo mediante secuencias reguladoras de la expresión génica como por ejemplo, el promotor de un gen de -gliadinas o el promotor del gen que codifica para una D-hordeína.Peer reviewedConsejo Superior de Investigaciones Científicas (España)B1 Patente sin examen previ

Genomic selection of new low-gliadin wheat lines.

Gluten proteins are responsible for the quality and properties of processed wheat products. However, consuming gluten proteins (gliadins and glutenins) from wheat, barley, and rye can cause celiac disease (CD) or non-celiac wheat sensitivity (NCWS) in genetically predisposed individuals. The immunogenic epitopes that trigger celiac disease are mainly present in the α-, β-, and γ-Gliadins. The only alternative currently available for this type of diseases is a strict, lifelong gluten-free diet. The use of genetic engineering techniques such as RNA interference (RNAi) for post-transcriptional regulation of α-gliadins [1] and gene editing using CRISPR/Cas for silencing the α-gliadins , has resulted in wheat lines (T. aestivum ssp. aestivum,) with reduced α-gliadin content. [2]. These methods produce offspring with silenced, deleted and/or edited gliadins, which can reduce patients' exposure to CD epitopes. To produce new lines with silenced α-gliadins, Francisco Barro's group at the Institute of Sustainable Agriculture (IASCSIC), crossed lines treated with CRISPR/Cas and RNAi and, using anther culture for the production of double haploid (DH) plants, they fixed the mutations of the offspring of these crosses into homozygous lines. (Unpublished). In this Master's thesis, a bioinformatics pipeline based on Next Generation Sequencing (NGS) amplicon sequencing (previously designed by Francisco Barro's group at the Institute for Sustainable Agriculture IAS-CSIC [3]) will be used to analyze the insertion and deletions (InDels) in target genes of lines treated by DH process. In addition, A-PAGE gels will be used to examine the protein profiles of these lines with the main objective of selecting those lines containing lower quantities of gliadins

Glyphosate-Resistant Parthenium hysterophorus in the Caribbean Islands: Non Target Site Resistance and Target Site Resistance in Relation to Resistance Levels

Peer reviewedGlyphosate has been the most intensely herbicide used worldwide for decades, and continues to be a single tool for controlling weeds in woody crops. However, the adoption of this herbicide in a wide range of culture systems has led to the emergence of resistant weeds. Glyphosate has been widely used primarily on citrus in the Caribbean area, but a study of resistance in the Caribbean islands of Cuba and the Dominican Republic has never been carried out. Unfortunately, Parthenium hysterophorus has developed glyphosate-resistance in both islands, independently. The resistance level and mechanisms of different P. hysterophorus accessions (three collected in Cuba (Cu-R) and four collected in the Dominican Republic (Do-R) have been studied under greenhouse and laboratory conditions. In in vivo assays (glyphosate dose causing 50% reduction in above-ground vegetative biomass and survival), the resistance factor levels showed susceptible accessions (Cu-S ≥ Do-S), low-resistance accessions (Cu-R3 Do-R2 > Cu-R2 > Do-R3 > Do-R4 > Cu-R3 >> Cu-S ≥ Do-S). Glyphosate was degraded to aminomethylphosphonic acid, glyoxylate and sarcosine by >88% in resistant accessions except in Cu-R3 and Do-R4 resistant accessions (51.12 and 44.21, respectively), whereas a little glyphosate (<9.32%) was degraded in both susceptible accessions at 96 h after treatment. There were significant differences between P. hysterophorus accessions in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) activity enzyme with and without different glyphosate rates. The R accessions showed values of between 0.026 and 0.21 μmol μg−1 TSP protein min−1 basal EPSPS activity values with respect to the S (0.024 and 0.025) accessions. The same trend was found in the EPSPS enzyme activity treated with glyphosate, where a higher enzyme activity inhibition (glyphosate μM) corresponded to greater resistance levels in P. hysterophorus accessions. One amino acid substitution was found at position 106 in EPSPS, consisting of a proline to serine change in Cu-R1, Do-R1 Do-R2. The above-mentioned results indicate that high resistance values are determined by the number of defense mechanisms (target-site and non-target-site resistance) possessed by the different P. hysterophorus accessions, concurrently.This work was funded by AGL2013-48946-C3-1-R and AGL2016-78944-R projects (Spain).Peer Reviewe

Evaluacion de Líneas de Triticale para la Producción de Bioetanol

La demanda de biocarburantes ha supuesto un incremento de la utilización de los cereales como materia prima para la producción de bioetanol. El triticale no se usa directamente para la dieta humana, por lo que se presenta como un cultivo alternativo a otros cereales. En este estudio se ha evaluado el potencial del triticale para la producción de bioetanol. Las líneas de triticale presentaron una buena adaptabilidad a las condiciones de cultivo del sur de España, encontrándose correlaciones significativas entre la producción en etanol y la fibra ADF, proteína, almidón, rendimiento e hidratos de carbono totales

Celiac Immunogenic Potential of α-Gliadin Epitope Variants from Triticum and Aegilops Species

