Identification and Quantitation of Amylase Trypsin Inhibitors Across Cultivars Representing the Diversity of Bread Wheat.

α-Amylase/trypsin inhibitors (ATIs) may have a role in nonceliac wheat sensitivity (NCWS) and celiac disease (CD), but the ATI content and diversity across a range of wheat cultivars are not well characterized. Discovery proteomics was used to detect ATIs across two wheat cultivars: Chara and Magenta. Comprehensive mapping of detected ATIs with the ATIs from the recently published wheat genome RefSeq v1.0 shows the presence of three major subclasses: monomeric (9%), dimeric (61%), and chloroform-methanol (CM) type (30%). Subsequently, the level of 18 ATI isoforms (63 peptides) grouped into four subtypes was monitored across 15 commercial wheat cultivars and the eight parental lines from a multiparent advanced-generation intercross (MAGIC) population using liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS). The ATI content of wheat cultivars Janz, Sunvale, Diamond Bird, and Longreach Scout was significantly lower than that of other wheat cultivars. The MAGIC parental cultivars Baxter and Xiaoyan 54 contain higher levels (∼115% relative to the average wheat ATI content), whereas cultivar Pastor contained the lowest levels (∼87%). Comprehensive sequence analysis, annotation, chromosomal locations, and epitope mapping enabled us to build an LC-MRM-MS method to monitor and quantify the immunostimulatory ATI proteins potentially related to NCWS, autoimmune diseases, and metabolic disorders. This provides an opportunity to select wheat cultivars with significantly lower levels of ATIs.

Catcher of the Rye: Detection of Rye, a Gluten-Containing Grain, by LC-MS/MS.

Rye, wheat, and barley contain gluten, proteins that trigger immune-mediated inflammation of the small intestine in people with celiac disease (CD). The only treatment for CD is a lifelong gluten-free diet. To be classified as gluten-free by the World Health Organization the gluten content must be below 20 mg/kg, but Australia has a more rigorous standard of no detectable gluten and not made from wheat, barley, rye, or oats. The purpose of this study was to devise an LC-MS/MS method to detect rye in food. An MS-based assay could overcome some of the limitations of immunoassays, wherein antibodies often show cross-reactivity and lack specificity due to the diversity of gluten proteins in commercial food and the homology between rye and wheat gluten isoforms. Comprehensive proteomic analysis of 20 rye cultivars originating from 12 countries enabled the identification of a panel of candidate rye-specific peptide markers. The peptide markers were assessed in 16 cereal and pseudocereal grains, and in 10 breakfast cereals and 7 snack foods. One of two spelt flours assessed was contaminated with rye at a level of 2%, and trace levels of rye were found in a breakfast cereal that should be gluten-free based on its labeled ingredients.

Proteomics: Tools of the Trade.

The proteome represents the total set of proteins produced by an organism or a system at a particular time or state, with proteomics being the study of the proteome. The proteome is a dynamic system wherein proteins are interconnected and serve to facilitate cellular processes in a concurrent and coordinated manner. Over the years, various biochemical and biophysical methods have been developed to elucidate the identities, structures and functions of proteins in order to understand their roles in complex biological systems. The success of proteomic approaches hinges on efficient protein extraction and sample preparation; however, these preliminary steps are often considered a bottleneck in proteomic workflows. Every biological sample is unique and complex, and sample processing needs to be tailored to the nature of the protein sample due to its vulnerability towards post-collection degradation and the complexity of its non-protein constituents. Sample pretreatment steps often employ buffers, solvents, salts and detergents that are not always compatible with the downstream analytical tools. This chapter will provide an overview of sample pretreatment techniques commonly used in conjunction with proteomics tools and discuss protein analysis methods. Such methods include the use of antibody-based techniques, separation sciences (e.g. chromatography, SDS-PAGE), detection methods (e.g. mass spectrometry) and structural techniques (e.g. NMR and X-ray crystallography).

Assessing the Utility of Multiplexed Liquid Chromatography-Mass Spectrometry for Gluten Detection in Australian Breakfast Food Products.

