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• W2029812675 abstract "Salvage pathways play an important role in providing nucleobases to cells, which are unable to synthesize sufficient amounts for their needs. Cellular uptake systems for pyrimidines have been described, but in higher eukaryotes, transporters for thymine and uracil have not been identified. Two plant transporters, AtUPS1 and PvUPS1, were recently identified as transporters for allantoin in Arabidopsis and French bean, respectively. However, Arabidopsis, in contrast to tropical legumes, uses mainly amino acids for long distance transport. Allantoin transport has not been described in the Brassicaceae. Thus, the physiological substrates of ureide permease (UPS) transporters in Arabidopsis may be compounds structurally related to allantoin. A detailed analysis of the substrate specificities of two members of the AtUPS family shows that AtUPS1 and AtUPS2 mediate high affinity uracil and 5-fluorouracil (a toxic uracil analogue) transport when expressed in yeast and Xenopus oocytes. Consistent with a function during germination and early seedling development, AtUPS1 expression is transiently induced during the early stages of seedling development followed by up-regulation of AtUPS2 expression. Arabidopsis ups2 insertion mutants and ups1 lines, in which transcript levels were reduced by post-transcriptional gene silencing, are more tolerant to 5-fluorouracil as compared with wild type plants. The results suggest that in Arabidopsis UPS transporters are the main transporters for uracil and potentially other nucleobases, whereas during evolution legumes may have taken advantage of the low selectivity of UPS proteins for long distance transport of allantoin. Salvage pathways play an important role in providing nucleobases to cells, which are unable to synthesize sufficient amounts for their needs. Cellular uptake systems for pyrimidines have been described, but in higher eukaryotes, transporters for thymine and uracil have not been identified. Two plant transporters, AtUPS1 and PvUPS1, were recently identified as transporters for allantoin in Arabidopsis and French bean, respectively. However, Arabidopsis, in contrast to tropical legumes, uses mainly amino acids for long distance transport. Allantoin transport has not been described in the Brassicaceae. Thus, the physiological substrates of ureide permease (UPS) transporters in Arabidopsis may be compounds structurally related to allantoin. A detailed analysis of the substrate specificities of two members of the AtUPS family shows that AtUPS1 and AtUPS2 mediate high affinity uracil and 5-fluorouracil (a toxic uracil analogue) transport when expressed in yeast and Xenopus oocytes. Consistent with a function during germination and early seedling development, AtUPS1 expression is transiently induced during the early stages of seedling development followed by up-regulation of AtUPS2 expression. Arabidopsis ups2 insertion mutants and ups1 lines, in which transcript levels were reduced by post-transcriptional gene silencing, are more tolerant to 5-fluorouracil as compared with wild type plants. The results suggest that in Arabidopsis UPS transporters are the main transporters for uracil and potentially other nucleobases, whereas during evolution legumes may have taken advantage of the low selectivity of UPS proteins for long distance transport of allantoin. Purine and pyrimidine nucleotides are building blocks for DNA and RNA synthesis and essential precursors for enzyme cofactors (NAD, FAD, 5-adenosylmethionin, thiamin), signal molecules (e.g. cyclic nucleotides or the phytohormone cytokinins), and plant secondary metabolites (e.g. caffeine). They can be synthesized de novo or obtained by recycling of nucleosides and nucleobases (salvage pathways) (1Moffatt B.A. Ashihara H. Somerville C.R. Meyerowitz E.M. The Arabidopsis Book. American Society of Plant Biologists, Rockville, MD2002: 1-20Google Scholar, 2van der Graaff E. Hooykaas P. Lein W. Lerchl J. Kunze R. Sonnewald U. Boldt R. Front Biosci. 