Poster: Metabolic Engineering

Abs # 1004: Screening the Arabidopsis thaliana knockout mutant collections for individuals displaying aberrant amino acid profiles

Presenter:	Hanes, Melinda S., msh175@psu.edu
Authors	Hanes, Melinda S. (A)   Campbell, Michael A. (A)
Affiliations:	(A): Penn State Erie

Although the synthesis and availability of amino acids dictates an organism’s growth and development, amino acid metabolism is poorly understood in plants, which are the source of essential amino acids. We have taken a biochemical approach to analyze amino acid levels found in knockout mutants of Arabidopsis thaliana containing T-DNA inserts in genes with high sequence similarities to enzymes in the biosynthetic pathway for the branched-chains (BCAAs), leucine, isoleucine, and valine. Using HPLC and fluorescent labeling of free amino acids, mutants were screened for aberrant profiles. Phenotypically, all the mutants appeared to be wild type. Among the 40 mutants, the amounts of BCAAs and phenylalanine were strongly conserved, confirming the importance of the BCAA levels. An increase of a non-amino acid amine was present in mutant 628_C04, a putative knockout of acetolactate synthase, and an increase in phenylalanine occurred in a putative knockout for branched-chain amino acid aminotransferase 5 (bcat5) (mutant 033104). Analysis of the progeny (F₁ generation), has shown that phenotype of bcat5 putative knockout segregates in a 1:1 ratio. The 40 mutants available in the public Arabidopsis thaliana mutant databases contain only three members with aberrant amine profiles. The aberrant profiles detected were not indicative of the specific enzymatic step putatively disrupted.