Identification of new catalytic and regulative components of purine nucleotide catabolism of Arabidopsis thaliana using a genetic screen.

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Plants are able to catabolize purine nucleotides completely to remobilize the nitrogen that is contained within the purine ring system. Substantial advances have been made recently in the genetic identification and biochemical characterization of enzymes that are involved in the degradation of purine nucleotides. Nonetheless, there are still several components of this metabolic route which remain unknown. In addition, nothing is known so far about the regulatory components of purine nucleotide catabolism.Such catalytic or regulative components will be identified through a genetic screen. A urate oxidase T-DNA insertion mutant of Arabidopsis thaliana will be mutagenized with ethyl methanesulfonate (EMS). Seeds from plants with a defect in urate oxidase do not complete the establishment of the seedling because they contain toxic levels of uric acid which accumulates during embryo development. This phenotype is suppressed in double mutants that additionally lack an enzyme upstream of urate oxidase in the degradation route of the purine nucleotides. Such mutants do not accumulate uric acid. In a genetic screen we will identify new suppressors which prevent the accumulation of uric acid in the urate oxidase background. This could be genes for enzymes of this metabolic route upstream of urate oxidase or regulative genes which are important for the expression of purine nucleotide catabolism. Alternatively, suppressors may be found which confer an increased tolerance to high levels of uric acid during seedling establishment. This group comprises genes whose identification may shed light on how uric acid exerts its negative effect on seedling establishment. Beyond plants, this may result in a deeper understanding of the toxic effects of uric acid on human metabolism as well.The identification of the suppressor genes will be followed by a comprehensive biochemical characterization of the corresponding proteins. Metabolomic analyses of nucleotide metabolism in mutants and double mutants will elucidate the function of the suppressors in vivo.