2004; 9: 1803-1816Crossref PubMed Scopus (31) Google Scholar). The enzymes involved in salvage pathways frequently act with cellular import systems for their substrates in a concerted manner. Thus, most unicellular organisms including prokaryotes, fungi, and cellular parasites possess specialized transporters for the capture of nucleosides and nucleobases from their environment, which are used for nucleotide synthesis with lower net energy cost compared with de novo synthesis. In some extreme cases obligate parasites have lost the capacity to produce nucleotides via de novo synthesis, and import of nucleobases or nucleosides represents the only route to nucleotide formation. Uptake systems for nucleosides and nucleobases are also present in multicellular organisms. Moreover, the expression of uptake and release systems in distinct animal cell types seems to play a crucial role in the recovery and the distribution of nucleobases and nucleosides passing tissue barriers, e.g. in the kidney, intestine, and placenta (3de Koning H. Diallinas G. Mol. Memb. Biol. 2000; 75: 75-94Google Scholar). These transport systems, therefore, play not only an important role in organism physiology but also in the pharmaco-dynamics of anti-tumor compounds, such as 5-FU 1The abbreviations used are: 5-FU, 5-fluorouracil; UPS, ureide permease; PUP, purine permease; ENT, equilibrative nucleoside transporter; PRT, purine-related transporter; NAT, nucleobase-ascorbate transporter; PTGS, post transcriptional gene silencing; Ura, uracil; Mes, 4-morpholineethanesulfonic acid; GUS, β-glucuronidase. (4Damaraju V.L. Damaraju S. Young J.D. Baldwin S.A. Mackey J. Sawyer M.B. Cass C.E. Oncogene. 2003; 22: 7524-7536Crossref PubMed Scopus (254) Google Scholar). In plant cells adenylates comprise the largest nucleotide pool followed in size by uridylates. UDP-sugars serve as activated intermediates in the synthesis of sucrose and cell wall polymers (5Wagner K.G. Backer A.I. Int. Rev. Cytol. 1992; 134: 1-84Crossref Scopus (68) Google Scholar). Of the free pyrimidine bases, plants apparently can only salvage uracil, whereas they are unable to salvage cytosine due to the lack of cytosine deaminase (5Wagner K.G. Backer A.I. Int. Rev. Cytol. 1992; 134: 1-84Crossref Scopus (68) Google Scholar). Cells often rely on salvage pathways when the activities of enzymes for de novo synthesis are low or absent (e.g. during early stages of seed germination) or when demand for nucleobases is high (e.g. when cells divide rapidly). Although salvaging of nucleobases and nucleosides predominates at the inception of germination, its importance declines when germination proceeds and de novo synthesis takes over (6Stasolla C. Katahira R. Thorpe T.A. Ashihara H. J. Plant Physiol. 2003; 160: 1-25Crossref Scopus (239) Google Scholar). Salvaging is often accompanied by transport of nucleobases and nucleosides from storage tissues, as was demonstrated for germinating castor bean (7Kombrink E. Beevers H. Plant Physiol. 1983; 73: 370-376Crossref PubMed Google Scholar). A high affinity transporter system for uracil with a Km of ∼40 μm was described (7Kombrink E. Beevers H. Plant Physiol. 1983; 73: 370-376Crossref PubMed Google Scholar). Despite the apparent physiological importance of high affinity transport of free pyrimidines, in particular of uracil, no transporters for these substrates have been identified in plants or other higher eukaryotes until now. A number of membrane protein families mediating transport of nucleobases or nucleosides into the cell have been described to date; they comprise (i) the nucleobase ascorbate transporters (NAT; Archaea, eubacteria, fungi, plants, and animals) (8Tsukaguchi H. Tokui T. Mackenzie B. Berger U.V. Chen X.Z. Wang Y. Brubaker R.F. Hediger M.A. Nature. 