Pyrimidine Salvage: Physiological Functions and Interaction with Chloroplast Biogenesis.

The synthesis of pyrimidine nucleotides, an essential process in every organism, is accomplished by de novo synthesis or by salvaging pyrimdines from e.g. nucleic acid turnover. Here, we identify two Arabidopsis (Arabidopsis thaliana) uridine/cytidine kinases, UCK1 and UCK2, which are located in the cytosol and are responsible for the majority of pyrimidine salvage activity in vivo. In addition, the chloroplast has an active uracil salvage pathway. Uracil phosphoribosyltransferase (UPP) catalyzes the initial step in this pathway and is required for the establishment of photosynthesis, as revealed by analysis of upp mutants. The upp knockout mutants are unable to grow photoautotrophically, and knockdown mutants exhibit a variegated phenotype, with leaves that have chlorotic pale areas. Moreover, the upp mutants did not show altered expression of chloroplast-encoded genes, but transcript accumulation of the LIGHT HARVESTING COMPLEX B nuclear genes LHCB1.2 and LHCB2.3 was markedly reduced. An active UPP homolog from Escherichia coli failed to complement the upp mutant phenotype when targeted to the chloroplast, suggesting that the catalytic function of UPP is not the important factor for the chloroplast phenotype. Indeed, the expression of catalytically inactive Arabidopsis UPP, generated by introduction of point mutations, did complement the upp chloroplast phenotype. These results suggest that UPP has a vital function in chloroplast biogenesis unrelated to its catalytic activity and driven by a moonlighting function.

In vitro analyses of mitochondrial ATP/phosphate carriers from Arabidopsis thaliana revealed unexpected Ca(2+)-effects.

BACKGROUND: Adenine nucleotide/phosphate carriers (APCs) from mammals and yeast are commonly known to adapt the mitochondrial adenine nucleotide pool in accordance to cellular demands. They catalyze adenine nucleotide--particularly ATP-Mg--and phosphate exchange and their activity is regulated by calcium. Our current knowledge about corresponding proteins from plants is comparably limited. Recently, the three putative APCs from Arabidopsis thaliana were shown to restore the specific growth phenotype of APC yeast loss-of-function mutants and to interact with calcium via their N-terminal EF--hand motifs in vitro. In this study, we performed biochemical characterization of all three APC isoforms from A. thaliana to gain further insights into their functional properties. RESULTS: Recombinant plant APCs were functionally reconstituted into liposomes and their biochemical characteristics were determined by transport measurements using radiolabeled substrates. All three plant APCs were capable of ATP, ADP and phosphate exchange, however, high preference for ATP-Mg, as shown for orthologous carriers, was not detectable. By contrast, the obtained data suggest that in the liposomal system the plant APCs rather favor ATP-Ca as substrate. Moreover, investigation of a representative mutant APC protein revealed that the observed calcium effects on ATP transport did not primarily/essentially involve Ca(2+)-binding to the EF-hand motifs in the N-terminal domain of the carrier. CONCLUSION: Biochemical characteristics suggest that plant APCs can mediate net transport of adenine nucleotides and hence, like their pendants from animals and yeast, might be involved in the alteration of the mitochondrial adenine nucleotide pool. Although, ATP-Ca was identified as an apparent import substrate of plant APCs in vitro it is arguable whether ATP-Ca formation and thus the corresponding transport can take place in vivo.

Apoplastic Nucleoside Accumulation in Arabidopsis Leads to Reduced Photosynthetic Performance and Increased Susceptibility Against Botrytis cinerea.

Interactions between plant and pathogen often occur in the extracellular space and especially nucleotides like ATP and NAD have been identified as key players in this scenario. Arabidopsis mutants accumulating nucleosides in the extracellular space were generated and studied with respect to susceptibility against Botrytis cinerea infection and general plant fitness determined as photosynthetic performance. The mutants used are deficient in the main nucleoside uptake system ENT3 and the extracellular nucleoside hydrolase NSH3. When grown on soil but not in hydroponic culture, these plants markedly accumulate adenosine and uridine in leaves. This nucleoside accumulation was accompanied by reduced photosystem II efficiency and altered expression of photosynthesis related genes. Moreover, a higher susceptibility toward Botrytis cinerea infection and a reduced induction of pathogen related genes PR1 and WRKY33 was observed. All these effects did not occur in hydroponically grown plants substantiating a contribution of extracellular nucleosides to these effects. Whether reduced general plant fitness, altered pathogen response capability or more direct interactions with the pathogen are responsible for these observations is discussed.

Nucleobase and nucleoside transport and integration into plant metabolism.

Nucleotide metabolism is an essential process in all living organisms. Besides newly synthesized nucleotides, the recycling (salvage) of partially degraded nucleotides, i.e., nucleosides and nucleobases serves to keep the homeostasis of the nucleotide pool. Both types of metabolites are substrates of at least six families of transport proteins in Arabidopsis thaliana (Arabidopsis) with a total of 49 members. In the last years several members of such transport proteins have been analyzed allowing to present a more detailed picture of nucleoside and nucleobase transport and the physiological function of these processes. Besides functioning in nucleotide metabolism it turned out that individual members of the before named transporters exhibit the capacity to transport a wide range of different substrates including vitamins and phytohormones. The aim of this review is to summarize the current knowledge on nucleobase and nucleoside transport processes in plants and integrate this into nucleotide metabolism in general. Thereby, we will focus on those proteins which have been characterized at the biochemical level.

Biochemical characterization and structure-function relationship of two plant NCS2 proteins, the nucleobase transporters NAT3 and NAT12 from Arabidopsis thaliana.

