Identification and characterization cytosolic nucleoside monophosphate phosphatases of plants

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Plant genomes code for a large number of phosphatases, but the physiological function of only a few is known. The difficulties in determining the in vivo function of phosphatases result among others from the fact that the substrate specificities in vitro are often low and that the enzymes belong to (functionally redundant) protein families.In plant nucleotide metabolism, the phosphatases that convert mononucleotides into the corresponding nucleosides in vivo are also still unknown. However, it is clear that such phosphatases must exist, because plants are able to completely degrade nucleotides.In this project, cytosolic mononucleotide phosphatases for both purine and pyrimidine nucleotides will be identified and biochemically characterized, and their in vivo position and function in metabolism will be investigated.Pyrimidine mononucleotide phosphatases from yeast were chosen as the starting point because they have a demonstrated role in nucleotide metabolism in vivo and because homologous enzymes occur in plants. The subcellular localization and substrate specificity of five homologous phosphatases from Arabidopsis thaliana were investigated. All enzymes were present in the cytosol. One enzyme dephosphorylated exclusively the purine nucleotide xanthosine monophosphate (XMP) with high catalytic efficiency. The existence of a plant XMP phosphatase (XMPP) has already been postulated by us on the basis of metabolite studies on Arabidopsis mutants. The biochemistry of the Arabidopsis XMPP will be comprehensively studied. The position and function of XMPP in metabolism will be investigated using metabolite analyses of mutants. The XMPP of common bean (Phaseolus vulgaris) will also be characterized and the gene will be mutated in transgenic roots / nodules using gene scissors. Metabolite analyses on xmpp nodules will clarify whether XMPP plays an important role in the generation of intermediates in purine catabolism (the ureides), which are used to export fixed nitrogen from the nodules. For this purpose, also the expression domain of the XMPP gene in the nodule will be examined.The remaining four phosphatases and a further phosphatase with homology to a human nucleotidase showed a high specificity for pyrimidine mononucleotides. These enzymes will be biochemically characterized in detail. Metabolite analyses in mutants and possibly multiple mutants will be performed in order to elucidate the physiological functions of these enzymes and to investigate their role in vivo, for example upon increased nucleotide degradation in the dark. Promoter-reporter studies will be performed to elucidate in which cell types and tissues the phosphatase genes are active. By integrating these data, a first model for the role of pyrimidine mononucleotide-specific phosphatases in nucleotide metabolism will be developed.