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J. Biol. Chem., Vol. 279, Issue 27, 28182-28186, July 2, 2004

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Nitrate Reductase Activity Is Required for Nitrate Uptake into Fungal but Not Plant Cells^*

Shiela E. Unkles, Rongchen Wang, Ye Wang¶, Anthony D. M. Glass¶, Nigel M. Crawford, and James R. Kinghorn||

From the School of Biology, University of St. Andrews, St. Andrews KY16 9TH, United Kingdom, the Section of Cell and Developmental Biology, Division of Biological Sciences, University of California at San Diego, La Jolla, California 92093-0116, and the ^¶Department of Botany, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada

The ability to transport net nitrate was conferred upon transformant cells of the non-nitrate-assimilating yeast Pichia pastoris after the introduction of two genes, one encoding nitrate reductase and the other nitrate transport. It was observed that cells of this lower eukaryote transformed with the nitrate transporter gene alone failed to display net nitrate transport despite having the ability to produce the protein. In addition, loss-of-function nitrate reductase mutants isolated from several nitrate-assimilating fungi appeared to be unable to accumulate nitrate. Uptake assays using the tracer showed that nitrate influx is negligible in cells of a nitrate reductase null mutant. In parallel studies using a higher eukaryotic plant, Arabidopsis thaliana, loss-of-function nitrate reductase strains homozygous for both NIA1 insertion and NIA2 deletion were found to have no detectable nitrate reductase mRNA or nitrate reductase activity but retained the ability to transport nitrate. The reasons for these fundamental differences in nitrate transport into the cells of representative members of these two eukaryotic kingdoms are discussed.

Received for publication, April 9, 2004 , and in revised form, April 29, 2004.

Note Added in Proof—Details of the RIP-generated nitrate assimilation defective mutants in N. crassa have been published recently, Gao-Rubinelli, F., and Marzluf, G. A. (2004) Biochem. Genet. 42, 21–34.

^* This work was supported by the Biotechnology and Biology Research Council (United Kingdom) (to J. R. K.), the Natural Sciences and Engineering Research Council (Canada) (to A. D. M. G.) and the National Institutes of Health Grant GM40672 (to N. M. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed. Tel.: 44-1334-463344; Fax: 44-1334-463366; E-mail: jrk{at}st-andrews.ac.uk.

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