Genetic diversity of durum wheat (Triticum turgidum ssp. durum) to mitigate abiotic stress: Drought, heat, and their combination.

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.

Study of the genetic variability of durum wheat (Triticum durum Desf.) in the face of combined stress: water and heat.

The devastating effects and extent of abiotic stress on cereal production continue to increase globally, affecting food security in several countries, including Tunisia. Heat waves and the scarcity of rainfall strongly affect durum wheat yields. The present study aims to screen for tolerance to combined water and heat stresses in durum wheat at the juvenile stage. Three combined treatments were tested, namely: T0 (100% field capacity (FC) at 24 °C), T1 (50% FC at 30 °C), and T2 (25% FC at 35 °C). The screening was carried out based on morphological, physiological, and biochemical criteria. The results showed that the combined stress significantly affected all the measured parameters. The relative water content (RWC) decreased by 37.6% under T1 compared to T0. Quantum yield (Fv/m) and photosynthetic efficiency (Fv/0) decreased under severe combined stress (T2) by 37.15% and 37.22%, respectively. Under T2 stress, LT increased by 63.7%. A significant increase in osmoprotective solutes was also observed, including proline, which increased by 154.6% under T2. Correlation analyses of the combination of water and heat stress confirm that the traits RWC, chlorophyll b content, Fv/m, proline content, Fv/0 and leaf temperature can be used as reliable screening criteria for the two stresses combined. The principal component analysis highlighted that Aouija tolerates the two levels of stresses studied, while the genotypes Karim and Hmira are the most sensitive. The results show that the tolerance of durum wheat to combined water and heat stress involves several adaptation mechanisms proportional to the stress intensity.

Unraveling the celiac disease-related immunogenic complexes in a set of wheat and tritordeum genotypes: implications for low-gluten precision breeding in cereal crops.

The development of low-gluten immunogenic cereal varieties is a suitable way to fight the increment of pathologies associated with the consumption of cereals. Although RNAi and CRISPR/Cas technologies were effective in providing low-gluten wheat, the regulatory framework, particularly in the European Union, is an obstacle to the short- or medium-term implementation of such lines. In the present work, we carried out a high throughput amplicon sequencing of two highly immunogenic complexes of wheat gliadins in a set of bread and durum wheat, and tritordeum genotypes. Bread wheat genotypes harboring the 1BL/1RS translocation were included in the analysis and their amplicons successfully identified. The number of CD epitopes and their abundances were determined in the alpha- and gamma-gliadin amplicons, including 40k-γ-secalin ones. Bread wheat genotypes not containing the 1BL/1RS translocation showed a higher average number of both alpha- and gamma-gliadin epitopes than those containing such translocation. Interestingly, alpha-gliadin amplicons not containing CD epitopes accounted for the highest abundance (around 53%), and the alpha- and gamma-gliadin amplicons with the highest number of epitopes were present in the D-subgenome. The durum wheat and tritordeum genotypes showed the lowest number of alpha- and gamma-gliadin CD epitopes. Our results allow progress in unraveling the immunogenic complexes of alpha- and gamma-gliadins and can contribute to the development of low-immunogenic varieties within precision breeding programs, by crossing or by CRISPR/Cas gene editing.

The opportunity of using durum wheat landraces to tolerate drought stress: screening morpho-physiological components.

Local genetic resources could constitute a promising solution to overcome drought stress. Thus, eight (8) durum wheat landraces and one improved variety were assessed for drought tolerance in pots under controlled conditions. Three water treatments were tested: control (100 % of the field capacity (FC)), medium (50 % FC) and severe (25 % FC) stress. The assessment was carried out at the seedling stage to mimic stress during crop set-up. Results showed that increased water stress led to a decrease in biomass and morpho-physiological parameters and an increase in antioxidant enzyme activities. Severe water stress decreased the chlorophyll fluorescence parameters, relative water content (RWC) and water potential of the investigated genotypes by 56.45, 20.58, 50.18 and 139.4 %, respectively. Besides, the phenolic compounds content increased by 169.2 % compared to the control. Catalase and guaiacol peroxidase activities increased 17 days after treatment for most genotypes except Karim and Hmira. A principal component analysis showed that the most contributed drought tolerance traits were chlorophyll fluorescence parameters, RWC and electrolyte conductivity. Unweighted pair group method with arithmetic mean clustering showed that the landraces Aouija, Biskri and Hedhba exhibited a higher adaptive response to drought stress treatments, indicating that water stress-adaptive traits are included in Tunisian landraces germplasm.

Identification of RNAi hypoallergic bread wheat lines for wheat-dependent exercise-induced anaphylaxis patients.

Wheat-dependent exercise-induced anaphylaxis (WDEIA) is one of the most severe forms of wheat allergy. It occurs in patients when they exercise after ingesting wheat-containing foods. Nowadays, the only possible alternative for WDEIA patients is to avoid such foods. This study investigated the potential of six RNA of interference (RNAi) wheat lines with low-prolamin content as alternatives for WDEIA patients. For that purpose, a high performance-liquid chromatography (HPLC) analysis was performed to evaluate differences in gluten protein fractions among these lines. Next, western blots were conducted to measure the immunoglobulin E (IgE) reactivity to wheat proteins in sera from five WDEIA patients. Additionally, monoclonal antibodies (moAb) recognition sites and the IgE binding sites were searched in all peptides identified by LC-MS/MS after protein digestion. The results showed a 61.4%-81.2% reduction in the gliadin content of the RNAi lines, accompanied by an increase in their high-molecular weight (HMW) glutenin content compared to the wild type bread wheat line (WT). In all cases, the reduction in gliadin content correlated with a decrease in IgE reactivity observed in the sera of WDEIA patients, highlighting the E82 and H320 lines. These two RNAi lines exhibited a ≤90% reduction in IgE reactivity. This reduction could be attributed to an absence of IgE binding sites associated with α- and ω5-gliadins, which were present in the WT. Overall, these lines offer a potential alternative for foodstuff for individuals with WDEIA.

