title: Analysis of Loss-of-Function Mutants in Aspartate Kinase and Homoserine Dehydrogenase Genes Points to Complexity in the Regulation of Aspartate-Derived Amino Acid Contents.
abstract: Biosynthesis of aspartate (Asp)-derived amino acids lysine (Lys), methionine (Met), threonine (Thr), and isoleucine involves monofunctional Asp kinases (AKs) and dual-functional Asp kinase-homoserine dehydrogenases (AK-HSDHs). Four-week-old loss-of-function Arabidopsis (Arabidopsis thaliana) mutants in the AK-HSDH2 gene had increased amounts of Asp and Asp-derived amino acids, especially Thr, in leaves. To explore mechanisms behind this phenotype, we obtained single mutants for other AK and AK-HSDH genes, generated double mutants from ak-hsdh2 and ak mutants, and performed free and protein-bound amino acid profiling, transcript abundance, and activity assays. The increases of Asp, Lys, and Met in ak-hsdh2 were also observed in ak1-1, ak2-1, ak3-1, and ak-hsdh1-1. However, the Thr increase in ak-hsdh2 was observed in ak-hsdh1-1 but not in ak1-1, ak2-1, or ak3-1. Activity assays showed that AK2 and AK-HSDH1 are the major contributors to overall AK and HSDH activities, respectively. Pairwise correlation analysis revealed positive correlations between the amount of AK transcripts and Lys-sensitive AK activity and between the amount of AK-HSDH transcripts and both Thr-sensitive AK activity and total HSDH activity. In addition, the ratio of total AK activity to total HSDH activity negatively correlates with the ratio of Lys to the total amount of Met, Thr, and isoleucine. These data led to the hypothesis that the balance between Lys-sensitive AKs and Thr-sensitive AK-HSDHs is important for maintaining the amounts and ratios of Asp-derived amino acids.
authors.affiliation: Department of Biological Sciences, Western Michigan University, Kalamazoo, Michigan 49008-5410.
authors.forename: Teresa J
authors.identifier: http://orcid.org/0000-0002-8396-8388
authors.initials: TJ
authors.lastname: Clark
authors.affiliation: Department of Biological Sciences, Western Michigan University, Kalamazoo, Michigan 49008-5410 yan.1.lu@wmich.edu.
authors.forename: Yan
authors.identifier: http://orcid.org/0000-0002-3374-7376
authors.initials: Y
authors.lastname: Lu
mesh_terms: D000596:Amino Acids / Q000235:genetics* / Q000378:metabolism; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D001222:Aspartate Kinase / Q000235:genetics* / Q000378:metabolism; D001224:Aspartic Acid / Q000235:genetics* / Q000378:metabolism; D001225:Aspartokinase Homoserine Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D015151:Immunoblotting; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008715:Methionine / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053719:Tandem Mass Spectrometry; D013912:Threonine / Q000235:genetics / Q000378:metabolism
chemical_list: D000596:Amino Acids; D029681:Arabidopsis Proteins; D007527:Isoenzymes; D013912:Threonine; D001224:Aspartic Acid; D008715:Methionine; D001222:Aspartate Kinase; D001225:Aspartokinase Homoserine Dehydrogenase; D008239:Lysine