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.

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.

Perspectives on Future Protein Production.

An increasing world population, rising affluence, urbanization, and changing eating habits are all contributing to the diversification of protein production. Protein is a building block of life and is an essential part of a healthy diet, providing amino acids for growth and repair. The challenges and opportunities for production of protein-rich foods from animals (meat, dairy, and aquaculture), plant-based sources (pulses), and emerging protein sources (insects, yeast, and microalgae) are discussed against the backdrop of palatability, nutrition, and sustainability.

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.

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.

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.

Proteome Analysis of Hordein-Null Barley Lines Reveals Storage Protein Synthesis and Compensation Mechanisms.

Hordeins are the major barley seed storage proteins and are elicitors of celiac disease. Attempts to reduce the hordein level in barley have been made; however, the resultant pleiotropic effects are less understood. Here, data-independent acquisition mass spectrometry was used to measure proteome-wide abundance differences between wild-type and single hordein-null barley lines. Using comparative quantitative proteomics, we detected proteome-wide changes (∼59%) as a result of the specific reduction in hordein proteins. The comparative analysis and functional annotation revealed an increase in non-gluten storage proteins, such as globulins and lipid transfer proteins, and proteins rich in essential amino acids in the null lines. This study yields an informative molecular portrait of the hordein-null lines and the underlying mechanisms of storage protein biosynthesis. This study indicates the extent to which protein content can be manipulated without biological consequence, and we envision its wide-scale application for studying modified crops.

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.

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.

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.

Hordein Accumulation in Developing Barley Grains.

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 6 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 was reduced while remaining members of the same family were not affected. The N-terminal signal region was removed by proteolysis during co-translation. In addition to a suite of previously characterized hordeins, two novel barley B-hordein isoforms 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 comprise the hordein family and the final hordein concentration affects the quality of baked and brewed products. It is therefore important to study the accumulation of hordeins as this knowledge may assist plant breeding for improved health outcomes (by minimizing triggering of detrimental immune responses), nutrition and food processing properties.

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).

Targeted proteomics to monitor the extraction efficiency and levels of barley α-amylase trypsin inhibitors that are implicated in non-coeliac gluten sensitivity.

Plant defense protein α-amylase trypsin inhibitors (ATIs) have been proposed as one of the triggers of non-coeliac gluten sensitivity, however there have been no focused studies on their optimal extraction and quantitation from cereal grains. The efficiency of extraction is of utmost interest for the downstream detection and characterisation. In the present study, three extraction buffers and two modified protocols were investigated using LC-MRM-MS in order to examine their ability to efficiently and repeatably extract ATIs from selected barley cultivars. Initially, three extraction buffers IPA/DTT, urea and Tris-HCl were used to extract ATIs from two selected barley cultivars, Commander and Hindmarsh. The results obtained from the preliminary study showed that IPA/DTT and urea-based buffer extraction could yield ∼70% and ∼45% more ATIs, respectively than a buffer based on Tris-HCl extraction, with all methods showing high repeatability (CV < 15%). A multi-step protocol, employing IPA/DTT and urea improved the extraction efficiency in comparison to the single buffer extraction protocols (p<0.0001). When solutions from parallel extractions using IPA/DTT and urea were combined, the results were comparable (p = 0.99) with a sequential multi-step IPA/DTT-urea protocol. However, the repeatability of the combined process was compromised, as discerned by greater variation (CV>30%). The optimised sequential two-step extraction protocol was successfully used to extract and quantify ATIs from 12 barley cultivars. LC-MS analysis revealed that cv Yagan and cv Scope contain the higher levels (∼143% relative to the average barley ATI content), whereas cultivars Fleet (61%), Baudin (77%) and Commander (79%) contained the lowest levels. The libraries of ATIs identified and the quantitative methods described here provide a foundation for the future application of MS-based quantitative methodologies to detect and quantify ATIs in barley varieties and in food products.

Optimisation of protein extraction for in-depth profiling of the cereal grain proteome.