Coeliac disease (CD) is an autoimmune disorder triggered by the ingestion of gluten that is associated with gastrointestinal issues, including diarrhea, abdominal pain, and malabsorption. Gluten is a general name for a class of cereal storage proteins of wheat, barley, and rye that are notably resistant to gastrointestinal digestion. After ingestion, immunogenic peptides are subsequently recognized by T cells in the gastrointestinal tract. The only treatment for CD is a life-long gluten-free diet. As such, it is critical to detect gluten in diverse food types, including those where one would not expect to find gluten. The utility of liquid chromatography-mass spectrometry (LC-MS) using cereal-specific peptide markers to detect gluten in heavily processed food types was assessed. A range of breakfast products, including breakfast cereals, breakfast bars, milk-based breakfast drinks, powdered drinks, and a savory spread, were tested. No gluten was detected by LC-MS in the food products labeled gluten-free, yet enzyme-linked immunosorbent assay (ELISA) measurement revealed inconsistencies in barley-containing products. In products containing wheat, rye, barley, and oats as labeled ingredients, gluten proteins were readily detected using discovery proteomics. Panels comprising ten cereal-specific peptide markers were analyzed by targeted proteomics, providing evidence that LC-MS could detect and differentiate gluten in complex matrices, including baked goods and milk-based products.

Comparing Multiple Reaction Monitoring and Sequential Window Acquisition of All Theoretical Mass Spectra for the Relative Quantification of Barley Gluten in Selectively Bred Barley Lines.

Celiac disease (CD) is a disease of the small intestine that occurs in genetically susceptible subjects triggered by the ingestion of cereal gluten proteins for which the only treatment is strict adherence to a life-long gluten-free diet. Barley contains four gluten protein families, and the existence of barley genotypes that do not accumulate the B-, C-, and D-hordeins paved the way for the development of an ultralow gluten phenotype. Using conventional breeding strategies, three null mutations behaving as recessive alleles were combined to create a hordein triple-null barley variety. Proteomics has become an invaluable tool for characterization and quantification of the protein complement of cereal grains. In this study multiple reaction monitoring (MRM) mass spectrometry, viewed as the gold standard for peptide quantification, was compared to the data-independent acquisition strategy known as SWATH-MS (sequential window acquisition of all theoretical mass spectra). SWATH-MS was comparable (p < 0.001) to MRM-MS for 32/33 peptides assessed across the four families of hordeins (gluten) in eight barley lines. The results of SWATH-MS analysis further confirmed the absence of the B-, C-, and D-hordeins in the triple-null barley line and showed significantly reduced levels ranging from <1% to 16% relative to wild-type (WT) cv Sloop for the minor γ-hordein class. SWATH-MS represents a valuable tool for quantitative proteomics based on its ability to generate reproducible data comparable with MRM-MS, but has the added benefits of allowing reinterrogation of data to improve analytical performance, ask new questions, and in this case perform quantification of trypsin-resistant proteins (C-hordeins) through analysis of their semi- or nontryptic fragments.

Creation of the first ultra-low gluten barley (Hordeum vulgare L.) for coeliac and gluten-intolerant populations.

Coeliac disease is a well-defined condition that is estimated to affect approximately 1% of the population worldwide. Noncoeliac gluten sensitivity is a condition that is less well defined, but is estimated to affect up to 10% of the population, and is often self-diagnosed. At present, the only remedy for both conditions is a lifelong gluten-free diet. A gluten-free diet is often expensive, high in fat and low in fibre, which in themselves can lead to adverse health outcomes. Thus, there is an opportunity to use novel plant breeding strategies to develop alternative gluten-free grains. In this work, we describe the breeding and characterization of a novel ultra-low gluten (ULG) barley variety in which the hordein (gluten) content was reduced to below 5 ppm. This was achieved using traditional breeding strategies to combine three recessive alleles, which act independently of each other to lower the hordein content in the parental varieties. The grain of the initial variety was shrunken compared to wild-type barleys. We implemented a breeding strategy to improve the grain size to near wild-type levels and demonstrated that the grains can be malted and brewed successfully. The ULG barley has the potential to provide novel healthy foods and beverages for those who require a gluten-free diet.

Engineering high α-amylase levels in wheat grain lowers Falling Number but improves baking properties.

