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,Dominique Van Der Straeten

doi:10.1074/jbc.m701158200

Abstract

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.

Highlights

Folates play a crucial role as cofactors in a number of onecarbon transfer reactions
No rescue was observed when the two constructs were introduced separately. These results indicate that the cytHPPK/dihydropteroate synthase (DHPS) gene is covering functional hydroxymethyldihydropterin pyrophosphokinase (HPPK) and DHPS domains
The existence of a cytosolic isoform of HPPK/DHPS raises the question as to whether it plays a particular role in planta, different from that of its mitochondrial counterpart, and whether the presence of the cytosolic enzyme is common in higher plants

Summary

The abbreviations used are

H4FGlun, tetrahydrofolate; mitHPPK/DHPS, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/7,8-dihydropteroate synthase; cytHPPK/DHPS, cytosolic HPPK/DHPS; p-ABA, p-aminobenzoic acid; p-ABAG, p-ABA-glutamate; H2FGlu, dihydrofolate; DHFS, dihydrofolate synthase; DHFR, dihydrofolate reductase; DHNA, dihydroneopterin aldolase; RACE, rapid amplification of cDNA ends; GFP, green fluorescent protein; 5-CHO-H4FGlu, 5-formyltetrahydrofolate; GUS, ␤-glucuronidase; RT, reverse transcription; T-DNA, transfer DNA. Chondria are a key compartment for folate synthesis in the yeast Saccharomyces cerevisiae, because the trifunctional enzyme supporting dihydroneopterin aldolase (DHNA), HPPK, and DHPS activities was recently shown to be associated with the mitochondrial membranes [12]. The first gene obviously coded for a protein-orthologue of the earlier characterized mitochondrial HPPK/DHPS from pea (mitHPPK/DHPS), the second gene apparently lacked the potential transit peptide, suggesting a cytosolic localization of the enzyme (cytHPPK/DHPS). Its role in H4FGlun biosynthesis as well as in stress response is discussed

EXPERIMENTAL PROCEDURES

RESULTS

Findings

DISCUSSION