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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.

[EN] The present invention relates to a transgenic wheat event, which we have designated T258, that leads to reduced gliadin content, as well as cells, seeds and plants that comprise the event. The invention also relates to methods for detecting the presence of the T258 event, probes and primers for use in the method and methods of breeding with T258 to produce wheat with reduced gliadin content., [FR] La présente invention concerne un événement de blé transgénique, que nous avons désigné T258, qui conduit à une teneur réduite en gliadine, ainsi que des cellules, des graines et des plantes qui comprennent l'événement. L'invention concerne également des procédés de détection de la présence de l'événement T258, des sondes et des amorces destinées à être utilisées dans le procédé et des procédés de multiplication avec T258 pour produire un blé présentant une teneur réduite en gliadine.

Weed community structure, including composition and taxonomic and functional diversity, may explain variability in crop quality, adding to the variability accounted for 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 an important part of 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 also selected 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 in designing sustainable weed management practices.

Wheat gluten proteins are responsible for the bread-making properties of the dough but also for triggering important gastrointestinal disorders. Celiac disease (CD) affects approximately 1% of the population in Western countries. The only treatment available is the strict avoidance of gluten in the diet. Interference RNA (RNAi) is an excellent approach for the down-regulation of genes coding for immunogenic proteins related to celiac disease, providing an alternative for the development of cereals suitable for CD patients. In the present work, we report a comparative study of the stimulatory capacity of seven low-gluten RNAi lines differing in grain gluten and non-gluten protein composition, relevant for CD and other gluten pathologies. Peripheral blood mononuclear cells (PBMCs) of 35 patients with active CD were included in this study to assess the stimulatory response induced by protein extracts from the RNAi lines. Analysis of the proliferative response and interferon-gamma (INF-γ) release of PBMCs demonstrated impaired stimulation in response to all RNAi lines. The lower response was provided by lines with a very low content of α- and γ-gliadins, and low or almost devoid of DQ2.5 and p31–43 α-gliadin epitopes. The non-gluten protein seems not to play a key role in PBMC stimulation.

La obtención de plantas doblehaploides (DH) cobra una especial importancia cuando se usa en combinación con las técnicas de edición génica como CRISPR/Cas9 ya que nos permite obtener líneas homocigóticas en una sola generación. Bobwhite es el cultivar modelo utilizado para obtener plantas editadas de trigo panadero con bajo contenido en gluten (Sánchez-León y col 2018). La eficiencia de Bobwhite para la producción de plantas DH mediante cultivo de anteras es baja. En este estudio se ha aplicado el protocolo descrito por Castillo et al. (2015) para la obtención de plantas DH de 9 cruzamientos F1, en los que intervienen al menos una línea CRISPR/Cas9 de Bobwhite. Los resultados obtenidos en este estudio confirman que la capacidad de este cultivar para el cultivo de anteras es baja. Uno de los factores más limitantes es el bajo porcentaje de regeneración, debido a una mala calidad de los embriones. Además, los bajos porcentajes de duplicación cromosómica espontánea, con valores inferiores al 25% en la mitad de los cruzamientos, han hecho necesario la aplicación de colchicina para inducir la duplicación cromosómica. El albinismo no fue un factor limitante ya que en ninguno de los cruzamientos se obtuvieron más de un 25% de plantas albinas. Se observó una gran variabilidad para la respuesta al cultivo de anteras entre todos los cruzamientos. El menor número de plantas verdes/100 anteras (1,38) se obtuvo a partir de un cruzamiento entre dos líneas de Bobwhite, siendo todas haploides. El cruzamiento de Bobwhite x Arthur Nick produjo el mayor número de plantas verdes/100 anteras (10) y un 50 % de autoduplicación. Sin embargo, en el cruzamiento de Bobwhite por el otro cultivar de interés agronómico, Anza, se obtuvieron valores similares al resto de los cruzamientos.

Gluten proteins, major determinants of the bread-making quality of wheat, are related to several digestive disorders. Advances in plant genetic breeding have allowed the production of wheat lines with very low gliadin content through the use of RNAi and gene editing technologies. In this review, we carried out a comprehensive study of the application of these cutting-edge technologies towards the development of wheat lines devoid of immunogenic gluten, and their genetic, nutritional and clinical characterization. One line, named E82, showed outstanding nutritional properties, with very low immunogenic gluten and a low stimulation capacity of T-cells from celiac patients. Moreover, a clinical trial with non-celiac wheat sensitivity (NCWS) patients showed that the consumption of bread made with this E82 low gliadin line induced positive changes in the gut microbiota composition.

Plant proteases play a crucial role in many different biological processes along the plant life cycle. One of the most determinant stages in which proteases are key protagonists is the plant germination through the hydrolysis and mobilization of other proteins accumulated in seeds and cereal grains. The most represented proteases in charge of this are the cysteine proteases group, including the C1A family known as papain-like and the C13 family also called legumains. In cereal species such as wheat, oat or rye, gluten is a very complex mixture of grain storage proteins, which may affect the health of sensitive consumers like celiac patients. Since gluten proteins are suitable targets for plant proteases, the knowledge of the proteases involved in storage protein mobilization could be employed to manipulate the amount of gluten in the grain. Some proteases have been previously found to exhibit promising properties for their application in the degradation of known toxic peptides from gluten. To explore the variability in gluten-degrading capacities, we have now analyzed the degradation of gluten from different wheat cultivars using several cysteine proteases from barley. The wide variability showed highlights the possibility to select the protease with the highest potential to alter grain composition reducing the gluten content. Consequently, new avenues could be explored combining genetic manipulation of proteolytic processes with silencing techniques to be used as biotechnological tools against gluten-related disorders.