Late maturity α-amylase (LMA) and preharvest sprouting (PHS) are genetic defects in wheat. They are both characterized by the expression of specific isoforms of α-amylase in particular genotypes in the grain prior to harvest. The enhanced expression of α-amylase in both LMA and PHS results in a reduction in Falling Number (FN), a test of gel viscosity, and subsequent downgrading of the grain, along with a reduced price for growers. The FN test is unable to distinguish between LMA and PHS; thus, both defects are treated similarly when grain is traded. However, in PHS-affected grains, proteases and other degradative process are activated, and this has been shown to have a negative impact on end product quality. No studies have been conducted to determine whether LMA is detrimental to end product quality. This work demonstrated that wheat in which an isoform α-amylase (TaAmy3) was overexpressed in the endosperm of developing grain to levels of up to 100-fold higher than the wild-type resulted in low FN similar to those seen in LMA- or PHS-affected grains. This increase had no detrimental effect on starch structure, flour composition and enhanced baking quality, in small-scale 10-g baking tests. In these small-scale tests, overexpression of TaAmy3 led to increased loaf volume and Maillard-related browning to levels higher than those in control flours when baking improver was added. These findings raise questions as to the validity of the assumption that (i) LMA is detrimental to end product quality and (ii) a low FN is always indicative of a reduction in quality. This work suggests the need for a better understanding of the impact of elevated expression of specific α-amylase on end product quality.

Suppression of glucan, water dikinase in the endosperm alters wheat grain properties, germination and coleoptile growth.

Starch phosphate ester content is known to alter the physicochemical properties of starch, including its susceptibility to degradation. Previous work producing wheat (Triticum aestivum) with down-regulated glucan, water dikinase, the primary gene responsible for addition of phosphate groups to starch, in a grain-specific manner found unexpected phenotypic alteration in grain and growth. Here, we report on further characterization of these lines focussing on mature grain and early growth. We find that coleoptile length has been increased in these transgenic lines independently of grain size increases. No changes in starch degradation rates during germination could be identified, or any major alteration in soluble sugar levels that may explain the coleoptile growth modification. We identify some alteration in hormones in the tissues in question. Mature grain size is examined, as is Hardness Index and starch conformation. We find no evidence that the increased growth of coleoptiles in these lines is connected to starch conformation or degradation or soluble sugar content and suggest these findings provide a novel means of increasing coleoptile growth and early seedling establishment in cereal crop species.

Proteomic profiling of 16 cereal grains and the application of targeted proteomics to detect wheat contamination.

Global proteomic analysis utilizing SDS-PAGE, Western blotting and LC-MS/MS of total protein and gluten-enriched extracts derived from 16 economically important cereals was undertaken, providing a foundation for the development of MS-based quantitative methodologies that would enable the detection of wheat contamination in foods. The number of proteins identified in each grain correlated with the number of entries in publicly available databases, highlighting the importance of continued advances in genome sequencing to facilitate accurate protein identification. Subsequently, candidate wheat-specific peptide markers were evaluated by multiple-reaction monitoring MS. The selected markers were unique to wheat, yet present in a wide range of wheat varieties that represent up to 80% of the bread wheat genome. The final analytical method was rapid (15 min) and robust (CV < 10%), showed linearity (R(2) > 0.98) spanning over 3 orders of magnitude, and was highly selective and sensitive with detection down to 15 mg/kg in intentionally contaminated soy flour. Furthermore, application of this technology revealed wheat contamination in commercially sourced flours, including rye, millet, oats, sorghum, buckwheat and three varieties of soy.

Engineering α-amylase levels in wheat grain suggests a highly sophisticated level of carbohydrate regulation during development.

Wheat starch degradation requires the synergistic action of different amylolytic enzymes. Our spatio-temporal study of wheat α-amylases throughout grain development shows that AMY3 is the most abundant isoform compared with the other known α-amylases. Endosperm-specific over-expression of AMY3 resulted in an increase of total α-amylase activity in harvested grains. Unexpectedly, increased activity did not have a significant impact on starch content or composition but led to an increase of soluble carbohydrate (mainly sucrose) in dry grain. In AMY3 overexpression lines (A3OE), germination was slightly delayed and triacylglycerol (TAG) content was increased in the endosperm of mature grain. Despite increased AMY3 transcript and protein content throughout grain development, alterations of α-amylase activity and starch granule degradation were not detected until grain maturation, suggesting a post-translational inhibition of α-amylase activity in the endosperm during the starch filling period. These findings show unexpected effects of a high level of α-amylase on grain development and composition, notably in carbon partitioning and TAG accumulation, and suggest the presence of a hitherto unknown regulatory pathway during grain filling.

