Molecular and physiological analysis of Arabidopsis mutants defective in cytosolic or chloroplastic aspartate aminotransferase

Arabidopsis mutants deficient in cytosolic (AAT2) or chloroplastic (AAT3) aspartate (Asp) aminotransferase were characterized at the molecular and physiological levels. All of the ethyl methane sulfonate- or nitrosomethylurea-generated mutants are missense mutations, as determined by sequencing of the ASP2 gene from the cytosolic aat2 mutants (aat2-1, aat2-2, aat2-4, and aat2-5) and the ASP5 gene from the chloroplastic aat3 mutants (aat3-1, aat3-2, and aat3-4). A T-DNA insertion mutant in cytosolic AAT2 (aat2-T) was also identified. All the cytosolic aat2 and chloroplastic aat3 mutants have less than 6% AAT2 and less than 3% AAT3 activity, respectively, as determined by the native gel assay; however, none are nulls. The metabolic and physiological affect of these mutations in AAT isoenzymes was determined by measuring growth and amino acid levels in the aat mutants. Two aat2 mutants (aat2-2 and aat2-T) show reduced root length on Murashige and Skoog medium. For aat2-2, this growth defect is exaggerated by Asp supplementation, suggesting a defect in Asp metabolism. Amino acid analysis of the aat mutants showed alterations in levels of Asp and/or Asp-derived amino acids in several aat2 alleles. Two aat2 mutants show dramatic decreases in Asp and asparagine levels in leaves and/or siliques. As such, the cytosolic AAT2 isoenzyme appears to serve a nonredundant function in plant nitrogen metabolism of Asp and Asp-derived amino acids.