1999; 399: 70-75Crossref PubMed Scopus (727) Google Scholar, 9Argyrou E. Sophianopoulou V. Schultes V. Diallinas G. Plant Cell. 2001; 13: 953-964Crossref PubMed Scopus (62) Google Scholar), (ii) the purine-related transporter family (PRT; Archaea, bacteria, fungi, and plants but not in animals) (10de Montigny J. Straub M.L. Wagner R. Bach M.L. Chevallier M.R. Yeast. 1998; 14: 1051-1059Crossref PubMed Scopus (10) Google Scholar), (iii) the purine-related permeases (PUP; only in plants) (11Gillissen B. Bürkle L. André B. Kühn C. Rentsch D. Brandl B. Frommer W.B. Plant Cell. 2000; 12: 291-300Crossref PubMed Scopus (156) Google Scholar, 12Bürkle L. Cedzich A. Döpke C. Stransky H. Okumoto S. Gillissen B. Kühn C. Frommer W.B. Plant J. 2003; 34: 13-26Crossref PubMed Scopus (174) Google Scholar), (iv) the ureide permeases (UPS; only in plants) (13Desimone M. Catoni E. Ludewig U. Hilpert M. Schneider A. Kunze R. Tegeder M. Frommer W.B. Schumacher K. Plant Cell. 2002; 14: 847-856Crossref PubMed Scopus (84) Google Scholar, 14Pélissier H.C. Frerich A. Desimone M. Schumacher K. Tegeder M. Plant Physiol. 2004; 134: 664-675Crossref PubMed Scopus (63) Google Scholar), (v) the concentrative nucleoside transporters (fungi and animals but not in plants) (15Cass C.E. Young J.D. Baldwin S.A. Biochem. Cell Biol. 1998; 76: 761-770Crossref PubMed Scopus (168) Google Scholar), and (vi) the equilibrative nucleoside transporters (ENT; fungi and animals and plants) (16Hyde R.J. Cass C.E. Young J.D. Baldwin S.A. Mol. Memb. Biol. 2001; 18: 53-63Crossref PubMed Google Scholar, 17Möhlmann T. Mezher Z. Schwerdtfeger H. Neuhaus E. FEBS Lett. 2001; 509: 370-374Crossref PubMed Scopus (45) Google Scholar). Only two of these families include members with demonstrated capacity to transport uracil and other free pyrimidines; NAT, e.g. UraA in Escherichia coli (18Andersen P.S. Frees D. Fast R. Mygind B. J. Bacteriol. 1995; 177: 2008-2013Crossref PubMed Google Scholar), and PRT, e.g. FUR4 in Saccharomyces cerevisiae (19Jund R. Weber E. Chevallier M.R. Eur. J. Biochem. 1988; 171: 417-424Crossref PubMed Scopus (83) Google Scholar). However, the investigated NAT homologues in animals mediate Na+-coupled ascorbate transport, and maize Lpe1 (leaf permease), the only NAT protein characterized so far in plants, transports xanthine and uric acid (9Argyrou E. Sophianopoulou V. Schultes V. Diallinas G. Plant Cell. 2001; 13: 953-964Crossref PubMed Scopus (62) Google Scholar). PRT homologues are not present in higher eukaryotes with the exception of an expressed Arabidopsis protein with unknown function (At5g03555). Recently, members of the UPS family mediating allantoin transport were identified in Arabidopsis and French bean (13Desimone M. Catoni E. Ludewig U. Hilpert M. Schneider A. Kunze R. Tegeder M. Frommer W.B. Schumacher K. Plant Cell. 2002; 14: 847-856Crossref PubMed Scopus (84) Google Scholar, 14Pélissier H.C. Frerich A. Desimone M. Schumacher K. Tegeder M. Plant Physiol. 2004; 134: 664-675Crossref PubMed Scopus (63) Google Scholar). Allantoin is an important transport form of organic nitrogen in legumes, whereas Arabidopsis is considered to preferentially transport amino acids. Therefore, the substrate specificities of AtUPS1 and AtUPS2, related members of the UPS family from Arabidopsis, were analyzed in detail. Using heterologous expression in yeast and in Xenopus oocytes, it was demonstrated that pyrimidines are excellent substrates for both AtUPS1 and AtUPS2. The affinity for uracil was several-fold higher than for allantoin. In plants, expression of AtUPS1 and AtUPS2 was high during periods of increased demand of nucleotides, i.e. early seedling development, indicating a function in salvage pathways, e.g. by the utilization of pyrimidines from seed storage tissue. In planta, AtUPS function as major transporters for uracil analogs and potentially uracil itself, as demonstrated by the increased tolerance to 5-FU of plants in which AtUPS1 was inhibited