Poster: Metabolic engineering
Abs #
284: Threonine aldolase, a previously uninvestigated component of plant amino acid metabolism
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Presenter: |
Joshi, Vijay J, vjj5@cornell.edu |
Authors | Joshi, Vijay J (A) Jander, Georg (A) | | Affiliations: |
(A): Boyce Thompson Institute for Plant Research
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Amino acids are not only the building blocks of proteins, but also serve as precursors for other important plant metabolites and constitute an essential part of human and animal diets. Nevertheless, many aspects of plant amino acid metabolism remain unknown. A mutant with a 23-fold increase in seed threonine was discovered in a screen for Arabidopsis with altered seed amino acid levels. Genetic mapping and DNA sequencing identified a missense mutation in a putative threonine aldolase (AT1G08630) as the underlying genetic basis of this phenotype. Threonine aldolase, which catalyzes the reversible interconversion of threonine and glycine + acetaldehyde, has not been studied previously in Arabidopsis or any other plant. Wild type, but not mutant, cDNA clones rescued the glycine auxotrophy of a yeast gly1, shm1, shm2 mutant. In vitro assays with extracts from the transformed yeast strain showed threonine aldolase activity, with an apparent Km for threonine of 7.0 mM for the Arabidopsis enzyme.
Analysis of the Arabidopsis genome identified a second putative threonine aldolase (AT3G04520), which also rescued the yeast glycine auxotrophy. Semi-quantitative RT-PCR experiments demonstrated that the two threonine aldolases have overlapping, but not identical expression patterns, indicating different roles in plant metabolism. Analysis of a T-DNA insertion in the AT3G04520 threonine aldolase showed that this gene may be essential for the life of the plant. It was not possible to isolate a homozygous T-DNA insertion mutant, and 25% of the seedlings from seeds of heterozygous plants abort early in development. Ongoing and future work with Arabidopsis threonine aldolases will reveal the role that these previously uninvestigated enzymes play in plant amino acid metabolism.
