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Nucleoside degradation and salvage are important metabolic pathways but hardly understood in plants. Recent work on human pathogenic protozoans like Leishmania and Trypanosoma substantiates an essential function of nucleosidase activity. Plant nucleosidases are related to those from protozoans and connect the pathways of nucleoside degradation and salvage. Here, we describe the cloning of such an enzyme from Arabidopsis thaliana, Uridine-Ribohydrolase 1 (URH1) and the characterization by complementation of a yeast mutant. Furthermore, URH1 was synthesized as a recombinant protein in Escherichia coli. The pure recombinant protein exhibited highest hydrolase activity for uridine, followed by inosine and adenosine, the corresponding K(m) values were 0.8, 1.4, and 0.7 mM, respectively. In addition, URH1 was able to cleave the cytokinin derivative isopentenyladenine-riboside. Promoter beta-glucuronidase fusion studies revealed that URH1 is mainly transcribed in the vascular cells of roots and in root tips, guard cells, and pollen. Mutants expressing the Arabidopsis enzyme or the homolog from rice (Oryza sativa) exhibit resistance toward toxic fluorouridine, fluorouracil, and fluoroorotic acid, providing clear evidence for a pivotal function of URH1 as regulative in pyrimidine degradation. Moreover, mutants with increased and decreased nucleosidase activity are delayed in germination, indicating that this enzyme activity must be well balanced in the early phase of plant development.

When grown in short day conditions and at low light, leaves of Arabidopsis plants with mutations in the genes encoding two plastidial ATP/ADP transporters (so-called null mutants) spontaneously develop necrotic lesions. Under these conditions, the mutants also display light-induced accumulation of H(2)O(2) and constitutive expression of genes for copper/zinc superoxide dismutase 2 and ascorbate peroxidase 1. In the light phase, null mutants accumulate high levels of phototoxic protoporphyrin IX but have only slightly reduced levels of Mg protoporphyrin IX. The physiological changes are associated with reduced magnesium-chelatase activity. Since the expression of genes encoding any of the three subunits of magnesium-chelatase is similar in wild type and null mutants, decreased enzyme activity is probably due to post-translational modification which might be due to limited availability of ATP in plastids during the night. Surprisingly, the formation of necrotic lesions was absent when null mutants were grown either in long days and low light intensity or in short days and high light intensity. We ascribe the lack of lesion phenotype to increased nocturnal ATP supply due to glycolytic degradation of starch which may lead to additional substrate-level phosphorylation in the stroma. Thus, nocturnal import of ATP into chloroplasts represents a crucial, previously unknown process that is required for controlled chlorophyll biosynthesis and for preventing photooxidative damage.

Arabidopsis (Arabidopsis thaliana) possesses two isoforms of plastidic ATP/ADP transporters (AtNTT1 and AtNTT2) exhibiting similar biochemical properties. To analyze the function of both isoforms on the molecular level, we examined the expression pattern of both genes by northern-blot analysis and promoter-beta-glucuronidase fusions. AtNTT1 represents a sugar-induced gene mainly expressed in stem and roots, whereas AtNTT2 is expressed in several Arabidopsis tissues with highest accumulation in developing roots and young cotyledons. Developing lipid-storing seeds hardly contained AtNTT1 or -2 transcripts. The absence of a functional AtNTT1 gene affected plant development only slightly, whereas AtNTT2T-DNA, AtNTT1-2T-DNA, and RNA interference (RNAi) plants showed retarded plant development, mainly characterized by a reduced ability to generate primary roots and a delayed chlorophyll accumulation in seedlings. Electron microscopic examination of chloroplast substructure also revealed an impaired formation of thylakoids in RNAi seedlings. Moreover, RNAi- and AtNTT1-2T-DNA plants showed reduced accumulation of the nuclear-encoded protein CP24 during deetiolation. Under short-day conditions reduced plastidic ATP import capacity correlates with a substantially reduced plant growth rate. This effect is absent under long-day conditions, strikingly indicating that nocturnal ATP import into chloroplasts is important. Plastidic ATP/ADP transport activity exerts significant control on lipid synthesis in developing Arabidopsis seeds. In total we made the surprising observation that plastidic ATP/ADP transport activity is not required to pass through the complete plant life cycle. However, plastidic ATP/ADP-transporter activity is required for both an undisturbed development of young tissues and a controlled cellular metabolism in mature leaves.