by PTGS or in plants that carry T-DNA insertions in AtUPS2. UPS1 and UPS2 Expression Constructs—AtUPS2 was amplified by PCR from a cDNA library from 5-day-old seedlings from Arabidopsis thaliana (ecotype Col-0) with the primers 5′-ATAGGATCCATCCATTTAGAGCCCGAGAAT-3′ and 5′-ATATCTAGATTACTTTCTATGTCCAGAAGA-3′ and cloned into pCR4TopoBlunt (Invitrogen). Sequencing revealed mutations of A to T (at position 276 of the open reading frame) and A678 to G not resulting in an altered protein sequence. In addition, T308 was mutated to C, causing a valine to alanine exchange. This mutation was reverted by site-directed mutagenesis. The AtUPS1-coding sequence was PCR-amplified from pFL61UPS1 (13Desimone M. Catoni E. Ludewig U. Hilpert M. Schneider A. Kunze R. Tegeder M. Frommer W.B. Schumacher K. Plant Cell. 2002; 14: 847-856Crossref PubMed Scopus (84) Google Scholar) and cloned into pCR4TopoBlunt. The AtUPS1-coding sequences were BamHI/XbaI excised from pCR4TopoBluntUPS1 and cloned into the oocyte expression vector pOO2 (20Ludewig U. von Wiren N. Frommer W.B. J. Biol. Chem. 2002; 277: 13548-13555Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar) cut by BamHI/XbaI. The UPS1 oocyte expression construct was described previously (13Desimone M. Catoni E. Ludewig U. Hilpert M. Schneider A. Kunze R. Tegeder M. Frommer W.B. Schumacher K. Plant Cell. 2002; 14: 847-856Crossref PubMed Scopus (84) Google Scholar). The UPS2 coding sequence was BamHI/XhoI-excised from pOO2UPS2 and subcloned into the yeast expression vector pDR199. pDR199UPS1 was derived by amplifying UPS1 from the cDNA library with primers 5′-CCCAGCCTTGAAAATAATTAAAATTTATAATTTTAAAAGATAAAAGAGAGATAGAAAGAT-G-3′ and 5′-AAGCTGGATCGCTCGAGTCGACTGCAGGCCGCCCGGGCCGTCATTTTCTATGTCCCGAGGAAG-3′. The PCR product together with the EcoRI/BamHI-linearized vector pDR199 was introduced into a yeast dal4 dal5 knock out strain (13Desimone M. Catoni E. Ludewig U. Hilpert M. Schneider A. Kunze R. Tegeder M. Frommer W.B. Schumacher K. Plant Cell. 2002; 14: 847-856Crossref PubMed Scopus (84) Google Scholar) by in vivo cloning (21Dohmen R.S. Strasser A.W. Honer C.B. Hollenberg C.P. Yeast. 1991; 7: 691-692Crossref PubMed Scopus (319) Google Scholar). After selection on uracil-free medium, plasmids were isolated, and the cDNA insertion was verified by sequencing. Two T to C mutations (positions 997 and 1152 of the open reading frame), which do not alter the protein sequence, were found in the coding sequence. pMD200 was derived from pDR195 (22Rentsch D. Laloi M. Rouhara I. Schmelzer E. Delrot S. Frommer W.B. FEBS Lett. 1995; 370: 264-268Crossref PubMed Scopus (278) Google Scholar) by disruption of the URA3 gene by ApaI/NcoI digestion and introduction of the LEU2 gene. LEU2 was PCR-amplified with primers 5′-ATAGGGCCCGTGGGAATACTCAGGTATCG-3′ and 5′-ATACCATGGCTGGACGTAAACTCCTCTTC-3′ from pACT2 (BD Biosciences). The UPS1 (UPS2)-coding sequences were excised from pCR4TopoBluntUPS1 (pCR4TopoBluntUPS2) and cloned into pMD200; UPS1 was excised EcoRI, blunted, and introduced into the blunted NotI site of pMD200; UPS2 was excised by XbaI/NotI, XbaI was blunted, and the construct was introduced into pMD200 (BamHI-blunted/NotI). Expression in Yeast—The yeast dal4 dal5 mutant strain was transformed with pDR199 or pDR199UPS1 (pDR199UPS2) (23Gietz R.D. Schiestl R.H. Methods Mol. Cell Biol. 1995; 5: 255-269Google Scholar). Growth tests were performed on medium (synthetic defined medium without ammonium sulfate) with 5 mm allantoin. The yeast strain CEN.PK 2-1C (Euroscarf) was used to generate a fur4 knockout mutant strain by using a loxP-KanMX-loxP disruption cassette (24Güldner U. Heck S. Fiedler T. Beinhauer J. Hegemann J. Nucleic Acids Res. 1996; 24: 2519-2524Crossref PubMed Scopus (1362) Google Scholar). The fur4 mutant strain was selected on synthetic defined medium (with 38 mm ammonium sulfate, 200 mg/liter G418, 0.1 mm l-histidine, 0.1 mm l-tryptophan, 0.2 mm uridine), confirmed