ENT1 of Arabidopsis thaliana was the first member of the equilibrative nucleoside transporter (ENT) family to be identified in plants and characterized as a cellular, high-affinity nucleoside importer. Evidence is presented here for a tonoplast localization of ENT1 based on proteome data and Western blot analyses. Increased export of adenosine from reconstituted tonoplast preparations from 35S:ENT1 mutants compared with those from the wild type and ENT1-RNAi mutants support this view. Furthermore, increased vacuolar adenosine and vacuolar 2′3′-cAMP (an intermediate of RNA catabolism) contents in ENT1-RNAi mutants, but decreased contents of these metabolites in 35S:ENT1 over-expresser mutants, were observed. An up-regulation of the salvage pathway was detected in the latter mutants, leading to the conclusion that draining the vacuolar adenosine storage by ENT1 over-expression interferes with cellular nucleotide metabolism. As a consequence of the observed metabolic alterations 35S:ENT1 over-expresser mutants exhibited a smaller phenotypic appearance compared with wild-type plants. In addition, ENT1:RNAi mutants exhibited significantly lower in vitro germination of pollen and contained reduced internal and external ATP levels. This indicates that ENT1-mediated nucleosides, especially adenosine transport, is important for nucleotide metabolism, thus influencing growth and pollen germination.

Nucleosides are intermediates of nucleotide metabolism. Nucleotide de novo synthesis generates the nucleoside monophosphates AMP and UMP, which are further processed to all purine and pyrimidine nucleotides involved in multiple cellular reactions, including the synthesis of nucleic acids. Catabolism of these substances results in the formation of nucleosides, which are further degraded by nucleoside hydrolase to nucleobases. Both nucleosides and nucleobases can be exchanged between cells and tissues through multiple isoforms of corresponding transport proteins. After uptake into a cell, nucleosides and nucleobases can undergo salvage reactions or catabolism. Whereas energy is preserved by salvage pathway reactions, catabolism liberates ammonia, which is then incorporated into amino acids. Keeping the balance between nitrogen consumption during nucleotide de novo synthesis and ammonia liberation by nucleotide catabolism is essential for correct plant development. Senescence and seed germination represent situations in plant development where marked fluctuations in nucleotide pools occur. Furthermore, extracellular nucleotide metabolism has become an immensely interesting research topic. In addition, selected aspects of nucleoside transport in yeast, protists and humans are discussed.