Thesis Format
Monograph
Degree
Doctor of Philosophy
Program
Biology
Collaborative Specialization
Scientific Computing
Supervisor
Marsolais, Frédéric
2nd Supervisor
Bernards, Mark
Co-Supervisor
Abstract
Improving the quality of economically and nutritionally valuable legume crops, such as soybean, requires a comprehensive understanding of nitrogen metabolism. L-asparagine (Asn) is an important source of nitrogen stored and transported in higher plants, favoured in legumes due to its advantageous 2N:4C ratio. The catabolism of Asn in tissues occurs through two major pathways: deamidation and transamination. The transamination pathway employs an unidentified α-ketosuccinamate (α-KSM ) reducing dehydrogenase (α-KSDH), as well as an ω-amidase which has not been fully characterized. Additionally, α-ketosuccinamate and α-hydroxysuccinamate are potentially toxic metabolites generated in this pathway. The goal of this research is to identify α-KSDH in soybean and characterize the role of ω-amidase in the transamination pathway in Arabidopsis. Fractionation, ammonium sulfate precipitation, size exclusion chromatography, and LC-MS/MS were employed to sequentially purify and identify an α-KSDH that showed activity with α-KSM. Altogether, these techniques revealed that soybean hydroxyphenylpyruvate reductase (GmHPPR) and Arabidopsis hydroxypyruvate reductase 2 (AtHPR2) catalyze the reduction of α-KSM into α-HSM. Based on this activity, these are likely the enzymes responsible for α-KSDH function in vivo of their respective species, however in vivo studies are required to confirm this function. The broad substrate specificity of HPPR and HPR2 and its activity with 2-hydroxyacids indicate that its main role in vivo is to convert intermediary metabolites into metabolites that can be directly used for other important pathways within the plant. An ω-amidase T-DNA insertion loss of function mutant was found to be embryo lethal in Arabidopsis, cause shortened siliques in heterozygotes, and overall have a detrimental effect on reproduction. The mechanism of these effects is likely a shortage of methionine and polyamines, toxic metabolite accumulation, or a combination of the two. Like ω-amidase, HPPR is known as a “clean-up” enzyme, and it can be assumed that it also plays a role in mitigating the levels of toxic intermediary metabolites as a “repair” enzyme. This research has advanced our understanding of Asn metabolism in higher plants and may ultimately contribute to advancements in crop nitrogen use efficiency.
Summary for Lay Audience
Legumes such as soybeans are an important source of protein for humans. Like most plants, soybeans require nitrogen for growth and development. The availability of nitrogen in the soil significantly impacts photosynthesis, the pathway plants use to generate sugar—the food they require to grow. Nitrogen is carried throughout the plant in the form of different molecules. However, metabolic pathways in plants have not always evolved to make efficient use of nitrogen. We evaluate how efficient a plant is at using nitrogen as a source of energy with a metric termed nitrogen use efficiency (NUE). NUE is used to compare the input of nitrogen (the uptake of nitrogen by the plant) and output of nitrogen (crop yield). Asparagine, an amino acid originally discovered in asparagus, acts as a major storage molecule for nitrogen. When nitrogen is needed, asparagine is broken down into other molecules which are then directed into the metabolic pathways in need. These pathways can be responsible for mitigating environmental or pest stressors, regulating plant hormone signaling, and overall generating energy to grow. Occasionally, intermediate molecules within these pathways store nitrogen in a form inaccessible to the plant. On rare occasions, these pathways generate molecules which can be toxic to the plant. Among two enzymes involved in the breakdown of asparagine, one is not well characterized and one has yet to be identified. Additionally, two molecules within this pathway have been proposed to be toxic to plant health. Identifying and characterizing these enzymes allows us to understand their role in asparagine metabolism, whether they contribute positively to nitrogen cycling, and which plant metabolic pathways may be critical or detrimental. This research advances our understanding of asparagine metabolism in agriculturally important plants, which may ultimately contribute to advancements in crop nitrogen use efficiency. In an effort to meet the growing challenges that come with climate change, these advancements are essential in meeting the food demand of the near future.
Recommended Citation
Zaman, Raiyan D., "Characterization of Enzymes in the L-Asparagine Metabolism Pathway in Legumes" (2025). Electronic Thesis and Dissertation Repository. 10797.
https://ir.lib.uwo.ca/etd/10797