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Vromindolines, a family of oat starch-bound proteins responsible for the extremely soft endosperm of this cereal, are characterized by a tryptophan-rich domain and ten conserved cysteine residues, as it was observed in the starch-bound proteins from other cereals, and proved to be specific of the genus Avena. Two-dimensional electrophoresis of vromindolines from different oat species, both cultivated and wild, revealed that they are present in all genomes and ploidy levels, with no major differences in their molecular weight and electrophoretic mobility. PCR amplification and sequencing of genes coding for VIN-2 and VIN-3 indicated that both proteins are strongly conserved across the species analysed. Some differences were found between diploid and polyploid accessions at specific amino acid positions along the protein. In particular the C-genome accessions (A. clauda, A. eriantha, A. ventricosa) differed from the A-genome diploids (A. canariensis, A. damascena, A. longiglumis, A. nuda, A. strigosa) for some peculiar sequences. Tetraploids (A. barbata, A. magna and A. insularis) showed two sequences each for both proteins; the variability observed in them was similar to that found in A. sativa. The characteristics of these genes could help to clarify the genetic relationship among the various species of the genus Avena. Moreover, the knowledge of the genetic control of these proteins represent an important tool for the modulation of oat endosperm texture.

Seven cultivated Triticum species or subspecies ( Triticum monococcum , Triticum turgidum ssp dicoccum , T. turgidum ssp. durum , T. turgidum ssp turanicum , Triticum timopheevii , Triticum aestivum and Triticum zhukovskyi ) were compared for their contents of bioactive compounds, mainly 5-n-alkylresorcinols (ARs). Multivariate analysis of variance and principal component analysis were used to evaluate the differences in the phytochemical profiles and to establish the relationships among variables. Significant differences were observed for both total phenol (TP) and AR content. The highest AR level (377 μg/g) was observed in Triticum dicoccum , which also exhibited the highest variability for these compounds (298–436 μg/g). By contrast, the lowest AR content (286 μg/g) was found in Triticum durum . C21:0 was the main homologue chain in all the samples, its value ranging between 54.5% in T. durum and 41.2% in T. aestivum . The T. dicoccum and T. monococcum wheats had relatively low amounts of TP, whereas Triticum turanicum (215 mg/kg), T. timopheevii (250 mg/kg) and T. zhukovskyi (286 mg/kg) had approximately 3-fold higher TP levels. These results suggest that there are opportunities for breeding wheat varieties with superior health benefits and for promoting the use of ancient Triticum crops as novel sources of healthy food.

Peptic-tryptic (PT) digested prolamins from spelt wheat Triticum aestivum ssp. spelta and landraces ERSA 6 and ERSA 8 of farro wheat T. turgidum ssp. dicoccum were found to agglutinate K562(S) cells, and exert strong toxic effects on Caco-2/TC7 cells. Cytotoxicity of spelt prolamins against Caco-2/TC7 cells was greatly reduced by 10-mer peptide QQPQDAVQPF. By contrast, the PT digests from monoccum wheat (Triticum monococcum) and farro landraces Prometeo, L5563, L5540 and L5558 did not exhibit any negative effects on K562(S) and Caco-2/TC7 cells. Toxic genotypes ERSA 6 and ERSA 8 were found to share the same gliadin pattern, which was absent in inactive landraces. Monococcum, farro and spelt wheats differed from each other in their responses to antibodies specific for 13-mer cytotoxic sequence FPGQQQPFPPQQP and 10-mer peptide QQPQDAVQPF. This latter sequence was found to occur in high amounts in common wheat line FG, Phaseoulus vulgaris, Ph. coccineus and Lens culinaria.

Among cereals, Avena sativa is characterized by an extremely soft endosperm texture, which leads to some negative agronomic and technological traits. On the basis of the well-known softening effect of puroindolines in wheat kernel texture, in this study, indolines and their encoding genes are investigated in Avena species at different ploidy levels. Three novel 14 kDa proteins, showing a central hydrophobic domain with four tryptophan residues and here named vromindoline (VIN)-1,2 and 3, were identified. Each VIN protein in diploid oat species was found to be synthesized by a single Vin gene whereas, in hexaploid A. sativa, three Vin-1, three Vin-2 and two Vin-3 genes coding for VIN-1, VIN-2 and VIN-3, respectively, were described and assigned to the A, C or D genomes based on similarity to their counterparts in diploid species. Expression of oat vromindoline transgenes in the extra-hard durum wheat led to accumulation of vromindolines in the endosperm and caused an approximate 50 % reduction of grain hardness, suggesting a central role for vromindolines in causing the extra-soft texture of oat grain. Further, hexaploid oats showed three orthologous genes coding for avenoindolines A and B, with five or three tryptophan residues, respectively, but very low amounts of avenoindolines were found in mature kernels. The present results identify a novel protein family affecting cereal kernel texture and would further elucidate the phylogenetic evolution of Avena genus. © 2014 Springer-Verlag Berlin Heidelberg.