Fast-tracking development of homozygous transgenic cereal lines using a simple and highly flexible real-time PCR assay.

BACKGROUND: A crucial step in the evaluation of newly produced transgenic plants is the selection of homozygous plants. Here we describe an efficient and highly flexible real-time PCR-based method for the development of homozygous lines in plant models with complex (multiple) genomes and/or relatively long generation times (>3 months) using direct copy number determinations. RESULTS: An existing DNA extraction method was converted into a high-throughput plant leaf DNA extraction procedure yielding DNA suitable for real-time PCR analyses. Highly specific and efficient primer pairs were developed for a bread wheat reference gene (Epsilon Cyclase) and for standard sequence elements in the gene cassette routinely used for cereal transformations (an intron bridge and the Nopaline Synthase terminator). The real-time PCR assay reliably distinguished wheat plants with a single copy of the transgene from individuals with multiple copies or those lacking the transgene. To obtain homozygous lines carrying a unique insertion event as efficiently as possible, T0 plants (plants raised from transformed callus) with a single copy of the transgene were selected and their progeny screened for homozygous plants. Finally, the assay was adapted to work on rice. CONCLUSIONS: The ability to quickly, easily and accurately quantify the construct copy numbers, as provided by the real-time PCR assay, greatly improved the efficiency and reliability of the selection of homozygous transgenic plants in our case study. We were able to select homozygous plants in early generations, avoiding time-consuming methods such as large scale analysis of segregation patterns of descendants and/or Southern blotting. Additionally, the ability to specifically develop homozygous lines carrying a unique insertion event could be important in avoiding gene silencing due to co-suppression, and if needed assist in the selection of lines suitable for future deregulation. The same primer pairs can be used to quantify many different wheat transgenic events because the construct-specific primer pairs are targeted to standard sequence elements of the cereal gene cassettes, making the method widely applicable in wheat GM research. Moreover, because all procedures described here are standardized, the method may easily be adapted to vectors lacking the target regions used here and/or to other plant models.

Characterization of starch phosphorylases in barley grains.

BACKGROUND: Starch is synthesized in both leaves and storage tissues of plants. The role of starch syntheses and branching enzymes is well understood; however, the role of starch phosphorylase is not clear. RESULTS: A gene encoding Pho1 from barley was characterized and starch phosphorylases from both developing and germinating grain were characterized and purified. Two activities were detected: one with a molecular mass of 110 kDa and the other of 95 kDa. It was demonstrated through the use of antisera that the 110 kDa activity was located in the amyloplast and could correspond to the polypeptide encoded by the Pho1 gene cloned. The 95 kDa activity was localized to the cytoplasm, most strongly expressed in germinating grain, and was classified as a Pho2-type sequence. Using RNAi technology to reduce the content of Pho1 in the grain to less than 30% of wild type did not lead to any visible phenotype, and no dramatic alterations in the structure of the starch were observed. CONCLUSION: Two starch phosphorylase activities were identified and characterized in barley grains, and shown to be present during starch synthesis. However, their role in starch synthesis still remains to be elucidated.

Proteome Changes Resulting from Malting in Hordein-Reduced Barley Lines.

Hordeum vulgare L., commonly known as barley, is primarily used for animal feed and malting. The major storage proteins in barley are hordeins, known triggers of celiac disease (CD). Here, sequential window acquisition of all theoretical mass spectra (SWATH)-MS proteomics was employed to investigate the proteome profile of grain and malt samples from the malting barley cultivar Sloop and single-, double-, and triple hordein-reduced lines bred in a Sloop background. Using a discovery proteomics approach, 2688 and 3034 proteins were detected from the grain and malt samples, respectively. By utilizing label-free relative quantitation through SWATH-MS, a total of 2654 proteins have been quantified from grain and malt. The comparative analyses between the barley grain and malt samples revealed that the C-hordein-reduced lines have a more significant impact on proteome level changes due to malting than B- and D-hordein-reduced lines. Upregulated proteins in C-hordein-reduced lines were primarily involved in the tricarboxylic acid cycle and fatty acid peroxidation processes to provide more energy for seed germination during malting. By applying proteomics approaches after malting in hordein-reduced barley lines, we uncovered additional changes in the proteome driven by the genetic background that were not apparent in the sound grain. Our findings offer valuable insights for barley breeders and maltsters seeking to understand and optimize the performance of gluten-free grains in malt products.

