Summary 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.

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Optimizing the bread-making quality properties of flour is currently one of the main aims of the bakery industry. Therefore, this study has investigated the effects of N fertilization and fungicide application at wheat heading on the protein content (GPC), gluten composition and rheological properties of wheat. Field experiments were carried out in North-West Italy over a 3 year period, on a high protein cultivar of soft winter wheat. Grain samples were collected for each agronomic treatment at four ripening timings, from the milk stage to the final combine harvesting, and the contents of the different gluten fractions were quantified. The late N fertilization increased the GPC (+1.2%) and dough strength (W) (+22%) as a result of a similar enhancement of all the gluten protein fractions, while the fungicide application slightly reduced the GPC (−0.3%) and W (−4%), mainly because of a dilution of the gliadin content, due to the significantly higher grain yield (+8.6%) and thousand kernel weight (+5.5%). These agronomic practices did not modify the gluten composition significantly, expressed as the relative ratio between the gliadins (glia) and the high (HMW) and low (LMW) molecular weight glutenins (gs), and confirmed by the accumulation trend of the different protein fractions during ripening. The year resulted to have the most marked effect on the gluten protein fraction ratios and alveographic parameters. The lowest W was observed in 2015, and although the highest GPC was recorded for the same year, the lowest gs/glia ratio was also observed. Instead, 2016 showed the highest gs/glia and HMW-gs/LMW-gs (H/L) ratios, and also the highest P/L value (2.2). In 2015, a slightly higher temperature during the ripening stage resulted in a greater increase in the γ-gliadin enriched fraction than the α/β gliadin ones, and marked differences were noted in the rheological traits. This field experiment has highlighted the interactive role of environmental and agronomic factors on the content and quality of gluten proteins and their bread-making ability, thus making a further contribution to the development of an integrated crop strategy for the cultivation of high protein wheat in humid Mediterranean areas.

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.

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