Wheat is a staple cereal in the human diet. Despite its significance, an increasing percentage of the population suffers adverse reactions to wheat, which are triggered by wheat gluten, particularly the gliadin fractions. In this study, we employed CRISPR/Cas [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein] multiplexing to introduce targeted mutations into γ- and ω-gliadin genes of wheat, to produce lines deficient in one or both immunogenic gliadin fractions simultaneously. For this study, eight single guide RNAs (sgRNAs) were designed and combined into four plasmids to produce 59 modified wheat lines, of which 20 exhibited mutations in the target genes. Characterization of these lines through Sanger sequencing or next-generation sequencing revealed a complex pattern of InDels, including deletions spanning multiple sgRNAs. The mutations were transmitted to the offspring, and the analysis of homozygous derived lines by reverse-phase HPLC and monoclonal antibodies showed a 97.7% reduction in gluten content. Crossing these lines with other CRISPR/Cas lines deficient in the α-gliadins allowed multiple mutations to be combined. This work represents an important step forward in the use of CRISPR/Cas to develop gluten-free wheat.

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.

Drought and heat are the main abiotic constraints affecting durum wheat production. This study aimed to screen for tolerance to drought, heat, and combined stresses in durum wheat, at the juvenile stage under controlled conditions. Five durum wheat genotypes, including four landraces and one improved genotype, were used to test their tolerance to abiotic stress. After 15 days of growing, treatments were applied as three drought levels (100, 50, and 25% field capacity (FC)), three heat stress levels (24, 30, and 35°C), and three combined treatments (100% FC at 24°C, 50% FC at 30°C and 25% FC at 35°C). The screening was performed using a set of morpho-physiological, and biochemical traits. The results showed that the tested stresses significantly affect all measured parameters. The dry matter content (DM) decreased by 37.1% under heat stress (35°C), by 37.3% under severe drought stress (25% FC), and by 53.2% under severe combined stress (25% FC at 35°C). Correlation analyses of drought and heat stress confirmed that aerial part length, dry matter content, hydrogen peroxide content, catalase, and Glutathione peroxidase activities could be efficient screening criteria for both stresses. The principal component analysis (PCA) showed that only the landrace Aouija tolerated the three studied stresses, while Biskri and Hedhba genotypes were tolerant to drought and heat stresses and showed the same sensitivity under combined stress. Nevertheless, improved genotype Karim and the landrace Hmira were the most affected genotypes by drought, against a minimum growth for the Hmira genotype under heat stress. The results showed that combined drought and heat stresses had a more pronounced impact than simple effects. In addition, the tolerance of durum wheat to drought and heat stresses involves several adjustments of morpho-physiological and biochemical responses, which are proportional to the stress intensity.

Drought is a major challenge for the cultivation of durum wheat, a crucial crop for global food security. Plants respond to drought by adjusting their mineral nutrient profiles to cope with water scarcity, showing the importance of nutrient plasticity for plant acclimation and adaptation to diverse environments. Therefore, it is essential to understand the genetic basis of mineral nutrient profile plasticity in durum wheat under drought stress to select drought-tolerant varieties. The research study investigated the responses of different durum wheat genotypes to severe drought stress at the seedling stage. The study employed an ionomic, molecular, biochemical and physiological approach to shed light on distinct behaviors among different genotypes. The drought tolerance of SVEMS16, SVEVO, and BULEL was related to their capacity of maintaining or increasing nutrient's accumulation, while the limited nutrient acquisition capability of CRESO and S.CAP likely resulted in their susceptibility to drought. The study highlighted the importance of macronutrients such as SO₄^2-, NO₃^-, PO₄^3-, and K⁺ in stress resilience and identified variant-containing genes potentially influencing nutritional variations under drought. These findings provide valuable insights for further field studies to assess the drought tolerance of durum wheat genotypes across various growth stages, ultimately ensuring food security and sustainable production in the face of changing environmental conditions.

Temperature and nitrogen (N) availability are two important environmental factors that may produce important changes in grain composition during grain filling of bread wheat. In this study, four wheat lines with the down-regulation of gliadins by means of RNA interference (RNAi) have been characterized to determine the effect of thermal stress and N availability on grain weight and quality; with focus on gliadin and glutenin protein fractions. Grain weight was reduced with heat stress (HS) in all RNAi lines, whereas gliadin content was increased in the wild-types. With respect to gliadin content, RNAi lines responded to HS and N availability differently from their respective wild-types, except for ω-gliadin content, indicating a very clear stability of silencing under different environmental conditions. In a context of increased temperature and HS events, and in environments with different N availability, the RNAi lines with down-regulated gliadins seem well suited for the production of wheat grain with low gliadin content.

Gluten proteins are responsible for the wheat breadmaking quality. However, gluten is also related to human pathologies for which the only treatment is a gluten-free diet (GFD). GFD has gained popularity among individuals who want to reduce their gluten intake. Tritordeum is a cereal species that originated after crossing durum wheat with wild barley and differs from bread wheat in its gluten composition. In this work, we have characterized the immunogenic epitopes of tritordeum bread and results from a four-phase study with healthy adults for preferences of bread and alterations in the gut microbiota after consuming wheat bread, gluten-free bread, and tritordeum bread are reported. Tritordeum presented fewer peptides related to gluten proteins, CD-epitopes, and IgE binding sites than bread wheat. Participants rated tritordeum bread higher than gluten-free bread. Gut microbiota analysis revealed that the adherence to a strict GFD involves some minor changes, especially altering the species producing short-chain fatty acids. However, the short-term consumption of tritordeum bread does not induce significant changes in the diversity or community composition of the intestinal microbiota in healthy individuals. Therefore, tritordeum bread could be an alternative for healthy individuals without wheat-related pathologies who want to reduce their gluten consumption without harming their gut health.