RNAi silencing of wheat gliadins alters the network of transcription factors that regulate the synthesis of seed storage proteins toward maintaining grain protein levels.

Gluten proteins are responsible for the unique viscoelastic properties of wheat dough, but they also trigger the immune response in celiac disease patients. RNA interference (RNAi) wheat lines with strongly silenced gliadins were obtained to reduce the immunogenic response of wheat. The E82 line presents the highest reduction of gluten, but other grain proteins increased, maintaining a total nitrogen content comparable to that of the wild type. To better understand the regulatory mechanisms in response to gliadin silencing, we carried out a transcriptomic analysis of grain and leaf tissues of the E82 line during grain filling. A network of candidate transcription factors (TFs) that regulates the synthesis of the seed storage proteins (SSPs), α-amylase/trypsin inhibitors, lipid transfer proteins, serpins, and starch in the grain was obtained. Moreover, there were a high number of differentially expressed genes in the leaf of E82, where processes such as nutrient availability and transport were enriched. The source-sink communication between leaf and grain showed that many down-regulated genes were related to protease activity, amino acid and sugar metabolism, and their transport. In the leaf, specific proline transporters and lysine-histidine transporters were down- and up-regulated, respectively. Overall, the silencing of gliadins in the RNAi line is compensated mainly with lysine-rich globulins, which are not related to the proposed candidate network of TFs, suggesting that these proteins are regulated independently of the other SSPs. Results reported here can explain the protein compensation mechanisms and contribute to decipher the complex TF network operating during grain filling.

Comparative characterization of the gluten and fructan contents of breads from industrial and artisan bakeries: a study of food products in the Spanish market.

Background: The consumption of wheat/gluten is associated with adverse reactions for human health. Gluten and fructans are identified as the major compounds triggering and worsening adverse reactions to wheat, which are increasing, and as a consequence, avoidance of gluten/wheat is the common strategy of many individuals of the western population. Although bread is a product of daily consumption, there is a lack of information on the gluten and fructan contents and the influence of artisanal or industrial processes. Objective: The aim of this study is to carry out a comparative characterization between artisan bakeries and hypermarkets in Spain for gluten and fructan contents in daily sold breads. Design: A total of 48 types of bread highly consumed in Spain sold in artisan bakeries (long fermentation) and hypermarkets (short fermentations) were selected for comparing the gluten and fructan contents. Methods such as reverse phase-high performance liquid chromatography (RP-HPLC), R5 monoclonal antibody (moAb), and fructans protocols were used for the quantification of these compounds. Results: Great variation for the content of gluten and fructans has been found between all bread categories. Although breads produced using long fermentation (artisan bakeries) contain significantly lower gluten, they have higher fructans than those using short fermentations (hypermarkets). Durum wheat breads had the lowest content of gluten. Moreover, spelt breads from artisan bakeries had the lowest content of fructans but not those from hypermarkets. Discussion: In this study, we report the comparative characterizarion of the breads of the Spanish market. These food products presented variation in the amount of gluten and fructans, ligated in most of the cases to the nature of the providers: artisan bakeries against hypermarkets. Depending on the type of bread, the differences for the daily consumption of gluten and fructan can be 4.5 and 20 times, respectively. Conclusions: We found strong differences for gluten and fructan contents among breads. These information may contribute to designing strategies to improve the management of gluten and fructans in bread.

Consumption of Tritordeum Bread Reduces Immunogenic Gluten Intake without Altering the Gut Microbiota.

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.

A Bioinformatic Workflow for InDel Analysis in the Wheat Multi-Copy α-Gliadin Gene Family Engineered with CRISPR/Cas9.

The α-gliadins of wheat, along with other gluten components, are responsible for bread viscoelastic properties. However, they are also related to human pathologies as celiac disease or non-celiac wheat sensitivity. CRISPR/Cas was successfully used to knockout α-gliadin genes in bread and durum wheat, therefore, obtaining low gluten wheat lines. Nevertheless, the mutation analysis of these genes is complex as they present multiple and high homology copies arranged in tandem in A, B, and D subgenomes. In this work, we present a bioinformatic pipeline based on NGS amplicon sequencing for the analysis of insertions and deletions (InDels) in α-gliadin genes targeted with two single guides RNA (sgRNA). This approach allows the identification of mutated amplicons and the analysis of InDels through comparison to the most similar wild type parental sequence. TMM normalization was performed for inter-sample comparisons; being able to study the abundance of each InDel throughout generations and observe the effects of the segregation of Cas9 coding sequence in different lines. The usefulness of the workflow is relevant to identify possible genomic rearrangements such as large deletions due to Cas9 cleavage activity. This pipeline enables a fast characterization of mutations in multiple samples for a multi-copy gene family.

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.

Prolamin Content and Grain Weight in RNAi Silenced Wheat Lines Under Different Conditions of Temperature and Nitrogen Availability.

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.