Cereal grain proteomics can provide valuable information regarding plant growth, nutritional status, adaptation to environmental stresses, and the role that grain proteins play in health disorders, such as coeliac disease. In this study liquid chromatography-mass spectrometry was used to compare the barley proteome after extraction using seven protocols, with and without defatting and protein precipitation steps that aimed to remove interfering secondary metabolites. Tris-HCl and urea buffers yielded 1405 and 1483 proteins (~79% overlap) from barley (cv Sloop). Inclusion of a pre-extraction defatting step yielded 1336 (Tris-HCl) and 1286 (urea) proteins (~74% overlap). Whilst post-extraction TCA/acetone protein precipitation negatively impacted protein recovery, yielding 673 (Tris-HCl) and 734 (urea) proteins. Alcohol-based extraction yielded a lower number of proteins (645), but notably this extraction method co-extracted and enriched the gluten and α-amylase trypsin inhibitors. Based on these preliminary results, proteins were extracted from two selected cultivars of wheat, rye, barley and oats using three extraction protocols. Bioinformatic analyses of the identified proteins provide evidence that the choice of extraction buffer enriches different protein functional classes. The selection of the protein extraction protocol directly influences the identified cereal grain proteome composition, thus affecting the downstream biological interpretation of data. SIGNIFICANCE: LC-MS/MS and bioinformatics analysis revealed that both Tris-HCl and urea-based extraction yielded a similar suite of proteins from cereal grains with remarkable (70-80%) overlap. Yet the peptides derived from the proteins differed, rendering these extraction buffers complementary, in particular resulting in improved protein sequence coverage. The inclusion of commonly incorporated practices, such as pre-extraction defatting or post-extraction precipitation steps offered no benefit. The extraction method selected was noted to impact the downstream functional annotation results and biological interpretation.

Efficient Extraction and Digestion of Gluten Proteins.

Coeliac disease (CD) is a T-cell mediated autoimmune disorder triggered by ingestion of cereal gluten found in wheat (gliadins and glutenins), barley (hordeins), and rye (secalins). As the only treatment for CD is a lifelong gluten-free diet, the measurement of gluten in raw ingredients and processed food products is critical to protecting people with CD or gluten intolerance. The most commonly employed method is the enzyme-linked immunosorbent assay (ELISA), but more recently mass spectrometry has been employed wherein the extracted gluten proteins are digested to peptides that are then directly measured. To achieve the goal of accurate gluten quantitation, gluten must be efficiently extracted from the ingredient or food matrix and then digested to yield the peptides that are monitored by LC-MS. In this chapter, a rapid, simple, and reproducible protocol for extraction and digestion of gluten proteins is described.

Does Late Maturity Alpha-Amylase Impact Wheat Baking Quality?

Late maturity α-amylase (LMA) and pre-harvest sprouting (PHS) are both recognized as environmentally induced grain quality defects resulting from abnormally high levels of α-amylase. LMA is a more recently identified quality issue that is now receiving increasing attention worldwide and whose prevalence is now seen as impeding the development of superior quality wheat varieties. LMA is a genetic defect present in specific wheat genotypes and is characterized by elevated levels of the high pI TaAMY1 α-amylase, triggered by environmental stress during wheat grain development. TaAMY1 remains present in the aleurone through the harvest, lowering Falling Number (FN) at receival, causing a down-grading of the grain, often to feed grade, thus reducing the farmers' income. This downgrading is based on the assumption within the grain industry that, as for PHS, a low FN represents poor quality grain. Consequently any wheat line possessing low FN or high α-amylase levels is automatically considered a poor bread wheat despite there being no published evidence to date, to show that LMA is detrimental to end product quality. To evaluate the validity of this assumption a comprehensive evaluation of baking properties was performed from LMA prone lines using a subset of tall non-Rht lines from a multi-parent advanced generation inter-cross (MAGIC) wheat population grown at three different sites. LMA levels were determined along with quality parameters including end product functionality such as oven spring, bread loaf volume and weight, slice area and brightness, gas cell number and crumb firmness. No consistent or significant phenotypic correlation was found between LMA related FN and any of the quality traits. This manuscript provides for the first time, compelling evidence that LMA has limited impact on bread baking end product functionality.

