Fachgebiet Pflanzenphysiologie

Nucleoside transport and metabolism

Responsible Scientist: Dr. Torsten Möhlmann

Nucleotides are uniquely important since they represent building blocks of genetic information (DNA and RNA), represent major energy carriers and are also core elements of cofactors such as NAD, FAD, S-Adenosylmethionine or Coenzyme A which serve in essential biochemical reactions such as the synthesis of phospholipids and polysaccharides. Additionally, nucleotides are components of secondary metabolites like coffein, cAMP, cGMP and cytokinins. By reviewing the recently published work on nucleotide metabolism it becomes obvious that many facets of this important biochemical aspect of plant metabolism are still poorly understood. A reason for this is the complexity of a multitude of biochemical reactions which facilitate de novo synthesis, degradation and interconversion (partial degradation and recycling) of nucleotides, nucleosides and nucleobases. The recycling of nucleosides and nucleobases is known as salvage.

Möhlmann T, Bernard C, Hach S, Neuhaus HE (2010) Nucleoside transport and associated metabolism. Plant Biology 12, Supplement 1, 26-32

 

1. The plastidic nucleobase transporter PLUTO and the organization of pyrimidine de novo synthesis.

Nucleotide de novo synthesis is highly conserved among organisms and represents an absolutely essential biochemical pathway. This also holds true for plants. The subcellular localization of this pathway was reinvestigated with help of GFP-fusion constructs and it was found that the last three enzymes are not located in plastids as previously thought, but in the cytosol (DHOase and UMPsase) and mitochondria (DHODH). Therefore, we hypothesize that pyrimidine nucleotides are not synthesized de novo in plastids and instead need to be imported in form of precursors. This explains why plastid pyrimidine salvage is highly important. We believe that our results provide important new insights in this essential biochemical process and allow better understanding of underlying regulatory mechanisms.

In addition, a first plastid localized nucleobase transport protein, PLUTO, was identified and characterized in detail. This protein is capable of importing required uracil to plastids to supply pyrimidine salvage as pointed out above. PLUTO belongs to the family of Nucleobase-Cation-Symporters-1 (NCS1) and shares most conserved amino acids with a family member of Microbacterium liquefaciens MHP1, which was crystallized a high resolution recently. After expression of PLUTO in an Escherichia coli strain deficient in uraA, the bacterial uracil permease, the biochemical properties of PLUTO could be determined. PLUTO transports uracil, adenine and guanine with high affinities. The calculated KM value for uracil uptake is in good agreement with that determined by us on isolated intact plastids. Furthermore PLUTO transports adenine and guanine.

Witz S, Jung B, Fürst S, Möhlmann T (2012) De novo pyrimidine nucleotide synthesis mainly locates outside of plastids, but a novel nucleobase importer provides substrates for the essential salvage pathway. Plant Cell, early view.

 

2. The equlibrative nucleoside transporter (ENT) family

Members of the ENT family of nucleoside transporters typically exhibit 11 predicted transmembrane domains and catalyze transport energized by protons or an existing nucleoside concentration gradient. Members of this protein family mediate the distribution of purine and pyrimidine nucleosides (Möhlmann et al., 2001; Wormit et al., 2004) and this carrier group comprises eight members in Arabidopsis. Five of them, ENT1, 3, 4, 6, and 7 have so far been characterized at the biochemical level by heterologous expression in baker's yeast (Möhlmann et al., 2001; Wormit et al., 2004). All of these carriers exhibit broad substrate specificity and transport purine and pyrimidine nucleosides with apparent high affinities ranging from 3 μM to 90 μM.

Expression of nucleoside transporters ENT1 and ENT3, together with nucleoside import, was increased upon nitrogen limitation. Thereby a role for ENT3, which is expressed mainly in the vasculature of roots and leaves, as a major import route for nucleosides was supported (Figure 2, 3). Exogenously fed nucleosides were able to attenuate nitrogen starvation effects such as chlorophyll breakdown, anthocyanin accumulation, RNA breakdown and reduced levels of amino acids. In response to nucleoside supply, up-regulation of genes involved in nitrogen distribution in plants was observed. In addition, genes involved in nucleoside metabolism were identified as regulated upon nitrogen limitation. In summary, an overall beneficial effect of nucleoside supply to Arabidopsis seedlings, especially under limiting nitrogen conditions, was observed.

