Date of Award
Edith Cowan University
Doctor of Philosophy
School of Science
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.
Bahmani, M. (2023). Mass spectrometry-based proteomic analysis to characterise barley breeding lines. Edith Cowan University. https://doi.org/10.25958/5hkr-1w78