Using LC-MS to examine the fermented food products vinegar and soy sauce for the presence of gluten.

A strict, lifelong gluten-free (GF) diet is currently the only treatment for coeliac disease (CD). Vinegar and soy sauce are fermented condiments that often include wheat and/or barley. During fermentation cereal proteins are partially degraded by enzymes to yield peptide fragments and amino acids. Whether these fermented products contain intact or degraded gluten proteins and if they are safe for people with CD remains in question. LC-MS offers the benefit of being able to detect hydrolysed gluten that might be present in commercial vinegar and soy sauce products. LC-MS revealed the presence of gluten in malt vinegar, wherein the identified peptides derived from B-, D- and γ-hordein from barley, as well as γ-gliadin, and HMW- and LMW-glutenins from wheat that are known to contain immunopathogenic epitopes. No gluten was detected in the soy sauces examined despite wheat being a labelled ingredient indicating extensive hydrolysis of gluten during soy sauce production.

Liquid Chromatography-Mass Spectrometry Analysis Reveals Hydrolyzed Gluten in Beers Crafted To Remove Gluten.

During brewing, gluten proteins may be solubilized, modified, complexed, hydrolyzed, and/or precipitate. Gluten fragments that persist in conventional beers render them unsuitable for people with celiac disease (CD) or gluten intolerance. Barley-based beers crafted to remove gluten using proprietary precipitation and/or application of enzymes, e.g. prolyl endopeptidases (PEP) that degrade the proline-rich gluten molecules, are available commercially. Gluten measurement in fermented products remains controversial. The industry standard, a competitive ELISA, may indicate gluten values <20 mg/kg, which is deemed safe for people with CD. However, in this study, liquid chromatography-mass spectrometry analyses revealed gluten peptides derived from hydrolyzed fragments, many >30 kDa in size. Barley gluten (hordeins) were detected in all beers analyzed with peptides representing all hordein classes detected in conventional beers but also, alarmingly, in many gluten-reduced beers. It is evident that PEP digestion was incomplete in several commercial beers, and peptides comprising missed cleavages were identified, warranting further optimization of PEP application in an industrial setting.

Food for thought: Selecting the right enzyme for the digestion of gluten.

Gluten describes a complex mixture of proteins found in wheat, rye, barley and oats that pose a health risk to people affected by conditions such as coeliac disease and non-coeliac gluten sensitivity. Complete digestion of gluten proteins is of critical importance during quantitative analysis. To this end, chymotrypsin was investigated for its ability to efficiently and reproducibly digest specific classes of gluten in barley. Using proteomics a chymotryptic peptide marker panel was elucidated and subjected to relative quantification using LC-MRM-MS. Thorough investigation of peptide markers revealed robust and reproducible quantification with CVs <15% was possible, however a greater proportion of non-specific cleavage variants were observed relative to trypsin. The selected peptide markers were assessed to ensure their efficient liberation from their parent proteins. While trypsin remains the preferred enzyme for quantification of the avenin-like A proteins, the B-, D- and γ-hordeins, chymotrypsin was the enzyme of choice for the C-hordeins.

Comparison of Gluten Extraction Protocols Assessed by LC-MS/MS Analysis.

The efficiency of gluten extraction is of critical importance to the results derived from any analytical method for gluten detection and quantitation, whether it employs reagent-based technology (antibodies) or analytical instrumentation (mass spectrometry). If the target proteins are not efficiently extracted, the end result will be an under-estimation in the gluten content posing a health risk to people affected by conditions such as celiac disease (CD) and nonceliac gluten sensitivity (NCGS). Five different extraction protocols were investigated using LC-MRM-MS for their ability to efficiently and reproducibly extract gluten. The rapid and simple "IPA/DTT" protocol and related "two-step" protocol were enriched for gluten proteins, 55/86% (trypsin/chymotrypsin) and 41/68% of all protein identifications, respectively, with both methods showing high reproducibility (CV < 15%). When using multistep protocols, it was critical to examine all fractions, as coextraction of proteins occurred across fractions, with significant levels of proteins existing in unexpected fractions and not all proteins within a particular gluten class behaving the same.