What is in a beer? Proteomic characterization and relative quantification of hordein (gluten) in beer.

The suite of prolamin proteins present in barley flour was characterized in this study, in which we provide spectral evidence for 3 previously characterized prolamins, 8 prolamins with only transcript evidence, and 19 genome-derived predicted prolamins. An additional 9 prolamins were identified by searching the complete spectral set against an unannotated translated EST database. Analyses of wort, the liquid extracted from the mashing process during beer production, and beer were undertaken and a similar suite of prolamins were identified. We have demonstrated by using tandem mass spectrometry that hordeins are indeed present in beer despite speculation to the contrary. Multiple reaction monitoring (MRM) mass spectrometry was used for the rapid analyses of hordein in barley (Hordeum vulgare L.) beer. A selection of international beers were analyzed and compared to the results obtained with hordein deletion beers. The hordein deletion beers were brewed from grains carrying mutations that prevented the accumulation of either B-hordeins (Risø 56) or C-hordeins (Risø 1508). No intact C-hordeins were detected in beer, although fragments of C-hordeins were present in wort. Multiple reaction monitoring analysis of non-barley based gluten (hordein)-free beers targeting the major hordein protein families was performed and confirmed the absence of hordein in several gluten-free commercial beers.

Down-regulation of Glucan, Water-Dikinase activity in wheat endosperm increases vegetative biomass and yield.

A novel mechanism for increasing vegetative biomass and grain yield has been identified in wheat (Triticum aestivum). RNAi-mediated down-regulation of Glucan, Water-Dikinase (GWD), the primary enzyme required for starch phosphorylation, under the control of an endosperm-specific promoter, resulted in a decrease in starch phosphate content and an increase in grain size. Unexpectedly, consistent increases in vegetative biomass and grain yield were observed in subsequent generations. In lines where GWD expression was decreased, germination rate was slightly reduced. However, significant increases in vegetative growth from the two leaf stage were observed. In glasshouse pot trials, down-regulation of GWD led to a 29% increase in grain yield while in glasshouse tub trials simulating field row spacing and canopy development, GWD down-regulation resulted in a grain yield increase of 26%. The enhanced yield resulted from a combination of increases in seed weight, tiller number, spikelets per head and seed number per spike. In field trials, all vegetative phenotypes were reproduced with the exception of increased tiller number. The expression of the transgene and suppression of endogenous GWD RNA levels were demonstrated to be grain specific. In addition to the direct effects of GWD down-regulation, an increased level of α-amylase activity was present in the aleurone layer during grain maturation. These findings provide a potentially important novel mechanism to increase biomass and grain yield in crop improvement programmes.

Down-Regulation of FAD2-1 Gene Expression Alters Lysophospholipid Composition in the Endosperm of Rice Grain and Influences Starch Properties.

Small quantities of lipids accumulate in the white rice grains. These are grouped into non-starch lipid and starch lipid fractions that affect starch properties through association with starch. Lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) are two major lipid classes in the two fractions. Using high-oleic rice grains, we investigated the fatty-acid composition in flour and starch by LC-MS and evaluated its impact on starch properties. In the wild-type grain, nearly 50% of fatty acids in LPC and LPE were palmitic acid (C16:0), over 20% linoleic acid (C18:2) and less than 10% oleic acid (C18:1). In the high-oleic rice grain, C18:1 increased at the expense of C18:2 and C16:0. The compositional changes in starch lipids suggest that LPC and LPE are transported to an amyloplast with an origin from endoplasmic reticulum-derived PC and PE during endosperm development. The high-dissociation temperature of the amylose-lipid complex (ALC) and restricted starch swelling power in the high-oleic rice starch indicates that the stability of the ALC involving C18:1 is higher than that of C18:2 and C16:0. This study provides insight into the lipid deposition and starch properties of rice grains with optimized fatty-acid composition.