Coeliac disease is an autoimmune disorder triggered in genetically predisposed individuals by the ingestion of gluten proteins from wheat, barley and rye. The a-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 a-gliadin genes. Twenty-one mutant lines were generated, all showing strong reduction in a-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.

The study evaluated the symptoms, acceptance, and digestibility of bread made from transgenic low-gliadin wheat, in comparison with gluten free bread, in Non-coeliac gluten sensitivity (NCGS) patients, considering clinical/sensory parameters and gut microbiota composition. This study was performed in two phases of seven days each, comprising a basal phase with gluten free bread and an E82 phase with low-gliadin bread. Gastrointestinal clinical symptoms were evaluated using the Gastrointestinal Symptom Rating Scale (GSRS) questionnaire, and stool samples were collected for gluten immunogenic peptides (GIP) determination and the extraction of gut microbial DNA. For the basal and E82 phases, seven and five patients, respectively, showed undetectable GIPs content. The bacterial 16S rRNA gene V1-V2 hypervariable regions were sequenced using the Illumina MiSeq platform and downstream analysis was done using a Quantitative Insights into Microbial Ecology (QIIME) pipeline. No significant differences in the GSRS questionnaires were observed between the two phases. However, we observed a significantly lower abundance of some gut genera Oscillospira, Dorea, Blautia, Bacteroides, Coprococcus, and Collinsella, and a significantly higher abundance of Roseburia and Faecalibacterium genera during the E82 phase compared with the basal phase. The consumption of low-gliadin bread E82 by NCGS subjects induced potentially positive changes in the gut microbiota composition, increasing the butyrate-producing bacteria and favoring a microbial profile that is suggested to have a key role in the maintenance or improvement of gut permeability.

The study evaluated the symptoms, acceptance, and digestibility of bread made from transgenic low-gliadin wheat, in comparison with gluten free bread, in Non-coeliac gluten sensitivity (NCGS) patients, considering clinical/sensory parameters and gut microbiota composition. This study was performed in two phases of seven days each, comprising a basal phase with gluten free bread and an E82 phase with low-gliadin bread. Gastrointestinal clinical symptoms were evaluated using the Gastrointestinal Symptom Rating Scale (GSRS) questionnaire, and stool samples were collected for gluten immunogenic peptides (GIP) determination and the extraction of gut microbial DNA. For the basal and E82 phases, seven and five patients, respectively, showed undetectable GIPs content. The bacterial 16S rRNA gene V1-V2 hypervariable regions were sequenced using the Illumina MiSeq platform and downstream analysis was done using a Quantitative Insights into Microbial Ecology (QIIME) pipeline. No significant differences in the GSRS questionnaires were observed between the two phases. However, we observed a significantly lower abundance of some gut genera Oscillospira, Dorea, Blautia, Bacteroides, Coprococcus, and Collinsella, and a significantly higher abundance of Roseburia and Faecalibacterium genera during the E82 phase compared with the basal phase. The consumption of low-gliadin bread E82 by NCGS subjects induced potentially positive changes in the gut microbiota composition, increasing the butyrate-producing bacteria and favoring a microbial profile that is suggested to have a key role in the maintenance or improvement of gut permeability.

Some studies have suggested that the immunogenicity of oats depends on the cultivar. RP-HPLC has been proposed as a useful technique to select varieties of oats with reduced immunogenicity. The aim of this study was to identify both the avenin protein patterns associated with low gluten content and the available variability for the development of new non-toxic oat cultivars. The peaks of alcohol-soluble avenins of a collection of landraces and cultivars of oats have been characterized based on the RP-HPLC elution times. The immunotoxicity of oat varieties for patients with celiac disease (CD) has been tested using a competitive ELISA based on G12 monoclonal antibody. The oat lines show, on average, seven avenin peaks giving profiles with certain similarities. Based on this similarity, most of the accessions have been grouped into avenin patterns. The variability of RP-HPLC profiles of the collection is great, but not sufficient to uniquely identify the different varieties of the set. Overall, the immunogenicity of the collection is less than 20 ppm. However, there is a different distribution of toxicity ranges between the different peak patterns. We conclude that the RP-HPLC technique is useful to establish groups of varieties differing in degree of toxicity for CD patients.

Some studies have suggested that the immunogenicity of oats depends on the cultivar. RP-HPLC has been proposed as a useful technique to select varieties of oats with reduced immunogenicity. The aim of this study was to identify both the avenin protein patterns associated with low gluten content and the available variability for the development of new non-toxic oat cultivars. The peaks of alcohol-soluble avenins of a collection of landraces and cultivars of oats have been characterized based on the RP-HPLC elution times. The immunotoxicity of oat varieties for patients with celiac disease (CD) has been tested using a competitive ELISA based on G12 monoclonal antibody. The oat lines show, on average, seven avenin peaks giving profiles with certain similarities. Based on this similarity, most of the accessions have been grouped into avenin patterns. The variability of RP-HPLC profiles of the collection is great, but not sufficient to uniquely identify the different varieties of the set. Overall, the immunogenicity of the collection is less than 20 ppm. However, there is a different distribution of toxicity ranges between the different peak patterns. We conclude that the RP-HPLC technique is useful to establish groups of varieties differing in degree of toxicity for CD patients.