Background: High amylose starch has attracted particular interest because of its correlation with the amount of Resistant Starch (RS) in food. RS plays a role similar to fibre with beneficial effects for human health, providing protection from several diseases such as colon cancer, diabetes, obesity, osteoporosis and cardiovascular diseases. Amylose content can be modified by a targeted manipulation of the starch biosynthetic pathway. In particular, the inactivation of the enzymes involved in amylopectin synthesis can lead to the increase of amylose content. In this work, genes encoding starch branching enzymes of class II (SBEIIa) were silenced using the RNA interference (RNAi) technique in two cultivars of durum wheat, using two different methods of transformation (biolistic and Agrobacterium). Expression of RNAi transcripts was targeted to the seed endosperm using a tissue-specific promoter. Results: Amylose content was markedly increased in the durum wheat transgenic lines exhibiting SBEIIa gene silencing. Moreover the starch granules in these lines were deformed, possessing an irregular and deflated shape and being smaller than those present in the untransformed controls. Two novel granule bound proteins, identified by SDS-PAGE in SBEIIa RNAi lines, were investigated by mass spectrometry and shown to have strong homologies to the waxy proteins. RVA analysis showed new pasting properties associated with high amylose lines in comparison with untransformed controls. Finally, pleiotropic effects on other starch genes were found by semi-quantitative and Real-Time reverse transcription-polymerase chain reaction (RT-PCR). Conclusion: We have found that the silencing of SBEIIa genes in durum wheat causes obvious alterations in granule morphology and starch composition, leading to high amylose wheat. Results obtained with two different methods of transformation and in two durum wheat cultivars were comparable.

Both high- and low-molecular-weight glutenin subunits (LMW-GS) play the major role in determining the viscoelastic properties of wheat (Triticum aestivum L.) flour. To date there has been no clear correspondence between the amino acid sequences of LMW-GS derived from DNA sequencing and those of actual LMW-GS present in the endosperm. We have characterized a particular LMW-GS from hexaploid bread wheat, a major component of the glutenin polymer, which we call the 42K LMW-GS, and have isolated and sequenced the putative corresponding gene. Extensive amino acid sequences obtained directly for this 42K LMW-GS indicate correspondence between this protein and the putative corresponding gene. This subunit did not show a cysteine (Cys) at position 5, in contrast to what has frequently been reported for nucleotide-based sequences of LMW-GS. This Cys has been replaced by one occurring in the repeated-sequence domain, leaving the total number of Cys residues in the molecule the same as in various other LMW-GS. On the basis of the deduced amino acid sequence and literature-based assignment of disulfide linkages, a computer-generated molecular model of the 42K subunit was constructed.

Technological properties of wheat flours are largely governed by the functional properties of storage proteins, comprising single chain gliadins and disulphide bonded polymeric proteins (glutenins, triticins, HMW albumins). This study was conducted (a) to measure the rate of accumulation of different classes of proteins, (b) to monitor the rate of polymerization of the polymeric subunits, and (c) to investigate the origin of differences in the degree of polymerization arising from allelic differences at the Glu-D1 locus of HMW glutenin subunits. Developing grains from hexaploid wheat cultivars were collected at 3-d intervals between 7 d and 40 d after anthesis (DAA). The accumulation behaviour of polymeric proteins was determined by size exclusion-HPLC and of the constituent polypeptides (HMW glutenin subunits, LMW glutenin subunits, HMW albumin subunits) by a combination of SDS-PAGE, reversed phase-HPLC and immunoblotting. The relative time for peak accumulation of the albumins/gobulins, gliadins and polymeric proteins occurred in that order during grain filling. Deposition of HMW glutenin subunits, LMW glutenin subunits and HMW albumin subunits (β-amylases) began as early as 7 d after anthesis and progressed until maturity. Disulphide-linked aggregation (polymerization) of these subunits also commenced at 7 DAA. However, significant changes in size distribution of the polymers occurred only during the late stages of seed development which coincided with a rapid increase in the amounts of the glutenin subunits. The effects of HMW albumin subunits and LMW glutenin subunits on polymer size were relatively smaller than that of the HMW glutenin subunits. The wheat biotype with HMW glutenin subunits 5+10 (Glu-D1 d allele) accumulated larger polymers more quickly than the biotype with allelic subunits 2+12 (Glu-D1 a allele), this difference becoming apparent at 28 DAA which could be attributed to variation in the accumulation rate of HMW glutenin subunits.

Wheat and its derived foods are widespread, representing one of the main food sources globally. During the last decades, the incidence of disorders related to wheat has become a global issue for the human population, probably linked to the spread of wheat-derived foods. It has been ascertained that structural and metabolic proteins, like α-amylase/trypsin inhibitors (ATI), are involved in the onset of wheat allergies (bakers' asthma) and probably Non-Coeliac Wheat Sensitivity (NCWS). The ATI are a group of exogenous protease inhibitors, which are encoded by a multigene family dispersed over several chromosomes in durum and bread wheat. WTAI-CM3 and WTAI-CM16 subunits are considered among the main proteins involved in the onset of bakers' asthma and probably NCWS. A CRISPR-Cas9 multiplexing strategy was used to edit the ATI subunits WTAI-CM3 and WTAI-CM16 in the grain of the Italian durum wheat cultivar Svevo with the aim to produce wheat lines with reduced amount of potential allergens involved in adverse reactions. Using a marker gene-free approach, whereby plants are regenerated without selection agents, homozygous mutant plants without the presence of CRISPR vectors were obtained directly from T₀ generation. This study demonstrates the capability of CRISPR technology to knock out immunogenic proteins in a reduced time compared to conventional breeding programmes. The editing of the two target genes was confirmed either at molecular (sequencing and gene expression study) or biochemical (immunologic test) level. Noteworthy, as a pleiotropic effect, is the activation of the ATI 0.28 pseudogene in the edited lines.