by PCR, and subsequently transformed with pMD200 or pMD200UPS1 (pMD200UPS2). Growth tests were performed on medium with 0.2 mm uracil instead of uridine. Transport measurements with 14C-labeled allantoin were performed as previously described (13Desimone M. Catoni E. Ludewig U. Hilpert M. Schneider A. Kunze R. Tegeder M. Frommer W.B. Schumacher K. Plant Cell. 2002; 14: 847-856Crossref PubMed Scopus (84) Google Scholar). Expression in Xenopus laevis Oocytes—Oocytes were surgically removed from female X. laevis frogs. Follicular cell layers and connective tissues were removed by digesting with 2 mg/ml collagenase A (Roche Applied Science) for 1.5–2 h at 22 °C in a Ca2+-free solution composed of 82.5 mm NaCl, 2 mm KCl, 1 mm MgCl2, 5mm HEPES, pH 7.4. Oocytes were subsequently washed 5–8 times with ND96 solution (96 mm NaCl, 2 mm KCl, 1 mm MgCl2, 1.8 mm CaCl2, 5 mm HEPES, pH 7.4). Healthy oocytes at developmental stages V-VI were selected and maintained at 16 °C in ND96 solution supplied with 50 mg/liter gentamycin and 2.5 mm sodium pyruvate (ND96-G). Capped cRNAs were transcribed in vitro using the mMessage mMachine mRNA SP6 kit (Ambion) from linearized plasmids pOO2UPS1 and pOO2UPS2. RNA yields were estimated by agarose gel electrophoresis, and the concentrations were finally adjusted to ∼1 μg/μl with nuclease-free water. Oocytes were microinjected with 50 nl of cRNA (20–50 ng/oocyte) and incubated at 16 °C in ND96-G solution to allow the expression of AtUPS genes. Electrophysiology—Two-electrode voltage clamp was performed starting 2 days after injection. The standard perfusion solution (wash solution) contained 100 mm choline chloride, 2 mm CaCl2, 2 mm MgCl2 in 5 mm Mes-Tris, pH 5.5. Substances were dissolved in this solution to final concentrations of 200 μm, and the pH values were adjusted to 5.5 with Mes or Tris. For voltage-current analysis, currents were recorded while clamping the oocytes at –100 mV or by applying an I/V protocol. The I/V protocol consisted of 200-ms pulses of voltages ranging from –140 to 0 mV in steps of 20 mV. Substrate evoked currents were constant during these short pulses. Net currents induced by substrates were obtained by subtracting the background currents (measured with “wash solution” alone) before and after the addition of substrates. To avoid endogenous currents, which were slowly activated at more negative voltages, currents elicited at more negative voltages than –120 mV were not analyzed (in some cases only voltages in the range of 0 to –100 mV were analyzed). No differences between non-injected and water-injected oocytes were observed; thus, in most cases non-injected oocytes were used as controls. T-DNA Insertion Lines—A. thaliana ecotype Col-0 was used throughout this study. Seeds of the T-DNA insertion lines Salk_ 143013 (designated ups2-1) and Salk_044551 (designated ups2-2) were obtained from the Arabidopsis Biological Resource Center (Columbus, Ohio) (25Alonso J.M. Stepanova A.N. Leisse T.J. Kim C.J. Chen H. et al.Science. 2003; 301: 653-657Crossref PubMed Scopus (4127) Google Scholar). Homozygous plants were isolated from T3 plants and confirmed in the T4 generation. Genomic DNA was prepared as described (www.dartmouth.edu/~tjack/TAILDNAprep.html). Homozygous lines were screened by two sets of PCR. The first PCR was performed using gene-specific primers 5′-GTAGATAAGTTTTCCGTGACG-3′ and 5′-GAACCTAGCAAAGGTAGTGTC-3′ (Salk_044551) or 5′-CATTGCAAACATAACAGACG-3′ and 5′-GTATCTTTACATTCTCTGG-AC-3′ (Salk_143013). The second PCR was carried out with the T-DNA left border primer 5′-CTTTGACGTTGGAGTCCAC-3′ and the primer 5′-GAACCTAGCAAAGGTAGTGTC-3′ (Salk_044551) or 5′-GTATCTTTACATTCTCTGGAC-3′ (Salk_143013). Plants were considered homozygous if no PCR product was obtained by the first PCR reaction, and fragments of expected size confirming the T-DNA insertion were amplified by the second PCR reaction. Localization of T-DNA insertions was analyzed