Nucleobase ascorbate transporters (NATs), also known as Nucleobase:Cation-Symporter 2 (NCS2) proteins, belong to an evolutionary widespread family of transport proteins with members in nearly all domains of life. We present the biochemical characterization of two NAT proteins, NAT3 and NAT12 from Arabidopsis thaliana after their heterologous expression in Escherichia coli UraA knockout mutants. Both proteins were shown to transport adenine, guanine and uracil with high affinities. The apparent KM values were determined with 10.12µM, 4.85µM and 19.95µM, respectively for NAT3 and 1.74µM, 2.44µM and 29.83µM, respectively for NAT12. Competition studies with the three substrates suggest hypoxanthine as a further substrate of both transporters. Furthermore, the transport of nucleobases was markedly inhibited by low concentrations of a proton uncoupler indicating that NAT3 and NAT12 act as proton-nucleobase symporters. Transient expression studies of NAT-GFP fusion constructs revealed a localization of both proteins in the plasma membrane. Based on the structural information of the uracil permease UraA from E. coli, a three-dimensional experimentally validated homology model of NAT12 was created. The NAT12 structural model is composed of 14 TM segments and divided into two inverted repeats of TM1-7 and TM8-14. Docking studies and mutational analyses identified residues involved in NAT12 nucleobase binding including Ser-247, Phe-248, Asp-461, Thr-507 and Thr-508. This is the first study to provide insight into the structure-function of plant NAT proteins, which reveals differences from the other members of the NCS2 protein family.

Structure-function relationship of a plant NCS1 member--homology modeling and mutagenesis identified residues critical for substrate specificity of PLUTO, a nucleobase transporter from Arabidopsis.

Plastidic uracil salvage is essential for plant growth and development. So far, PLUTO, the plastidic nucleobase transporter from Arabidopsis thaliana is the only known uracil importer at the inner plastidic membrane which represents the permeability barrier of this organelle. We present the first homology model of PLUTO, the sole plant NCS1 member from Arabidopsis based on the crystal structure of the benzyl hydantoin transporter MHP1 from Microbacterium liquefaciens and validated by molecular dynamics simulations. Polar side chains of residues Glu-227 and backbones of Val-145, Gly-147 and Thr-425 are proposed to form the binding site for the three PLUTO substrates uracil, adenine and guanine. Mutational analysis and competition studies identified Glu-227 as an important residue for uracil and to a lesser extent for guanine transport. A differential response in substrate transport was apparent with PLUTO double mutants E227Q G147Q and E227Q T425A, both of which most strongly affected adenine transport, and in V145A G147Q, which markedly affected guanine transport. These differences could be explained by docking studies, showing that uracil and guanine exhibit a similar binding mode whereas adenine binds deep into the catalytic pocket of PLUTO. Furthermore, competition studies confirmed these results. The present study defines the molecular determinants for PLUTO substrate binding and demonstrates key differences in structure-function relations between PLUTO and other NCS1 family members.

De novo pyrimidine nucleotide synthesis mainly occurs outside of plastids, but a previously undiscovered nucleobase importer provides substrates for the essential salvage pathway in Arabidopsis.

Nucleotide de novo synthesis is highly conserved among organisms and represents an essential biochemical pathway. In plants, the two initial enzymatic reactions of de novo pyrimidine synthesis occur in the plastids. By use of green fluorescent protein fusions, clear support is provided for a localization of the remaining reactions in the cytosol and mitochondria. This implies that carbamoyl aspartate, an intermediate of this pathway, must be exported and precursors of pyrimidine salvage (i.e., nucleobases or nucleosides) are imported into plastids. A corresponding uracil transport activity could be measured in intact plastids isolated from cauliflower (Brassica oleracea) buds. PLUTO (for plastidic nucleobase transporter) was identified as a member of the Nucleobase:Cation-Symporter1 protein family from Arabidopsis thaliana, capable of transporting purine and pyrimidine nucleobases. A PLUTO green fluorescent protein fusion was shown to reside in the plastid envelope after expression in Arabidopsis protoplasts. Heterologous expression of PLUTO in an Escherichia coli mutant lacking the bacterial uracil permease uraA allowed a detailed biochemical characterization. PLUTO transports uracil, adenine, and guanine with apparent affinities of 16.4, 0.4, and 6.3 µM, respectively. Transport was markedly inhibited by low concentrations of a proton uncoupler, indicating that PLUTO functions as a proton-substrate symporter. Thus, a protein for the absolutely required import of pyrimidine nucleobases into plastids was identified.

Pyrimidine degradation influences germination seedling growth and production of Arabidopsis seeds.

PYD1 (dihydropyrimidine dehydogenase) initiates the degradation of pyrimidine nucleobases and is located in plastids. In this study, a physiological analysis of PYD1 employing T-DNA knockout mutants and overexpressors was carried out. PYD1 knockout mutants were restricted in degradation of exogenously provided uracil and accumulated high uracil levels in plant organs throughout development, especially in dry seeds. Moreover, PYD1 knockout mutants showed delayed germination which was accompanied by low invertase activity and decreased monosaccharide levels. Abscisic acid (ABA) is an important regulator of seed germination, and ABA-responsive genes were deregulated in PYD1 knockout mutants. Together with an observed increased PYD1 expression in wild-type seedlings upon ABA treatment, an interference of PYD1 with ABA signalling can be assumed. Constitutive PYD1 overexpression mutants showed increased growth and higher seed number compared with wild-type and knockout mutant plants. During senescence PYD1 expression increased to allow uracil catabolism. From this it is concluded that early in development and during seed production PYD1 is needed to balance pyrimidine catabolism versus salvage.