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1.
Plant J 2007 Mar ; 5(49):855-64.
The fluorouridine insensitive 1 (fur1) mutant is defective in equilibrative nucleoside transporter 3 (ENT3), and thus represents an important pyrimidine nucleoside uptake system in Arabidopsis thaliana.
Traub M ,Flörchinger M ,Piecuch J ,Kunz HH ,Weise-Steinmetz A ,Deitmer JW ,Ekkehard Neuhaus H ,Möhlmann T ,
Abteilung Pflanzenphysiologie, Fachbereich Biologie, Technische Universitat Kaiserslautern, PO Box 3049, D-67653 Kaiserslautern, Germany.
The fluorouridine insensitive 1 (fur1) locus in Arabidopsis thaliana (L.) Heynh. has previously been identified in a screen for growth resistance towards the toxic compound fluorouridine. Mutation of this locus by ethylmethane sulfonate (EMS) allows mutants to grow on this uridine analogue. We identified that the A. thaliana equilibrative nucleoside transporter (AtENT3) was encoded by the fur1 locus. T-DNA insertional mutant plants for AtENT3 resemble the fur1 mutant phenotype: i.e. they grow on fluorouridine, and seedlings as well as leaf discs exhibit a markedly reduced uptake capacity for uridine and cytidine, but a less pronounced reduced uptake for adenosine and guanosine. These results indicate that AtENT3 is an important pyrimidine nucleoside transporter in Arabidopsis. In addition, we identified the mutation in fur1 as a single base-pair exchange, guanine --> adenine, leading to an amino acid exchange G --> R at position 281. Furthermore, we showed that this mutation is indeed responsible for the observed alterations in nucleoside transport in the fur1-1 line, because the introduction of this mutation in AtENT3 promoted fluorouridine resistance in yeast cells expressing this mutated protein. The biochemical characterization of AtENT3 expressed in Xenopus oocytes identified a proton-coupled concentrative mode of nucleoside transport, although this carrier possesses structural features characteristic for equilibrative nucleoside carriers.
Publication Type: Research Support, Non-U.S. Gov't;
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2.
Cell Res 2006 Apr ; 4(16):377-88.
Identification of AtENT3 as the main transporter for uridine uptake in Arabidopsis roots.
Chen KL ,Xu MX ,Li GY ,Liang H ,Xia ZL ,Liu X ,Zhang JS ,Zhang AM ,Wang DW ,
School of Life Sciences, Northwest Agricultural and Forestry University, Yangling 712100, China.
Previous studies have shown that Arabidopsis equilibrative nucleoside transporters (AtENTs) possess transport activities when produced in yeast cells and are differentially expressed in Arabidopsis organs. Herein, we report further analysis on the nucleoside transport activities and transcriptional patterns of AtENT members. The recombinant proteins of AtENTs 3, 6, and 7, but not those of AtENTs 1, 2, 4, and 8, were found to transport thymidine with high affinity. Contrary to previous suggestion that AtENT1 may not transport uridine, this work showed that recombinant AtENT1 was a pH-dependent and high-affinity transporter of uridine. When grown on MS plates, the AtENT3 knockout plants were more tolerant to the cytotoxic uridine analog 5-fluorouridine than wild-type plants and the knockout plants of AtENT1 or AtENT6. Consistent with this observation, the AtENT3 knockout line exhibited a significantly decreased ability to take up [(3)H]uridine via the roots when compared with wild-type plants and the plants with mutated AtENT1 or AtENT6. This indicates that AtENT3, but not AtENTs 1 and 6, is the main transporter for uridine uptake in Arabidopsis roots. The transcription of AtENTs 1, 3, 4, 6, 7, and 8 was regulated in a complex manner during leaf development and senescence. In contrast, the six AtENT members were coordinately induced during seed germination. This work provides new information on the transport properties of recombinant AtENT proteins and new clues for future studies of the in vivo transport activities and physiological functions of the different ENT proteins in Arabidopsis plants.
Publication Type: Comparative Study; Research Support, Non-U.S. Gov't;
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