The high global demand of wheat and its subsequent consumption arise from the physicochemical properties of bread dough and its contribution to the protein intake in the human diet. Gluten is the main structural complex of wheat proteins and subjects affected by celiac disease (CD) cannot tolerate gluten protein. Within gluten proteins, α-gliadins constitute the most immunogenic fraction since they contain the main T-cell stimulating epitopes (DQ2.5-glia-α1, DQ2.5-glia-α2, and DQ2.5-glia-α3). In this work, the celiac immunotoxic potential of α-gliadins was studied within Triticeae: diploid, tetraploid, and hexaploid species. The abundance and immunostimulatory capacity of CD canonical epitopes and variants (with one or two mismatches) in all α-gliadin sequences were determined. The results showed that the canonical epitopes DQ2.5-glia-α1 and DQ2.5-glia-α3 were more frequent than DQ2.5-glia-α2. A higher abundance of canonical DQ2.5-glia-α1 epitope was found to be associated with genomes of the BBAADD, AA, and DD types; however, the abundance of DQ2.5-glia-α3 epitope variants was very high in BBAADD and BBAA wheat despite their low abundance in the canonical epitope. The most abundant substitution was that of proline to serine, which was disposed mainly on the three canonical DQ2.5 domains on position 8. Interestingly, our results demonstrated that the natural introduction of Q to H at any position eliminates the toxicity of the three T-cell epitopes in the α-gliadins. The results provided a rational approach for the introduction of natural amino acid substitutions to eliminate the toxicity of three T-cell epitopes, while maintaining the technological properties of commercial wheats

Grain quality as Influenced by the structural properties of weed communities in Mediterranean wheat crops

Weed community structure, including composition, taxonomic and functional diversity, may explain variability in crop quality, adding to the variability accounted by management, climatic and genetic factors. Focusing on Mediterranean rainfed wheat crops, we sampled weed communities from 26 fields in Spain that were either organically or conventionally managed. Weed communities were characterized by their abundance and taxonomic, compositional and trait-based measures. Grain protein concentration and the glutenin to gliadin ratio were used as indicators of wheat grain quality. Linear mixed effects models were used to analyze the relationship between crop quality and weed community variables, while accounting for environmental factors. Nitrogen fertilization, previous crop and precipitation explained a large portion of the variation in wheat grain protein concentration (R2marginal = 0.39) and composition (R2marginal = 0.26). Weed community measures had limited effects on grain quality (increasing R2marginal of models by 1% on average). The weed effects were related to the composition and the functional structure of their communities, but not to their abundance. Environmental conditions promoting higher protein concentration were also selecting for weed species with competitive attributes, whereas the role of weed functional diversity depended on the functional trait and on the resource limiting crop grain quality. Understanding the mechanisms of weed effects on crop quality could aid on designing sustainable weed management practices.This research was supported by grants AGL2012-33736 and AGL2015-64130-R funded by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe”. D.J.G. was partially supported by the United States National Science Foundation (DUE 1758497 and DUE 1949969)Postprint (published version

Tritordeum breads are well tolerated with preference over gluten-free breads in non-celiac wheat-sensitive patients and its consumption induce changes in gut bacteria

[Backgound] The ingestion of wheat and other cereals are related to several gut disorders. The specific components responsible for non-celiac wheat-sensitivity (NCWS) may include gluten and other compounds. Tritordeum is a new cereal derived from crossing durum wheat with a wild barley species, which differs from bread wheat in its gluten composition. In the present work, we examined the response of NCWS patients to tritordeum bread Gastrointestinal symptoms as well as tritordeum acceptability, gluten immunogenic peptides excretion, and the composition and structure of the intestinal microbiota were evaluated.[Results] Gastrointestinal symptoms of the subjects showed no significant change between the gluten-free bread and the tritordeum bread. Participating subjects rated tritordeum bread higher than the gluten-free bread. Analysis of the bacterial gut microbiota indicated that tritordeum consumption does not alter the global structure and composition of the intestinal microbiota, and only a few changes in some butyrate-producing bacteria were observed. [Conclusions] All the results derived from acceptability, biochemical and microbiological tests suggest that tritordeum may be tolerated by a sub-set of NCWS sufferers who do not require strict exclusion of gluten from their diet. © 2020 Society of Chemical Industry.This research was funded by Junta de Andalucía, Consejería de Conocimiento, Investigación y Universidad, project AT-5985 and the European Regional Development Fund (FEDER)

Oral Consumption of Bread from an RNAi Wheat Line with Strongly Silenced Gliadins Elicits No Immunogenic Response in a Pilot Study with Celiac Disease Patients

Celiac disease (CD) is a genetically predisposed, T cell-mediated and autoimmune-like disorder caused by dietary exposure to the storage proteins of wheat and related cereals. A gluten-free diet (GFD) is the only treatment available for CD. The celiac immune response mediated by CD4+ T-cells can be assessed with a short-term oral gluten challenge. This study aimed to determine whether the consumption of bread made using flour from a low-gluten RNAi wheat line (named E82) can activate the immune response in DQ2.5-positive patients with CD after a blind crossover challenge. The experimental protocol included assessing IFN-γ production by peripheral blood mononuclear cells (PBMCs), evaluating gastrointestinal symptoms, and measuring gluten immunogenic peptides (GIP) in stool samples. The response of PBMCs was not significant to gliadin and the 33-mer peptide after E82 bread consumption. In contrast, PBMCs reacted significantly to Standard bread. This lack of immune response is correlated with the fact that, after E82 bread consumption, stool samples from patients with CD showed very low levels of GIP, and the symptoms were comparable to those of the GFD. This pilot study provides evidence that bread from RNAi E82 flour does not elicit an immune response after a short-term oral challenge and could help manage GFD in patients with CD.This research was funded by The Spanish Ministry of Science and Innovation, Agencia Estatal de Investigación (Project PID2019-110847RB-I00), Consejería de Transformación Económica, Industria, Conocimiento y Universidades, Junta de Andalucía (Project P20_01005), and “ERDF A way of making Europe”, by the “European Union”