Identification of AtENT3 as the main transporter for uridine uptake in Arabidopsis roots

Abstract

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.

Nucleobase and nucleoside transport and integration into plant metabolism

“…FUR1 mutants and AtENT3 T-DNA mutants are completely resistant against the toxic pyrimidine nucleoside analog 5-fluorouridine (5-FU). This lead to the identification of AtENT3 as the major pyrimidine importer in Arabidopsis seedlings (Chen et al, 2006; Traub et al, 2007). Since NSH3—an extracellular purine specific nucleoside hydrolase—cleaves inosine and adenosine purines can still be imported in form of nucleobases in AtENT3 mutants (Jung et al, 2011).…”

“…Gene expression of all six AtENTs could be observed during seed germination and development. Whereas the transcript level of AtENT1 remained relatively constant, the transcript of AtENT3, 4, 6, 7, and 8 steadily increased while germination proceeded (Chen et al, 2006). Expression analysis with 10 day old seedlings indicated relatively high transcript levels for AtENT1 and AtENT3 whereas the transcript of all other AtENTs was markedly lower (Cornelius et al, 2012).…”

Equilibrative nucleoside transporter 1 (ENT1) is critical for pollen germination and vegetative growth in Arabidopsis

“…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. 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; Li et al , 2003; Wormit et al , 2004; Chen et al , 2006). 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.…”

AtAzg1 and AtAzg2 comprise a novel family of purine transporters in Arabidopsis

“…None of the 12 identified Arabidopsis NAT loci are yet functionally characterized. Equilibrative nucleoside transporters (ENT) found in plants, protozoans and mammals, act primarily as nucleoside transporters but can also transport nucleobases adenine, guanine, xanthine and hypoxanthine or cytokinin [7][8][9][10][11][12][13]. Eight identified ENT loci are present in the Arabidopsis genome.…”