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J. Biol. Chem., Vol. 282, Issue 14, 10749-10761, April 6, 2007

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Cytosolic Hydroxymethyldihydropterin Pyrophosphokinase/Dihydropteroate Synthase from Arabidopsis thaliana

A SPECIFIC ROLE IN EARLY DEVELOPMENT AND STRESS RESPONSE^*

Sergei Storozhenko, Oscar Navarrete, Stéphane Ravanel, Veerle De Brouwer^¶, Peter Chaerle, Guo-Fang Zhang^¶, Olivier Bastien, Willy Lambert^¶, Fabrice Rébeillé, and Dominique Van Der Straeten¹

From the Unit of Plant Hormone Signaling and Bio-imaging, Department of Molecular Genetics, Ghent University, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium, the Laboratoire de Physiologie Cellulaire Végétale, Département de Réponse et Dynamique Cellulaire, CEA-Grenoble, F-38054 Grenoble Cédex 9, France, and the ^¶Laboratory of Toxicology, Ghent University, Harelbekestraat 72, B-9000 Gent, Belgium

In plants, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/7,8-dihydropteroate synthase (mitHPPK/DHPS) is a bifunctional mitochondrial enzyme, which catalyzes the first two consecutive steps of tetrahydrofolate biosynthesis. Mining the Arabidopsis genome data base has revealed a second gene encoding a protein that lacks a potential transit peptide, suggesting a cytosolic localization of the isoenzyme (cytHPPK/DHPS). When the N-terminal part of the cytHPPK/DHPS was fused to green fluorescent protein, the fusion protein appeared only in the cytosol, confirming the above prediction. Functionality of cytHPPK/DHPS was addressed by two parallel approaches: first, the cytHPPK/DHPS was able to rescue yeast mutants lacking corresponding activities; second, recombinant cytHPPK/DHPS expressed and purified from Escherichia coli displayed both HPPK and DHPS activities in vitro. In contrast to mitHPPK/DHPS, which was ubiquitously expressed, the cytHPPK/DHPS gene was exclusively expressed in reproductive tissue, more precisely in developing seeds as revealed by histochemical analysis of a transgenic cytHPPK/DHPS promoter-GUS line. In addition, it was observed that expression of cytHPPK/DHPS mRNA was induced by salt stress, suggesting a potential role of the enzyme in stress response. This was supported by the phenotype of a T-DNA insertion mutant in the cytHPPK/DHPS gene, resulting in lower germination rates as compared with the wild-type upon application of oxidative and osmotic stress.

Received for publication, February 7, 2007

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AJ866732 [GenBank] .

^* This work was supported by Ghent University (Bijzonder Onderzoeksfonds GOA 1251204). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence should be addressed: Tel.: 32-9-264-5185; Fax: 32-9-264-5333; E-mail: dominique.vanderstraeten{at}ugent.be.

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