A-PAGE fractionation of starch granule proteins from 63 bread wheat cultivars with contrasting grain texture characteristics revealed two prominent polypeptides and three minor ones, approximately 15 kDa in size. These proteins were found to be encoded by genes on the short arm of chromosome 5D. The two major friabilin components were assumed to correspond to puroindolines a and b (pinA and pinB), as suggested by PCR amplification of genes coding for pinA, glycine-type or serine-type pinB. Two electrophoretic patterns for pinA (presence vs absence) and three patterns for pinB were obtained by A-PAGE. In cultivars with pinA (allele Pina-D1a), pinB was found to be encoded by wild-type Pinb-D1a, serine-type Pinb-D1b or by the novel glycine-type b1 allele. Cultivars lacking pinA (allele Pina-D1b) were shown to contain eitherPinb-D1a or the novel b2 allele, both alleles coding for glycine-type pinB. The intensity of pinB in A-PAGE gels was found to be associated with grain hardness as determined by the SKCS method. Cultivars lacking pinA had the highest SKCS values, suggesting that both pinA and pinB may affect grain texture. In the presence of pinA, cultivars with wild-type allelePinb-D1a had soft grain texture, whereas those with alleles Pinb-D1b or b1 showed increased grain hardness. It is suggested that allele b1 affects the interaction of pinB with starch granules because of a sequence mutation different from the glycine-to-serine change.

Hordoindolines, the tryptophan-rich polypeptides affecting grain hardness in barley, appeared as three pairs of polypeptides in the acidic polyacrylamide gel electrophoresis (A-PAGE) and two-dimensional A-PAGE × SDS-PAGE patterns of starch-granule proteins from 18 barley cultivars. On capillary RP-HPLC/nESI-MS/MS spectrometry, one pair of polypeptides was found to correspond to hordoindoline A (HINA), one to hordoindoline B1 (HINB1) and one to hordoindoline B2 (HINB2), the two polypeptides of each pair deriving from post-translational cleavage of a native hordoindoline at different positions at the N-terminus and/or C-terminus. Amongst the barley cultivars analyzed, cvs Hart and Sundance, which were claimed to be unique in lacking the Hina gene coding for HINA, revealed similar Hina coding sequences and accumulated hordoindoline HINA on their starch granules. The amount of total hordoindolines (HINA + HINB1 + HINB2) on the starch granules, as quantified by densitometric scanning of A-PAGE gels, was comparable with that of puroindolines (PINA + PINB) in soft-textured wheat. By contrast, the amount of B-type hordoindolines (HINB1 and HINB2 combined) was 50 % lower than that of PINB, suggesting that the absence of barley cultivars with soft kernels is likely due to the reduced amount of B-type hordoindolines accumulated on the starch granules. Approximately 22 and 27 % of the phenotypic variation for kernel hardness in 56 barley cultivars analyzed by the Single Kernel Characterization System (SKCS) were explained by differences in kernel weight and B-type hordoindoline level, respectively. By contrast, the outer husk of barley grain showed no effect on the SKCS index.

Background and Aim: In the present paper, the toxicity of prolamines derived from three cereals with a different genome was investigated in human colon cancer Caco-2/TC7 and human myelogenous leukemia K562(S) cells. The purpose of this study was to investigate if species from ancient wheat could be considered as healthy food crops devoid or poor in cytotoxic prolamines for celiac disease. Methods: Cytotoxicity was measured in terms of inhibition of cell growth, activation of apoptosis, release of nitric oxide (NO), detection of tissue transglutaminase (TG II) and alteration of transepithelial electrical resistance (TEER) on Caco-2/Tc7 and K562 (S) cell agglutination. Peptic-tryptic (PT) digest from bread wheat (T. aestivum S. Pastore) was used as a positive control. Results: PT digests of prolamins from spelt wheat (T. aestivum ssp. spelta) were found to exert toxic effects on Caco-2/TC7 cells and to agglutinate K562(S) cells. Increased amounts of NO and TG II expression were observed in Caco-2/TC7 cells exposed to 1 mg/mL of spelt prolamins, suggesting that spelt wheat can induce cellular mechanisms implicated in the pathogenesis of celiac disease. By contrast, the PT digests from monoccum wheat (Triticum monococcum) and farro wheat (T. turgidum ssp. dicoccum) did not exhibit any negative effects on Caco-2/TC7 and K562(S) cells. Conclusions: The results have shown a constant and significant toxic effect of spelt wheat which is not shared by the two other ancient cereals. Future studies on celiac intestinal organ cultures are needed to increase the prospects of breeding programs aimed at developing wheat cultivars potentially tolerated by most celiac patients.