Dissertation
CHARACTERIZATION OF FORMYLTETRAHYDROFOLATE LIGASE IN ARABIDOPSIS THALIANA AND INVESTIGATION OF ITS FUNCTIONAL ROLE IN THE ONE-CARBON METABOLIC PATHWAY
Washington State University
Doctor of Philosophy (PhD), Washington State University
01/2021
DOI:
https://doi.org/10.7273/000003135
Handle:
https://hdl.handle.net/2376/122784
Abstract
Formate is the simplest molecule that participates in one-carbon metabolism. In plants, formate is enzymatically or non-enzymatically produced by several reactions. An excess amount of formate harms cells by disrupting mitochondrial electron transport and energy production. In plant cells, two enzymes transform formate into non-toxic compounds: 1. In the cytosol, formyltetrahydrofolate ligase (FTHFL) is responsible for the entry of formate into one-carbon metabolism; and 2. In mitochondria, formate is oxidized by formate dehydrogenase (FDH) to produce CO2. This dissertation includes two studies; one investigates the role of AtFTHFL in one-carbon metabolism and the other investigates how AtFDH contributes to one-carbon metabolism.To investigate the physiological roles of AtFTHFL, we have identified and obtained two T-DNA insertion lines which were verified to be the knockdown mutants of AtFTHFL. Additionally, three independent lines of AtFTHFL overexpressors were generated. To study the biochemical role of AtFTHFL, we investigated catalytic parameters of the recombinant AtFTHFL and found that AtFTHFL displays higher affinity toward a polyglutamylated form in comparison between monoglutamylated H4PteGlun and pentaglutamylated H4PteGlun. We further confirmed that AtFTHFL is solely localized in the cytosol, as predicted using bioinformatics. We detected an elongated root phenotype and increased H4PteGlun +5,10-methylene-H4PteGlun in root tissues of the overexpression lines grown in tissue culture, but no visible changes in growth and development parameters, including fresh weight, dry weight, height and flowering time in either AtFTHFL knockdown or overexpression mutants for the aerial tissues. We conclude that AtFTFHL is not crucially required for plant growth and development; instead, AtFTHFL functions to detoxify formate by funneling it into one-carbon metabolism in the cytosol.
Next, we investigated the function of FDH in one-carbon metabolism. We collaborated with Dr. Andreas Weber who provided us with three AtFDH knockout mutants, which were generated via the CRISPR-Cas9 approach, and three AtFDH overexpression lines. Neither inactivating nor overexpressing AtFDH caused changes in formate or folate levels in the photosynthetic tissues (rosettes) of the transgenic plants. We hypothesize that we do not see changes in formate pool sizes in any of the transgenics because the plant cells contain a sufficient amount of AtFTHFL to regulate the formate pool size through changes in flux through AtFTHFL. In addition, we hypothesize that the one-carbon pool size is maintained at a constant level by adjusting the supply of one-carbon units at other entry points. This hypothesis will be investigated in the laboratory of Dr. Weber using metabolic flux analyses.
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Details
- Title
- CHARACTERIZATION OF FORMYLTETRAHYDROFOLATE LIGASE IN ARABIDOPSIS THALIANA AND INVESTIGATION OF ITS FUNCTIONAL ROLE IN THE ONE-CARBON METABOLIC PATHWAY
- Creators
- Sompop Saeheng
- Contributors
- Sanja Roje (Advisor)John Browse (Committee Member)Hanjo Hellmann (Committee Member)Asaph Cousins (Committee Member)
- Awarding Institution
- Washington State University
- Academic Unit
- Program in Molecular Plant Sciences
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Publisher
- Washington State University
- Number of pages
- 114
- Identifiers
- 99900651900301842
- Language
- English
- Resource Type
- Dissertation