Date of Award
2024
Document Type
Thesis - ECU Access Only
Publisher
Edith Cowan University
Degree Name
Doctor of Philosophy
School
School of Science
First Supervisor
Michelle Colgrave
Second Supervisor
Angela Juhasz
Third Supervisor
Rhonda Foley
Fourth Supervisor
Karam Singh
Abstract
Lupin is a dominant legume crop in Western Australia, with strong domestic and export markets. Due to its high protein and fibre content, coupled with low levels of carbohydrates and fat, lupin has garnered significant interest over other legumes. It is cultivated for a variety of food and animal feed applications. However, lupin products may pose health risks to consumers due to the presence of toxic quinolizidine alkaloids (QAs). Quinolizidine alkaloids serve as natural plant defence mechanisms, and they have wide industrial applications. However, they can be toxic to humans and animals if consumed above the industrial limits (200 mg/kg). The levels of alkaloids in sweet lupin species vary and can occasionally surpass safe consumption thresholds. An accurate and validated methodology for quantifying QAs in lupin seeds is important for monitoring their concentrations across different sweet lupin varieties. Furthermore, the factors contributing to variations in QA levels within lupin seeds demand additional exploration. Currently, most proteins implicated in the biosynthesis and transportation of QAs remain unidentified. This thesis comprises two parts: the first involves establishing a new method for QAs determination in lupin grain and tracking their concentrations across various lupin species and under different daylight durations. The second part explores proteins associated with alkaloid biosynthesis and transportation in lupin through proteomics analysis of lupin leaves, correlating with alkaloid levels.
Chapter 1 of the thesis presents an extensive literature review on the occurrence of QAs in lupin seeds, highlighting the primary gaps in current knowledge. Alkaloids serve a crucial role in defending lupins against insect attacks and enabling plants to adapt to various environmental changes. However, when QAs are present in high concentrations in lupin seeds, they pose toxicity risks to both humans and animals. Since alkaloids are synthesized in lupin leaves and then translocated to the seeds, it is possible to develop a "bittersweet" lupin accession. This accession would contain high levels of alkaloids in the leaves for protection while maintaining low levels in the seeds to ensure safety for consumption. To achieve this goal, there is a need for a precise and accurate method for analysing alkaloids in both lupin seeds and leaves. Additionally, there is a need to develop a proteomics approach to identify key proteins involved in the biosynthesis and transportation of alkaloids.
Chapter 2 of the thesis focuses on the development and validation of an LC-MS/MS method for the quantification of QAs in lupin seeds. This method facilitated the determination of alkaloid levels in seeds from major Lupinus species (Lupinus angustifolius, L. cosentinii, L. albus, L. luteus, and L. mutabilis), obtained from the Australian Grains Genebank. Demonstrating high accuracy and precision, with low limits of detections, the method enabled the identification of trace levels of QAs in individual lupin seeds. Subsequent QAs analysis of narrow-leafed lupin (NLL) accessions is presented in Chapter 3. Seeds from both commercial and wild NLL accessions were cultivated under greenhouse conditions, and their QAs contents were evaluated using the validated LC-MS/MS method. The analysis revealed a broad spectrum of QA levels and profiles among different NLL accessions. For instance, low-alkaloid accessions like Geebung exhibited QA levels below 10 mg/kg, whereas bitter accessions such as GRC5011A reached QA concentrations as high as 100,000 mg/kg. The study also found that the level of biological variation was greater in sweet accessions (CV > 100%) compared to bitter ones (CV < 30%).
In Chapter 4, the thesis explores the intra-plant variability of QA content across three NLL genotypes: Tanjil, Quillinock, and P22660. This analysis extended to assess QA levels in seeds from the same and different plants: within pods, between branches, within individual plant replicates, and across the mentioned accessions. The findings indicated that while variation in QA levels among seeds from the same pods within any given lupin plant was relatively low (CV < 30%) for all studied accessions, variability within a single replicate of a lupin plant could reach as high as 94%. This suggests that intra-plant variation in QA content can, at times, exceed the variation observed between different plant replicates. Additionally, the thesis examines the impact of extended daylight hours, a condition typical within a ‘speed breeding’ greenhouse, on the QA levels in seeds of the Tanjil cultivar. The data suggest that speed breeding growth conditions may influence QA levels in NLL accessions, highlighting a potential area for further research on how controlled environmental conditions can affect alkaloid biosynthesis in lupins.
Chapter 5 introduces a comprehensive multi-omics approach for analysing proteomics and QAs in lupin leaves. This methodology includes the extraction of proteins and alkaloids from the same tissue using a mixture of urea, thiourea, and CHAPS. The method was further validated for alkaloid quantification using LC-MS/MS. By employing the quantitative proteomics SWATH-MS technique and DIA-NN data analysis tool, 4,768 proteins were quantified in leaves of sweet and bitter NLL varieties. Through correlation analysis between protein levels and QA concentrations, the research elucidated connections within the proteome profiles, confirming previously identified proteins involved in QA biosynthesis. From all positively significant modules, we selected the top 351 protein groups, with VIP score > 1, which indicates a strong association between the biosynthesis and transport of QAs. Some proteins were specifically correlated with distinct alkaloid profiles, suggesting their involvement in the biosynthesis of individual alkaloids.
DOI
10.25958/y8wt-rw90
Access Note
Access to this thesis is embargoed until 6 August 2025
Recommended Citation
Khedr, T. (2024). Bitter sweet: Exploring alkaloid synthesis in lupin using LC-MS/MS. Edith Cowan University. https://doi.org/10.25958/y8wt-rw90