Analysis of the biochemical and genetic properties of alanine aminotransferase enzymes

• Author / Creator

  McAllister, Chandra H

• 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.

• Subjects / Keywords

  • Nitrogen
  • AlaAT
  • NUE
  • Enzyme kinetics

• Graduation date

  Fall 2014

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3PG1HV6K

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.