Genetic, biochemical, and metabolomic analysis of plant purine catabolism
Final Report Abstract
This project has led to major advances in the understanding of purine nucleotide catabolism in plants. It was demonstrated that the enzymes carrying out the finals steps of purine ring catabolism (the ureide degradation) are not only doing so in vitro but are also relevant in vivo in Arabidopsis. Furthermore, orthologs could be identified in soybean and functionally characterized. Also in this plant, ureide catabolism is performed by similar enzymes which could be shown in vitro and in vivo. There is no functional diversification of these enzymes in soybean although several isoforms exist and soya uses ureide catabolism for the special purpose of supplying the shoot with nitrogen under N2-fixing conditions. The generation of an allantoate amidohydrolase (AAH) mutant in soybean was abandoned because no suitable mutants were identified in a soybean TILLING population and the discovery of two AAH genes in the soybean genome made this goal difficult to achieve. Guanosine deaminase, a novel enzyme only present in plants, was characterized in the course of this work. It is required to generate xanthosine in vivo, showing that a direct metabolic connection between XMP and xanthosine does not exist and that guanosine plays a central role in purine nucleotide catabolism. This has implications for the current model of purine nucleotide catabolism and future research into the role of guanosine deaminase in soybean (for the production of ureides in nodules) and in purine alkaloid producing plants (like coffee or tea) has been triggered by these findings. First steps for a full (genetic and biochemical) identification of 5' nucleotidases in Arabidopsis have been made by cloning and functional testing of candidate proteins. First evidence was obtained that purine nucleotide catabolism contributes to the nitrogen supply for seedling establishment in Arabidopsis. However, the effects are rather subtle and experiments need to be repeated to test statistical significance. In the course of these experiments phenotypes of reduced germination and dark-stress susceptibility were discovered for the gsda mutant. These results will trigger research into the role of nucleotides as sugar stores. Based on transcriptome data, it has been speculated that allantoin accumulates during abiotic stress situations. This was tested and could not be verified. The suggested role of allantoin as protectant against abiotic stresses therefore seems unlikely. The molecular cause of the strong urate oxidase knockout phenotype in Arabidopsis was investigated. It could be shown that uric acid accumulates to high concentrations during embryo development leading to a defect in peroxisome maintenance. The local and temporal absence of peroxisomes in the uox mutant explains the observed phenotypes of failing germination and seedling establishment due to lack of cotyledon development. A novel connection between peroxisome maintenance and uric acid accumulation was drawn which may relevant beyond plants, for example in humans, where the molecular reasons for the toxicity of high serum uric acid levels are not fully understood.
Publications
- (2008). Identification, biochemical characterization, and subcellular localization of allantoate amidohydrolases from Arabidopsis and soybean. Plant Physiology 146,418-430
Andrea K. Werner, Imogen A. Sparkes, Tina Romels, Claus-Peter Witte
- (2010). Ureide catabolism in Arabidopsis thaliana and Escherichia coli. Nature Chemical Biology 6,19-21
Andrea K. Werner, Tina Remels, Claus-Peter Witte
- (2011). The biochemistry of nitrogen mobilization: purine ring catabolism. Trends in Plant Science 16, 381-387
Andrea K. Werner, Claus-Peter Witte
- (2013). Plant purine nucleotide catabolism employs a guanosine deaminase required for the generation of xanthosine in Arabidopsis. Plant Cell 25, 4101-4109
Kathleen Dahncke, Claus-Peter Witte
(See online at https://doi.org/10.1105/tpc.113.117184) - (2013). The ureide degrading reactions of purine ring catabolism employ one aminohydrolase and three amidohydrolases in Arabidopsis, soybean and rice. Plant Physiology 163, 672-681
Andrea K. Werner, Nieves Medina-Escobar, Monika Zulawski, Imogen A. Sparkes, Feng-Qiu Cao, Claus-Peter Witte
(See online at https://doi.org/10.1104/pp.113.224261) - Uric acid accumulation in an Arabidopsis urate oxidase mutant impairs seedling establishment by blocking peroxisome maintenance, The Plant Cell, July 2014
Oliver K. Hauck, Jana Scharnberg, Nieves Medina Escobar, Gerhard Wanner, Patrick Giavalisco, Claus-Peter Witte
(See online at https://doi.org/10.1105/tpc.114.124008)
