Mass spectrometry-based proteomic analysis to characterise barley breeding lines

Barley is a key ingredient in the malting and brewing industry, and it is the fourth most important crop being cultivated worldwide. The protein content of the barley grain is one of the main components determining the quality and nutritive value of the food and beverages prepared from barley. Mass spectrometry-based proteomic analysis is a valuable tool that can guide and inform plant breeding strategies and crop improvement programs. Understanding the proteome changes in barley grain under different growing locations, the impact of different environmental conditions and its relationship with malting characteristics have the potential to inform breeding programs to achieve high-quality malt. Moreover, hordeins, the major barley storage proteins, are among the known triggers of coeliac disease (CD). Therefore, investigating the changes in the overall grain proteome, especially hordeins provides valuable insight from a food safety perspective. This thesis focuses on the proteomic investigation of barley grain to understand differences due to genetic and environmental factors and how these differences impact end use application after food processing steps such as malting. In Chapter 2 of this thesis, the proteome and malting characteristics of three different barley genotypes grown in three different locations in Western Australia were measured by applying a bottom-up proteomics workflow. First, using discovery proteomics, 1,571 proteins were detected and in the next step, by applying a global proteome quantitation workflow, 920 proteins were quantified in barley samples. Data analysis revealed that growing location outweighed the impact of genetic background, and samples were clustered into two major groupings of northern and southern growing locations. Also, a relationship between proteome measurements and malting characteristics using weighted gene co-expression network analysis (WGCNA) were investigated. The statistical analysis showed that both the genotypes and the growing locations strongly correlate with changes in the proteomes and desirable traits such as malt yield. Finally, linking meteorological data with proteomic measurements revealed how high-temperature stress in northern regions affects the seed temperature tolerance during malting, resulting in a higher malt yield. In Chapter 3, a targeted proteomics approach was used to investigate the changes of hordein peptides after malting in grain samples of previously developed hordein-reduced barley lines, including a triple-hordein-reduced ultra-low gluten (ULG) barley line and their corresponding malt samples. Peptides representing hordein-like proteins, including B-, D- and γ-hordeins and avenin-like proteins (ALPs), were tracked using relative quantitation across single-, double-, and triple-hordein-reduced barley grain and malt samples. Further analysis showed that malting further reduced the quantity of B-, D- and -hordeins and ALPs in the ULG malt sample compared to the unmalted grain. Moreover, the detection and quantitation of globulin proteins in the experimental samples indicated a compensation mechanism of storage proteins leading to the biosynthesis of seed storage globulins (vicilin-like globulins) in the ULG-line derived grain and malt sample compared to the wild type. Taken together, these results suggest that the compensation effect enables the hordein-reduced ULG line to maintain the balance of overall N-rich reservoir accumulation. In Chapter 4, the impact of malting of barley grain was investigated by unbiased proteome comparison of the grain and malt. Using discovery proteomics, 2,688 proteins were detected in the barley grain and 3,034 proteins in the malt samples of which 807 proteins were unique to malt samples. Next, Gene Ontology (GO) enrichment analysis was performed on the unique proteins and revealed that “hydrolysing activity” was the most significant GO term enriched in malt over barley. By conducting quantitative proteomics using SWATH-MS, 2,654 proteins were quantified in the barley grain and malt samples. Based on their proteome level quantitation, the unsupervised clustering analysis showed two distinct clusters representing grain flour and malt samples. Moreover, a relationship between hordein-reduced backgrounds and proteome data was established. The results showed that the inclusion of C-hordein-reduced lines significantly impacted the proteome level changes in the grain and malt samples, more so than the inclusion of the B- and D-hordein-reduced lines. Furthermore, univariate analyses were performed to identify the differentially abundant proteins in each hordein-reduced background by comparing barley grain to malt samples. Finally, GO enrichment analysis was performed on the up- and down-regulated proteins detected from the pair-wise comparisons. GO enrichment analyses revealed that the up-regulated proteins in C-hordein-reduced lines were primarily involved in the small molecule metabolic process and provided more energy during malting to facilitate seed germination. Advancements in mass spectrometry-based proteomics approaches and cutting-edge bioinformatics tools have revolutionised protein detection and quantitation from model and non-model species, enabling us to obtain unprecedented views on changes in the barley grain proteomes at the molecular level. The results generated from this PhD project have further illustrated the underlying complex regulatory mechanisms controlling storage protein accumulation upon malting in barley grains. The approaches used and the insights gleaned have the potential to accelerate the development of new varieties with desired traits of interest. Specifically, the foundational knowledge and workflow developed from this thesis can be applied in the selection of unique germplasm by barley breeders for barley food and beverage applications

