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Hordein accumulation in developing barley grains

ABSTRACT: The temporal pattern of accumulation of hordein storage proteins in developing barley grains was studied by enzyme-linked immunosorbent assay (ELISA), western blot and liquid chromatography tandem mass spectrometry (LC-MS/MS). Hordein accumulation was compared to the pattern seen for two abundant control proteins, serpin Z4 (an early accumulator) and lipid transferase protein (LTP1, a late accumulator). Hordeins were detected from six days post-anthesis (DPA) and peaked at 30 DPA. Changes in fresh weight indicate that desiccation begins at 20 DPA and by 37 DPA fresh weight had decreased by 35%. ELISA analysis of hordein content, expressed on a protein basis, increased to a maximum at 30 DPA followed by a 17% decrease by 37 DPA. The accumulation of 39 tryptic and 29 chymotryptic hordein peptides representing all classes of hordein was studied by LC-MS/MS. Most peptides increased to a maximum at 30 DPA, and either remained at the maximum or did not decrease significantly. Only five tryptic peptides, members of the related B1- and γ1-hordeins decreased significantly by 21-51% at 37 DPA. Thus, the concentration of some specific peptides wasere reduced while remaining members of the same family were not affected. The N-terminal signal region was removed by proteolysis during co-translation. , evidenced by a notable absence of the predicted peptides despite near complete protein sequence coverage. In addition to a suite of previously characterised hordeins, two novel barley B-hordein isoforms and two avenin-like proteins (ALPs), mapping to wheat low molecular weight glutenins (LMW-GS-like B-hordeins), and two avenin-like proteins (ALPs) sharing homology with wheat ALPs, were identified. These identified isoforms have not previously been mapped in the barley genome. Cereal storage proteins provide significant nutritional content for human consumption and seed germination. In barley, the bulk of the storage proteins are due tocomprise the hordein family and the final hordein concentration affects the quality of baked and brewed products. Hordeins are members of the gluten protein family and consumption can trigger health conditions such as coeliac disease, non-coeliac gluten sensitivity, and gluten allergy. It is therefore important to study the accumulation of these proteinshordeins as this knowledge may assist plant breeding for improved health outcomes (by minimizing triggering of detrimental immune responses), nutrition and food reduced coeliac reactivity.processing properties.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Hordeum Vulgare (barley)

TISSUE(S): Endosperm

SUBMITTER: Michelle Colgrave

LAB HEAD: Michelle Lisa Colgrave

PROVIDER: PXD013331 | Pride | 2019-04-18

REPOSITORIES: Pride

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Project description:The consumption of wheat, rye and barley may cause adverse reactions to wheat such as celiac disease (CD), non-celiac gluten/wheat sensitivity (NCGS) or wheat allergy. The storage proteins (gluten) are known as major triggers, but also other functional protein groups such as α-amylase/trypsin-inhibitors or enzymes are possibly harmful for people suffering of adverse reactions to wheat. Gluten is widely used as a collective term for the complex protein mixture of wheat, rye or barley and can be subdivided into the following gluten protein types (GPTs): α-gliadins, γ-gliadins, ω5-gliadins, ω1,2-gliadins, high- (HMW-GS) and low-molecular-weight glutenin subunits (LMW-GS) of wheat, ω-secalins, HMW-secalins, γ-75k-secalins and γ-40k-secalins of rye and C-hordeins, γ-hordeins, B-hordeins and D-hordeins of barley. GPTs isolated from the flours are useful as reference materials for clinical studies, diagnostics or in food analyses and to elucidate disease mechanisms. A combined strategy of protein separation according to solubility followed by preparative reversed-phase high-performance liquid chromatography (RP-HPLC) was employed to purify the GPTs according to hydrophobicity. Due to the heterogeneity of gluten proteins and their partly polymeric nature, it is a challenge to obtain highly purified GPTs with only one protein group. Therefore, it is essential to characterize and identify the proteins and their proportions in each GPT. In this study, the complexity of gluten from wheat, rye, and barley was demonstrated by identification of the individual proteins employing an undirected proteomics strategy involving liquid chromatography-tandem mass spectrometry (LC-MS/MS) of tryptic and chymotryptic hydrolysates of the GPTs. Different protein groups were obtained and the relative composition of the GPTs was revealed. Multiple reaction monitoring (MRM) LC-MS/MS was used for the relative quantitation of the most abundant gluten proteins. These analyses also allowed the identification of known wheat allergens and CD-active peptides. Combined with functional assays, these findings may shed light on the mechanisms of gluten/wheat-related disorders and may be useful to characterize reference materials for analytical or diagnostic assays more precisely.

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Hordein accumulation in developing barley grains

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