by sequencing. Homozygous lines were subsequently taken for further investigations. PTGS Construct—A 521-bp AtUPS1 fragment (sense) was amplified by PCR from pFL61UPS1 using primers 5′-ACTAGTTAAAAGAGAGATAGAAAGATG-3′ and 5′-AAGCTTCTTTTGAAATTTTGGAGTTTG-3′, and a similar 521-bp fragment (antisense) was amplified using primers 5′-CTCGAGCTTTTGAAATTTTGGAGTTTG-3′ and 5′-TCTAGATAAAAGAGAGATAGAAAGATG-3′. An 882-bp fragment of the second intron of AtAAP6 (At5g49630) was amplified from BAC K6M13 using primers 5′-ATAAAGCTTATCCTATCTAGGTCAGATTCG-3′ and 5′-TCTAGAATACTCGAGACTTTTCTTCCTCCTGTTTAT-3′. All fragments were cloned into pCR4TopoBlunt and sequenced. The UPS1 antisense fragment was excised with XbaI/XhoI and ligated into the pCR4TopoBluntAAP6intron construct. The resulting construct was subsequently cleaved with HindIII/SpeI and the UPS1 sense fragment, excised by HindIII/SpeI, was introduced. The complete UPS1 sense-AAP6-intron-UPS1-antisense construct was subcloned into pCB302.3 (27Xiang C. Han P. Lutziger I. Wang K. Oliver D.J. Plant Mol. Biol. 1999; 40: 711-717Crossref PubMed Scopus (367) Google Scholar) using SpeI/XbaI. Promoter-GUS Constructs—The AtUPS1 (AtUPS2) promoter region comprising the 647-bp (721 bp) genomic DNA extending from the 3′-untranslated region of the preceding gene to ATG was PCR-amplified from genomic DNA (BAC T4M8) using primers 5′-ATAACTAGTCTTACAAGAACAGCAAGCTTT-3′ and 5′-ATAGGATCCCTTTCTATCTCTCTTTTATAT-3′ (5′-ATATCTAGAATGATCTTATCATAGTTGTAT-3′ and 5′-ATAGGATCCACAAGCTATGGCACCTCCTTT-3′), cloned into pCR4TopoBlunt, and sequenced. The AtUPS1 (AtUPS2) promoter was excised by SpeI/BamHI (XbaI/BamHI) and introduced into pCB308 (27Xiang C. Han P. Lutziger I. Wang K. Oliver D.J. Plant Mol. Biol. 1999; 40: 711-717Crossref PubMed Scopus (367) Google Scholar). Plant Transformation—pCB302.3 and pCB308 constructs were introduced into Agrobacterium strain GV3101 for plant transformation. Arabidopsis plants grown in the greenhouse were transformed with Agrobacterium using the floral dip method (28Clough S.J. Bent A.F. Plant J. 1998; 16: 735-743Crossref PubMed Google Scholar) and transgenic plants (T1 to T3 generation) were recovered by BASTA™ (phosphinothricin). For analysis of GUS activity three lines (T3) of the translational fusions of AtUPS1 or AtUPS2 promoter regions with the uidA gene were used for further investigation. Five randomly chosen T3 or T4 lines containing the UPS1-PTGS construct, three of which were homozygous and two heterozygous (based on BASTA resistance segregation), were analyzed for mRNA levels and phenotypic alterations. Plant Growth—Seeds were vernalized at 4 °C for 2 days before imbibition. Plants were grown in a Percival Scientific growth chamber under a 16 h of light/8 h of dark photoperiod at 22 °C and a light intensity of 100–150 microeinsteins m-2 s-1. Plates with MS medium (Invitrogen) supplemented with 1% (w/v) sucrose and 0.7% (w/v) phytagar (Invitrogen) were used for all experiments except for studies with N-(phosphonacetyl)-l-aspartate (1 mm) and exogenous pyrimidines (1 mm). In the latter studies seedlings were grown in liquid MS medium containing 1% (w/v) sucrose on a rotary shaker at 100 rpm. For growth assays on 5-FU, the medium was supplemented with 200 μm quantities of this compound. Promoter GUS assays—Plants were harvested from days 1 to 11 after imbibition and analyzed for β-glucuronidase activity (29Martin T. Wöhner R.V. Hummel S. Willmitzer L. Frommer W.B. Gallagher S.R. GUS Protocols. Academic Press, Inc., San Diego, CA1992: 23-43Crossref Google Scholar). Staining was performed for 15 h at 37 °C. For root sections, 6-day-old plants were prefixed in 50 mm phosphate buffer, pH 7.2, containing 1.4% formaldehyde. Plants were washed in 50 mm phosphate buffer, pH 7.2, followed by GUS staining for 8 h at 37 °C and subsequent fixation in 50 mm phosphate buffer, pH 7.2, containing 2% paraformaldehyde and 1% glutaraldehyde. Dehydration was performed in an ethanol series followed by embedding in hydroxyethyl methacrylate (Technovit 7100, Heraeus Kulzer). 