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 for a tonoplast localization of ENT1 comes from proteome data and Western blot analyses. Moreover, 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. These observations were accompanied by 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 (Figure 4).

Bernard C, Traub M, Kunz HH, Hach S, Trentmann O, Möhlmann T (2011) Equilibrative nucleoside transporter 1 (ENT1) is critical for pollen germination and vegetative growth in Arabidopsis. J. Exp. Bot. 62, 4627-37

Traub M, Flörchinger M, Piecuch J, Kunz H-H, Weise-Steinmetz A, Deitmer JW, Neuhaus HE, Möhlmann T (2007) The fur1 (fluorouridine insensitive 1) mutant is defective in equilibrative nucleoside transporter 3 (ENT3), thus representing an important pyrimidine nucleoside uptake system in Arabidopsis thaliana. Plant Journal 49, 855–864

Flörchinger M, Zimmermann M, Traub M, Neuhaus HE, Möhlmann T (2006) Adenosine stimulates anabolic metabolism in developing castor bean (Ricinus communis L.) cotyledons. Planta 223: 340-348

Wormit A, Traub M, Flörchinger M, Neuhaus HE, Möhlmann T (2004) Characterization of three novel members of the Arabidopsis thaliana equilibrative-nucleoside transporter (ENT) family. Biochem. J. 383: 19-26

Möhlmann T, Mezher Z, Schwerdtfeger G, Neuhaus HE (2001) Characterization of a concentrative type of adenosine transporter from Arabidopsis thaliana (ENT1,At) . FEBS Lett 509: 370-374

 

3. The nucleoside hydrolase (NSH) family

Nucleoside hydrolases are key enzymes in both nucleotide salvage and breakdown pathways, cleaving the nucleobase from the sugar, thereby allowing the base to be recycled as a nucleotide monophosphate, or being completely degraded. To examine nucleoside breakdown and salvage, a nucleosidase was characterized that acts on uridine and affects this key balance. NSH1 produced in E. coli was found to act on uridine and, with lower activity, on inosine, adenosine, and the cytokinin riboside isopentenyladenine-riboside, but not on cytidine (Jung et al., 2009). NSH1 was found to be a cytoplasmic enzyme expressed in root vasculature, root meristems, guard cells, and mature pollen cells. Plants with either reduced or enhanced NSH1 expression showed germination delays compared to wild-type, pointing to an overall alteration of pyrimidine nucleotide metabolism, since germination is a high-demand time for nucleotides. Further proteins studied are NSH2 and 3 which support NSH1 or act as extracellular, purine-specific nucleoside hydrolase, respectively.

Jung B, Hoffmann C, Möhlmann T (2011) Arabidopsis nucleoside hydrolases involed in intracellular and extracellular degradation of purines. Plant J. 65, 703-711

Jung B, Flörchinger M, Kunz H-H, Traub M, Wartenberg R, Jeblick W, Neuhaus HE, Möhlmann T (2009) Uridine-Ribohydrolase (URH) is a key regulator of uridine degradation. Plant Cell 21, 876–891

4. PYD1 initiates pyrimidine base catabolism

Pyd1 (dihydropyrimidine dehydogenase) initiates the degradation of pyrimidine nucleobases and is located in plastids. Pyd1 knockout mutants are restricted in degradation of exogenously provided uracil and accumulate high uracil levels in plant organs throughout development, especially in dry seeds. Moreover, Pyd1 knockout mutants show delayed germination (Figure 6) accompanied by low invertase activity and decreased monosaccharide levels. Absisic acid (ABA) is an important regulator of seed germination and ABA responsive genes are deregulated in Pyd1-knockout mutants. Together with an observed increased Pyd1 expression in wildtype seedlings an interference with ABA signaling in these plants can be assumed. Constitutive Pyd1 overexpression mutants germinate early and showed increased growth (Figure 6) and higher seed number compared to wildtype and knockout mutant plants. During senescence Pyd1 expression increased to allow uracil catabolism. From this we conclude that early in development and during seed production Pyd1 is needed to balance pyrimidine catabolism vs. salvage.

Cornelius S, Witz S, Rolletschek H, Möhlmann T (2011) Pyrimidine degradation influences germination seedling growth and production of Arabidopsis seeds. J. Exp. Bot. 62:5623-5632.

RIMB (Research Initiative Membrane Transport)

From 2002-2007 through The Arabidopsis Functional Genomics Network

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