Plant Physiol. Drug Metab Dispos
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Published on April 22, 2005; 10.1104/pp.105.060137

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Received January 28, 2005
Returned for revision February 28, 2005
Accepted March 4, 2005

Functional Characterization and Expression Analysis of a Gene, OsENT2, Encoding an Equilibrative Nucleoside Transporter in Rice Suggest a Function in Cytokinin Transport

Naoya Hirose , Nobue Makita , Tomoyuki Yamaya , and Hitoshi Sakakibara *

RIKEN (The Institute of Physical and Chemical Research) Plant Science Center, Tsurumi-ku, Yokohama 230-0045, Japan

* Corresponding author; email: sakaki{at}postman.riken.go.jp.

We identified four genes for potential equilibrative nucleoside transporters (ENTs) from rice (Oryza sativa; designated OsENT1 through OsENT4). Growth analysis of budding yeast (Saccharomyces cerevisiae) cells expressing OsENTs showed that OsENT2 transported adenosine and uridine with high affinity (adenosine, Km = 3.0 µM; uridine, Km = 0.7 µM). Purine or pyrimidine nucleosides and 2'-deoxynucleosides strongly inhibited adenosine transport via OsENT2, suggesting that OsENT2 possesses broad substrate specificity. OsENT2-mediated adenosine transport was resistant to the typical inhibitors of mammalian ENTs, nitrobenzylmercaptopurine ribonucleoside, dilazep, and dipyridamole. The transport activity was maximal at pH 5.0 and decreased slightly at lower as well as higher pH. In competition experiments with various cytokinins, adenosine transport by OsENT2 was inhibited by isopentenyladenine riboside (iPR). Direct measurements with radiolabeled cytokinins demonstrated that OsENT2 mediated uptake of iPR (Km = 32 µM) and trans-zeatin riboside (Km = 660 µM), suggesting that OsENT2 participates in iPR transport in planta. In mature plants, OsENT2 was predominantly expressed in roots. The OsENT2 promoter drove the expression of the {beta}-glucuronidase reporter gene in the scutellum during germination and in vascular tissues in germinated plants, suggesting a participation of OsENT2 in the retrieval of endosperm-derived nucleosides by the germinating embryo and in the long-distance transport of nucleosides in growing plants, respectively.




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