6-μm-thick sections were prepared with a LKB Ultratome and counterstained in purple by periodic acid-Schiff staining. Reverse Transcription-PCR Analysis—Total RNA from agar-grown seedlings of Arabidopsis was extracted (30Chang S. Puryear J. Cairney J. Plant Mol. Biol. Rep. 1993; 11: 113-116Crossref Scopus (2977) Google Scholar). cDNA synthesized with RevertAid™ H minus M murine leukemia virus reverse transcriptase (MBI) was used as template for PCR reactions. As an internal control a 641-bp fragment of the AtACT2 gene was amplified by PCR (26 cycles) using primers 5′-CGTACAACCGGTATTGTGCTGG-3′ and 5′-GGACCTGCCTCATCATACTCG-3′. For AtUPS1 (AtUPS2) primers 5′-CAATTTCTGCAAGCAACGGGTTAAC-3′ and 5′-ACAAGTGGAAGTGCCTGAACAGCG-3′ (5′-CATCAAAAGACCTGGAGACTAATG-3′ and 5′-CTCACAAGTGGAAGAGCCTGAACG-3′) were used to amplify a 481-bp (484 bp) fragment by PCR in 30 or 32 cycles. Total RNA was isolated from 9-day-old seedlings grown in liquid cultures using RNeasy (Qiagen), treated with DNase I, and reverse-transcribed. PCR (29 cycles each) of AtUPS1 (AtUPS2) templates with primers 5′-GCACAATAATCGGATTGGTG-3′ and 5′-ATGTTAAGTATCAGAGCAACTACAAATGC-3′ (5′-GTATCGTGCTTAGCCTCG-3′ and 5′-TGTTCCTATTTCGGTAGATGGACC-3′) produced a 187-bp (299 bp) fragment. For AtACT2 a 192-bp fragment was amplified by PCR (26 cycles) using primers 5′-TCCTCACTTTCATCAGCCG-3′ and 5′-ATTGGTTGAATATCATCAGCC-3′. AtUPS1 and –2 Mediate Allantoin Uptake—Out of the five members of the UPS family in Arabidopsis, AtUPS1 and AtUPS3 seem to have arisen from a recent gene duplication as well as AtUPS 2 and AtUPS4. To be able to compare the properties of these two groups, the AtUPS2 cDNA (At2g03530) was isolated from a seedling cDNA library. AtUPS2 showed 74% sequence identity to AtUPS1 on the amino acid level. The yeast mutant dal4 dal5, deficient in the uptake of allantoin and allantoic acid, previously used to isolate AtUPS1 (13Desimone M. Catoni E. Ludewig U. Hilpert M. Schneider A. Kunze R. Tegeder M. Frommer W.B. Schumacher K. Plant Cell. 2002; 14: 847-856Crossref PubMed Scopus (84) Google Scholar), was used to demonstrate that AtUPS2 was able to suppress the mutant phenotype in a growth assay using 5 mm allantoin as the sole source of nitrogen (Fig. 1A). Colonies of cells expressing AtUPS2 or AtUPS1 were several times bigger as those formed by control cells transformed with the empty vector. AtUPS1 colonies grew slightly faster than AtUPS2 colonies. In addition, uptake of [14C]allantoin, due to AtUPS2 expression, was directly shown. The uptake rate was ∼75% of the rate observed for AtUPS1-expressing yeast, whereas the uptake rates of dal4 dal5 yeast mutant cells transformed with the empty vector was ∼3% of the rate observed upon AtUPS1 or AtUPS2 expression (Supplemental Fig. 1). Thus, both proteins mediate allantoin transport. AtUPS1 and AtUPS2 Also Transport Uracil—Because there is no evidence that Arabidopsis uses allantoin as a long distance transport form of nitrogen, it was conceivable that in Arabidopsis UPS has a different physiological substrate. Previous studies had shown that AtUPS1 recognizes oxo derivatives of N-heterocycles (13Desimone M. Catoni E. Ludewig U. Hilpert M. Schneider A. Kunze R. Tegeder M. Frommer W.B. Schumacher K. Plant Cell. 2002; 14: 847-856Crossref PubMed Scopus (84) Google Scholar). Because the structure of uracil fits the criteria established previously, and since uracil transport in plants has been described but transporters have not been identified, we tested whether AtUPS1 or AtUPS2 would be able to mediate uracil uptake. The yeast mutant fur4, deficient in uracil uptake, grew on 0.2 mm uracil as the sole source of pyrimidines when expressing AtUPS1 or AtUPS2, demonstrating that both transporters mediate uracil uptake (Fig. 1B). Functional Expression of AtUPS1 and AtUPS2 in Xenopus Oocytes—To