Preparation and Characterization of Avenin-Enriched Oat Protein by Chill Precipitation for Feeding Trials in Celiac Disease.

The safety of oats for people with celiac disease remains unresolved. While oats have attractive nutritional properties that can improve the quality and palatability of the restrictive, low fiber gluten-free diet, rigorous feeding studies to address their safety in celiac disease are needed. Assessing the oat prolamin proteins (avenins) in isolation and controlling for gluten contamination and other oat components such as fiber that can cause non-specific effects and symptoms is crucial. Further, the avenin should contain all reported immunogenic T cell epitopes, and be deliverable at a dose that enables biological responses to be correlated with clinical effects. To date, isolation of a purified food-grade avenin in sufficient quantities for feeding studies has not been feasible. Here, we report a new gluten isolation technique that enabled 2 kg of avenin to be extracted from 400 kg of wheat-free oats under rigorous gluten-free and food grade conditions. The extract consisted of 85% protein of which 96% of the protein was avenin. The concentration of starch (1.8% dry weight), ß-glucan (0.2% dry weight), and free sugars (1.8% dry weight) were all low in the final avenin preparation. Other sugars including oligosaccharides, small fructans, and other complex sugars were also low at 2.8% dry weight. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of the proteins in these preparations showed they consisted only of oat proteins and were uncontaminated by gluten containing cereals including wheat, barley or rye. Proteomic analysis of the avenin enriched samples detected more avenin subtypes and fewer other proteins compared to samples obtained using other extraction procedures. The identified proteins represented five main groups, four containing known immune-stimulatory avenin peptides. All five groups were identified in the 50% (v/v) ethanol extract however the group harboring the epitope DQ2.5-ave-1b was less represented. The avenin-enriched protein fractions were quantitatively collected by reversed phase HPLC and analyzed by MALDI-TOF mass spectrometry. Three reverse phase HPLC peaks, representing ~40% of the protein content, were enriched in proteins containing DQ2.5-ave-1a epitope. The resultant high quality avenin will facilitate controlled and definitive feeding studies to establish the safety of oat consumption by people with celiac disease.

The different effects of starch synthase IIa mutations or variation on endosperm amylose content of barley, wheat and rice are determined by the distribution of starch synthase I and starch branching enzyme IIb between the starch granule and amyloplast stroma.

KEY MESSAGE: The distribution of starch synthase I and starch branching enzyme IIb between the starch granule and amyloplast stroma plays an important role in determining endosperm amylose content of cereal grains. Starch synthase IIa (SSIIa) catalyses the polymerisation of intermediate length glucan chains of amylopectin in the endosperm of cereals. Mutations of SSIIa genes in barley and wheat and inactive SSIIa variant in rice induce similar effects on the starch structure and the amylose content, but the severity of the phenotypes is different. This study compared the levels of transcripts and partitioning of proteins of starch synthase I (SSI) and starch branching enzyme IIb (SBEIIb) inside and outside the starch granules in the developing endosperms of these ssIIa mutants and inactive SSIIa variant. Pleiotropic effects on starch granule-bound proteins suggested that the different effects of SSIIa mutations on endosperm amylose content of barley, wheat and rice are determined by the distribution of SSI and SBEIIb between the starch granule and amyloplast stroma in cereals. Regulation of starch synthesis in ssIIa mutants and inactive SSIIa variant may be at post-translational level or the altered amylopectin structure deprives the affinity of SSI and SBEIIb to amylopectin.

Molecular classification of barley (Hordeum vulgare L.) mutants using derivative NIR spectroscopy.

Near-infrared reflectance (NIR) spectroscopy was used in the characterization of grain morphology mutants of barley ( Hordeum vulgare L.) in relation to grain nitrogen (N) content and protein composition. Derivative spectroscopy provided spectra with enhanced resolution, allowing wavelengths to be identified with clear differences in contribution from associated chemical bonds. Comparisons of fourth-derivative spectra of wholemeal flour from high-N grains with flour from low-N grains identified wavelengths at which there were statistically significant differences between the groups. Their importance was independently confirmed by step-up regression using these wavelengths to generate an equation predicting N content (R(2) = 0.98). Fourth-derivative spectral comparisons also allowed novel biochemical differences to be predicted. Visual assessment of the spectra of all mutants revealed a variable region (1470-1520 nm, corresponding to N-H stretch vibrations) that allowed two extreme sets to be defined. The protein extracted from these two sets differed markedly in hordein content.

The Arabidopsis thaliana vernalization response requires a polycomb-like protein complex that also includes VERNALIZATION INSENSITIVE 3.

In Arabidopsis thaliana, the promotion of flowering by cold temperatures, vernalization, is regulated via a floral-repressive MADS box transcription factor, FLOWERING LOCUS C (FLC). Vernalization leads to the epigenetic repression of FLC expression, a process that requires the polycomb group (PcG) protein VERNALIZATION 2 (VRN2) and the plant homeodomain protein VERNALIZATION INSENSITIVE 3 (VIN3). We demonstrate that the repression of FLC by vernalization requires homologues of other Polycomb Repressive Complex 2 proteins and VRN2. We show in planta that VRN2 and VIN3 are part of a large protein complex that can include the PcG proteins FERTILIZATION INDEPENDENT ENDOSPERM, CURLY LEAF, and SWINGER. These findings suggest a single protein complex is responsible for histone deacetylation at FLC and histone methylation at FLC in vernalized plants. The abundance of the complex increases during vernalization and declines after plants are returned to higher temperatures, consistent with the complex having a role in establishing FLC repression.