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Date of Award
Doctor of Philosophy (PhD)
In Arabidopsis thaliana, the acyl acid amido synthetase family of Gretchen Hagen 3 (GH3) enzymes conjugate amino acids to plant hormones. AtGH3.5 conjugates both indole-3-acetic acid (IAA) and salicylic acid (SA) to modulate auxin and pathogen response pathways. To understand the molecular basis for activity of AtGH3.5, we determined the x-ray crystal structure of the enzyme in complex with IAA and AMP. Biochemical analysis demonstrates that the substrate preference of AtGH3.5 is wider than originally described and includes the natural auxin phenylacetic acid (PAA) and the potential SA precursor benzoic acid (BA). Residues that determine IAA versus BA substrate preference were identified. The dual functionality of AtGH3.5 is unique to this enzyme, even though multiple IAA-conjugating GH3 proteins share nearly identical acyl acid binding sites. In planta analysis of IAA, PAA, SA, and BA and their respective aspartyl-conjugates were determined in wild-type and overexpression lines of A. thaliana. This study suggests that AtGH3.5 conjugates auxins (i.e., IAA and PAA) and benzoates (i.e., SA and BA) to mediate crosstalk between different metabolic pathways, which broadens the potential roles for GH3 acyl acid amido synthetases in plants.
The substrates of many of the GH3 enzymes are unknown. Using a biochemical approach, we identified the acyl acid and amino acid substrates of AtGH3.15 as the auxin precursor indole-3-butyric acid (IBA) and glutamine. This is the first AtGH3 identified to have activity with IBA and not the main auxin, indole-3-acetic acid (IAA). To understand the molecular basis for this specificity, we determined the X-ray crystal structure of AtGH3.15 in complex with AMP and IBA was modeled into the acyl acid binding site. Mutations in the acyl acid binding site were generated to further explore IBA binding specificity. The size of the acyl acid binding pocket and the location of indole binding, likely, facilities substrate specificity in AtGH3.15. T-DNA insertions in AtGH3.15 and 35S-FLAG-AtGH3.15 overexpression lines show hypersensitivity and resistance respectively in root elongation and lateral root density assays when grown on IBA. Transcript analysis of AtGH3.15:YFP lines show significant expression in seedlings, roots, and parts of siliques. This work suggests AtGH3.15 is unique in the GH3 proteins studied thus far in its role in regulating IBA levels in auxin homeostasis
The ability of an AtGH3 to conjugate herbicides has not been reported. We show AtGH3.15 has the biochemical capability to conjugate glutamine to the auxinic herbicide 4-(2,4-dichlorophenoxy) butyric acid (2,4-DB). To understand the molecular basis for this activity, we determined the X-ray crystal structure of the enzyme in complex with 2,4-DB. Mutations were made in the acyl acid binding site of AtGH3.15 to further explore 2,4-DB binding. T-DNA insertions in AtGH3.15 and 35S-FLAG-AtGH3.15 overexpression lines show hypersensitivity and resistance respectively in root elongation assays when grown on 2,4-DB. These findings highlight the possibility of GH3 proteins having a role in herbicide detoxification that could be used in genetic engineering of 2,4-DB resistant crops.
Chair and Committee
Joseph M. Jez
Ram Dixit, Barbara N. Kunkel, Sona Pandey, Lucia C. Strader,
Sherp, Ashley Marie, "Investigating the Role of GH3 Acyl Acid Amido Synthetases in Plant Hormone Homeostasis" (2017). Arts & Sciences Electronic Theses and Dissertations. 1235.
Available for download on Monday, June 26, 2119