Perennial Ryegrass Contains Gluten-Like Proteins That Could Contaminate Cereal Crops.

Background: To ensure safe consumption of gluten-free products, there is a need to understand all sources of unintentional contamination with gluten in the food chain. In this study, ryegrass (Lolium perenne), a common weed infesting cereal crop, is analysed as a potential source of gluten-like peptide contamination. Materials and Methods: Ten ryegrass cultivars were analysed using shotgun proteomics for the presence of proteins from the prolamin superfamily. A relative quantitative assay was developed to detect ryegrass gluten-like peptides in comparison with those found in 10 common wheat cultivars. Results: A total of 19 protein accessions were found across 10 cultivars of ryegrass for the protein families of PF00234-Tryp_alpha_amyl, PF13016-Gliadin, and PF03157-Glutenin_HMW. Protein and peptide homology searches revealed that gliadin-like peptides were similar to avenin and gamma-gliadin peptides. A total of 20 peptides, characteristic of prolamin superfamily proteins, were selected for liquid chromatography mass spectrometry (LC-MS) with multiple reaction monitoring (MRM). Only two of the monitored peptides were detected with high abundance in wheat, and all others were detected in ryegrass. Glutenin and alpha-amylase/trypsin inhibitor peptides were reported for the first time in ryegrass and were noted to be conserved across the Poaceae family. Conclusion: A suite of gluten-like peptides were identified using proteomics that showed consistent abundance across ryegrass cultivars but were not detected in wheat cultivars. These peptides will be useful for differentiating wheat gluten contamination from ryegrass gluten contamination.

Proteome and Nutritional Shifts Observed in Hordein Double-Mutant Barley Lines.

Lysine is the most limiting essential amino acid in cereals, and efforts have been made over the decades to improve the nutritional quality of these grains by limiting storage protein accumulation and increasing lysine content, while maintaining desired agronomic traits. The single lys3 mutation in barley has been shown to significantly increase lysine content but also reduces grain size. Herein, the regulatory effect of the lys3 mutation that controls storage protein accumulation as well as a plethora of critically important processes in cereal seeds was investigated in double mutant barley lines. This was enabled through the generation of three hordein double-mutants by inter-crossing three single hordein mutants, that had all been backcrossed three times to the malting barley cultivar Sloop. Proteome abundance measurements were integrated with their phenotype measurements; proteins were mapped to chromosomal locations and to their corresponding functional classes. These models enabled the prediction of previously unknown points of crosstalk that connect the impact of lys3 mutations to other signalling pathways. In combination, these results provide an improved understanding of how the mutation at the lys3 locus remodels cellular functions and impact phenotype that can be used in selective breeding to generate favourable agronomic traits.

Rice with Multilayer Aleurone: A Larger Sink for Multiple Micronutrients.

Diet-related noncommunicable diseases impose a heavy burden on human health worldwide. Rice is a good target for diet-related disease prevention strategies because it is widely consumed. Liu et al. (Proc Natl Acad Sci USA 115(44):11327-11332, 2018. https://doi.org/10.1073/pnas.1806304115 ) demonstrated that increasing the number of cell layers and thickness of putative aleurone in ta2-1 (thick aleurone 2-1) mutant rice enhances simultaneously the content of multiple micronutrients. However, the increases of aleurone-associated nutrients were not proportional to the increases in the aleurone thickness. In this study, first, cytohistological analyses and transmission electron microscopy demonstrated that the multilayer in ta2-1 exhibited aleurone cell structural features. Second, we detected an increase in insoluble fibre and insoluble bound-phenolic compounds, a shift in aleurone-specific neutral non-starch polysaccharide profile, enhancement of phytate and minerals such as iron, zinc, potassium, magnesium, sulphur, and manganese, enrichment of triacylglycerol and phosphatidylcholine but slight reduction in free fatty acid, and an increase in oleic fatty acid composition. These findings support our hypothesis that the expanded aleurone-like layers in ta2-1 maintained some of the distinctive aleurone features and composition. We provide perspectives to achieve even greater filling of this expanded micronutrient sink to provide a means for multiple micronutrient enhancements in rice.