test the substrate specificities of AtUPS1 and AtUPS2 more directly, both transporters were functionally expressed in X. laevis oocytes. Expression of AtUPS1 induced inward currents in oocytes bathed in buffer with allantoin in a flow system. When clamped at –100 mV, significant inward currents were also recorded from AtUPS2-expressing oocytes upon the addition of allantoin (Fig. 2A), whereas no significant currents were detected in non-injected control oocytes (Fig. 2, B and C). To define the substrate specificity in detail, a large number of compounds were tested. Criteria for the selection of potential substrates were (i) molecules belonging to the group of nucleobases and derivatives, (ii) compounds transported by other nucleobase transporters, and (iii) compounds with similar structural features. Currents were induced by cyclic purine degradation products and pyrimidines. In case of allantoin, hydantoin-, cytosine-, thymine- or uracil-induced currents remained stable or slowly decreased until washout of the substrates. However, when flushed with xanthine, the evoked current decreased within 10 s to a lower steady state level (Fig. 2A). A similar effect was found for uric acid (not shown). Despite lower current levels, AtUPS1 behaved in a manner similar to AtUPS2. Clamping oocytes to –100 mV for longer periods resulted in small shifts of the base line (Fig. 2A). To minimize errors conferred by this process, further investigations were done using brief substrate flushing periods and the I/V protocol described under “Experimental Procedures.” The allantoin-induced currents recorded from AtUPS1- or AtUPS2-expressing oocytes increased in response to further hyperpolarization of the membrane potential (Fig. 2C) and displayed clear voltage dependence. Steady state currents induced by different substances were plotted as a percentage of those caused by allantoin set to 100% (Fig. 3, A and B). The cyclic purine degradation products uric acid and xanthine induced currents in AtUPS1- and AtUPS2-expressing oocytes (∼60 and 50% for AtUPS1, 75 and 40% for AtUPS2, respectively). All pyrimidines and their tested derivatives (cytosine, uracil, thymine, dihydrouracil, and 5-FU) induced currents of different magnitude (Fig. 3A). Slight differences were found between AtUPS1 and AtUPS2 in response to thymine and cytosine (∼92 and 105% for thy" @default.
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• W2029812675 title "UPS1 and UPS2 from Arabidopsis Mediate High Affinity Transport of Uracil and 5-Fluorouracil" @default.
• W2029812675 cites W1517698775 @default.
• W2029812675 cites W1850569493 @default.
• W2029812675 cites W1979189272 @default.
• W2029812675 cites W1998467122 @default.
• W2029812675 cites W2001696162 @default.
• W2029812675 cites W2017181437 @default.
• W2029812675 cites W2031211089 @default.
• W2029812675 cites W2034206097 @default.
• W2029812675 cites W2042717435 @default.
• W2029812675 cites W2044729422 @default.
• W2029812675 cites W2047219099 @default.
• W2029812675 cites W2050526911 @default.
• W2029812675 cites W2056196476 @default.
• W2029812675 cites W2063226474 @default.
• W2029812675 cites W2069675427 @default.
• W2029812675 cites W2082498303 @default.
• W2029812675 cites W2084431957 @default.
• W2029812675 cites W2092686336 @default.
• W2029812675 cites W2098115340 @default.
• W2029812675 cites W2099791906 @default.
• W2029812675 cites W2111137841 @default.
• W2029812675 cites W2116122104 @default.
• W2029812675 cites W2118998865 @default.
• W2029812675 cites W2124558093 @default.
• W2029812675 cites W2125046636 @default.
• W2029812675 cites W2137479668 @default.
• W2029812675 cites W2146347493 @default.
• W2029812675 cites W2148260406 @default.
• W2029812675 cites W2149406354 @default.
• W2029812675 cites W232916448 @default.
• W2029812675 cites W4248646040 @default.
• W2029812675 cites W71705854 @default.
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