Proteome phenotypes discriminate the growing location and malting traits in field-grown barley

Barley is one of the key cereal grains for malting and brewing industries. However, climate variability and unprecedented weather events can impact barley yield and end-product quality. The genetic background and environmental conditions are key factors in defining the barley proteome content and malting characteristics. Here, we measure the barley proteome and malting characteristics of three barley lines grown in Western Australia, differing in genetic background and growing location, by applying liquid chromatography-mass spectrometry (LC-MS). Using data-dependent acquisition LC-MS, 1571 proteins were detected with high confidence. Quantitative data acquired using sequential window acquisition of all theoretical (SWATH) MS on barley samples resulted in quantitation of 920 proteins. Multivariate analyses revealed that the barley lines\u27 genetics and their growing locations are strongly correlated between proteins and desired traits such as the malt yield. Linking meteorological data with proteomic measurements revealed how high-temperature stress in northern regions affects seed temperature tolerance during malting, resulting in a higher malt yield. Our results show the impact of environmental conditions on the barley proteome and malt characteristics; these findings have the potential to expedite breeding programs and malt quality prediction

From grain to malt: Tracking changes of ultra-low-gluten barley storage proteins after malting

Barley (Hordeum vulgare L.) is a major cereal crop produced globally. Hordeins, the major storage proteins in barley, can trigger immune responses leading to celiac disease or symptoms associated with food allergy. Here, proteomics approaches were employed to investigate the proteome level changes of grain and malt from the malting barley cultivar, Sloop, and single-, double- and triple hordein-reduced lines. The triple hordein-reduced line is an ultra-low gluten barley cultivar, Kebari®. Using discovery proteomics, 2,688 and 3,034 proteins in the barley and malt samples were detected respectively. Through the application of targeted proteomics, a significant reduction in the quantity of B-, D-, and γ-hordeins, as well as avenin-like proteins, was observed in the ultra-low gluten malt sample. A compensation mechanism was observed evidenced by increased biosynthesis of seed storage globulins, specifically vicilin-like globulins. Overall, this study has provided insights into protein compositional changes after malting in celiac-friendly barley varieties

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

Application of mass spectrometry-based proteomics to barley research

Barley (Hordeum vulgare) is the fourth most cultivated crop in the world in terms of production volume, and it is also the most important raw material of the malting and brewing industries. Barley belongs to the grass (Poaceae) family and plays an important role in food security and food safety for both humans and livestock. With the global population set to reach 9.7 billion by 2050, but with less available and/or suitable land for agriculture, the use of biotechnology tools in breeding programs are of considerable importance in the quest to meet the growing food gap. Proteomics as a member of the “omics” technologies has become popular for the investigation of proteins in cereal crops and particularly barley and its related products such as malt and beer. This technology has been applied to study how proteins in barley respond to adverse environmental conditions including abiotic and/or biotic stresses, how they are impacted during food processing including malting and brewing, and the presence of proteins implicated in celiac disease. Moreover, proteomics can be used in the future to inform breeding programs that aim to enhance the nutritional value and broaden the application of this crop in new food and beverage products. Mass spectrometry analysis is a valuable tool that, along with genomics and transcriptomics, can inform plant breeding strategies that aim to produce superior barley varieties. In this review, recent studies employing both qualitative and quantitative mass spectrometry approaches are explored with a focus on their application in cultivation, manufacturing, processing, quality, and the safety of barley and its related products

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

Proteomics, physiological, and biochemical analysis of cross tolerance mechanisms in response to heat and water stresses in soybean.

Water stress (WS) and heat stress (HS) have a negative effect on soybean plant growth and crop productivity. Changes in the physiological characteristics, proteome, and specific metabolites investigated on molecular and cellular functions were studied in two soybean cultivars exposed to different heat and water stress conditions independently and in combination. Leaf protein composition was studied using 2-DE and complemented with MALDI TOF mass spectrometry. While the two cultivars displayed genetic variation in response to water and heat stress, thirty-nine proteins were significantly altered in their relative abundance in response to WS, HS and combined WS+HS in both cultivars. A majority of these proteins were involved in metabolism, response to heat and photosynthesis showing significant cross-tolerance mechanisms. This study revealed that MED37C, a probable mediator of RNA polymerase transcription II protein, has potential interacting partners in Arabidopsis and signified the marked impact of this on the PI-471938 cultivar. Elevated activities in antioxidant enzymes indicate that the PI-471938 cultivar can restore the oxidation levels and sustain the plant during the stress. The discovery of this plant's development of cross-stress tolerance could be used as a guide to foster ongoing genetic modifications in stress tolerance