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Analysis of the biochemical and genetic properties of alanine aminotransferase enzymes Open Access


Other title
enzyme kinetics
Type of item
Degree grantor
University of Alberta
Author or creator
McAllister, Chandra H
Supervisor and department
Allen Good (Biological Sciences)
Examining committee member and department
Robert Mullen (Biological Sciences, University of Guelph)
Allen Good (Biological Sciences)
Enrico Scarpella (Biological Sciences)
Martin Srayko (Biological Sciences)
Uwe Hacke (Renewable Resources)
Department of Biological Sciences
Plant Biology
Date accepted
Graduation date
Doctor of Philosophy
Degree level
Quantitatively, nitrogen (N) has shown to be one of the most important nutrients for proper plant growth and development, and a key limiting factor in crop productivity. In the last 40 years, there has been a significant increase in the production of synthetic N-fertilizers, as well as an increase in the overall application of these N-fertilizers to crops and subsequently considerable negative impacts on the environment. In order to mitigate N compounds released into the environment while maintaining crop yields to feed a growing world population, there is a need for plants that more effectively uptake, assimilate, and mobilize applied N. While advances in plant breeding practices and agricultural technologies have increased greatly due to Norman Borlaug’s “Green Revolution” in the 1960’s, the selection of traits associated with efficiency of nutrient uptake and usage was greatly ignored because of excessive fertilizer applications during this time. Consequently, there is a need for crop plants with increased nutrient efficiency, specifically nitrogen use efficiency (NUE). Alanine aminotransferase (AlaAT), is a pyridoxal-5’-phosphate-dependent (PLP) enzyme that catalyzes the reversible transfer of an amino group from alanine to 2-oxoglutarate to produce glutamate and pyruvate, or vice versa. It has been well documented in both greenhouse and field studies that tissue-specific over-expression of AlaAT from barley (Hordeum vulgare, HvAlaAT) results in a significant increase in plant NUE in both canola and rice. While the physical phenotypes associated with over-expression of HvAlaAT have been well characterized, specifically in regards to NUE, the role this enzyme plays in vivo to create a more N efficient plant remains unknown. Furthermore, the importance of HvAlaAT and not other AlaAT homologues in creating this phenotype has not yet been explored. iii To address the role of HvAlaAT in NUE, the in vitro KM values of a selection of AlaAT variants was analyzed. Based on this kinetic analysis, several of these enzymes were chosen to be expressed, both tissue specifically and constitutively, in Arabidopsis thaliana Col-0 background and alaat1;2 (alaat1-1;alaat2-1) knockout background. The analysis and comparison here of both the physical and physiological properties of AlaAT transgenic plants revealed significant differences between plants expressing different AlaAT enzymes in regard to externally applied factors as well as differences in internal C and N concentrations. The analysis reported herein indicates that, the over-expression of AlaAT variants other than HvAlaAT in crop plants could further increase the NUE phenotype(s) previously observed.
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
Citation for previous publication
McAllister, C. H., Beatty, P. H. and Good, A. G. (2012) Engineering nitrogen use efficient crop plants: the current status. Plant Biotech. J. 10: 1011–1025.McAllister, C. H., Facette, M., Holt, A. and Good, A. G. (2013) Analysis of the enzymatic properties of a broad family of alanine aminotransferases. PloS One, 8: e55032. doi: 10.1371/journal/pone.0055132.

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