Tritordeum as an Innovative Alternative to Wheat: A Comparative Digestion Study on Bread.

Tritordeum results from the crossbreeding of a wild barley (Hordeum chilense) species with durum wheat (Triticum turgidum spp. turgidum). This hexaploid crop exhibits agronomic and rheological characteristics like soft wheat, resulting in an innovative raw material to produce baked goods. We applied a gel-based proteomic approach on refined flours to evaluate protein expression differences among two widespread tritordeum cultivars (Aucan and Bulel) taking as the reference semolina and flour derived from a durum and a soft wheat cvs, respectively. The products of in vitro digestion of model breads were analyzed to compare bio-accessibility of nutrients and mapping tritordeum bread resistant peptides. Significant differences among the protein profiles of the four flours were highlighted by electrophoresis. The amino acid bio-accessibility and the reducing sugars of tritordeum and wheat breads were comparable. Tritordeum cvs had about 15% higher alpha-amino nitrogen released at the end of the duodenal simulated digestion than soft wheat (p < 0.05). Bulel tritordeum flour, bread and digested bread had about 55% less R5-epitopes compared to the soft wheat. Differences in protein expression found between the two tritordeum cvs reflected in diverse digestion products and allergenic and celiacogenic potential of the duodenal peptides. Proteomic studies of a larger number of tritordeum cvs may be successful in selecting those with good agronomical performances and nutritional advantages.

The effect of nitrogen fertilization on the expression of protein in wheat and tritordeum varieties using a proteomic approach.

Wheat, an essential ingredient for several bakery preparations, is also responsible for gluten-related diseases in sensitive subjects. The effect of the N fertilization rate (80 vs 160 kg N ha-1) on gluten protein expression profile has been evaluated considering two soft wheats (landrace and modern) and one tritordeum cultivar (cv), grown in the same experimental field in North Italy. The proteins of refined flour were characterized through advanced proteomic approaches, including chromatography (RP-HPLC) and electrophoresis. A static model system was used to simulate in vitro digestion and the digestome peptides were examined by mass spectrometry and in silico approaches, to investigate the celiac and allergenic sequences. The CD-toxic epitopes in the digested samples were quantified by means of a R5 ELISA assay. The N fertilization rate increased the grain protein content, but it did not lead to any difference in gluten composition, with exception of glu/glia ratio in the modern wheat cv. Moreover, the gluten composition and the occurrence of toxic/allergenic epitopes varied to a great extent, according mostly to the genotype. A lower immunoreactivity, determined using R5 ELISA, was detected for the digested tritordeum flours than for the landrace (-51%) or modern (-58%) cvs, while no significant difference was observed for the N rates between each genotype. In silico analysis showed that tritordeum has fewer CD epitopes belonging to the ω-gliadins and a lower LMW-GS than the landrace or modern cv. Tritordeum presented fewer α-gliadin allergenic epitopes than the modern wheat cv. The lower frequency of celiac epitopes in tritordeum, compared to the old and the modern wheat, is probably due to the absence of a D genome.

Assessment of Cas12a-mediated gene editing efficiency in plants.

The CRISPR/Cas12a editing system opens new possibilities for plant genome engineering. To obtain a comparative assessment of RNA-guided endonuclease (RGEN) types in plants, we adapted the CRISPR/Cas12a system to the GoldenBraid (GB) modular cloning platform and compared the efficiency of Acidaminococcus (As) and Lachnospiraceae (Lb) Cas12a variants with the previously described GB-assembled Streptococcus pyogenes Cas9 (SpCas9) constructs in eight Nicotiana benthamiana loci using transient expression. All three nucleases showed drastic target-dependent differences in efficiency, with LbCas12 producing higher mutagenesis rates in five of the eight loci assayed, as estimated with the T7E1 endonuclease assay. Attempts to engineer crRNA direct repeat (DR) had little effect improving on-target efficiency for AsCas12a and resulted deleterious in the case of LbCas12a. To complete the assessment of Cas12a activity, we carried out genome editing experiments in three different model plants, namely N. benthamiana, Solanum lycopersicum and Arabidopsis thaliana. For the latter, we also resequenced Cas12a-free segregating T2 lines to assess possible off-target effects. Our results showed that the mutagenesis footprint of Cas12a is enriched in deletions of -10 to -2 nucleotides and included in some instances complex rearrangements in the surroundings of the target sites. We found no evidence of off-target mutations neither in related sequences nor somewhere else in the genome. Collectively, this study shows that LbCas12a is a viable alternative to SpCas9 for plant genome engineering.