Deciphering the role of adenosine nucleosidases (ADNs) - closing a gap in purine and cytokinin metabolism

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Adenosine nucleosidase (ADN) is an enzyme of purine metabolism catalyzing the hydrolysis of purine/cytokinin nucleosides to the corresponding bases. Although the activity of this enzyme has been described in several plants, no ADN encoding genes were identified to date. In our preliminary work, we identified the first plant ADN genes (PpADN1, -2 and -3) from the moss Physcomitrella patens as well as from Arabidopsis thaliana (AtADN1 and -2). For the gene product of PpADN1 we proofed the hydrolysis of adenosine and the plant hormone isopentenyladenosine (cytokinin) to the corresponding bases. The project deals with the elucidation of protein properties and the function of ADN in plants using the model organisms Physcomitrella and Arabidopsis. Intensive studies on enzyme kinetics and substrate specificity will be carried out on recombinant ADN proteins. The regulation and substrate inducibility of ADN transcription will be studied in Physcomitrella using RT-PCR. Expression of ADN-GFP fusion proteins in plants will be used to determine the subcellular localisation of ADNs. Further important information will derive from T-DNA insertion mutants of Arabidopsis, which will be analysed for adenosine and cytokinin metabolism. It is aimed to determine in how far ADNs can be used to activate cytokinins by their conversion of low active ribosides to highly active cytokinin bases. Characterisation of ADNs will close a gap in the metabolic pathways of purine/cytokinin interconversion and the expected advances are likely to open applications for crop plant improvement and biotechnology.