Cytosolic HPPK/DHPS from Arabidopsis thaliana: A specific role in stress response.
Résumé
In plants, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/7,8- dihydropteroate synthase (mitHPPK/DHPS) is a bifunctional mitochondrial enzyme, which catalyses the first two consecutive steps of tetrahydrofolate biosynthesis. Mining the Arabidopsis genome database 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 GFP, 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 E. 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 to the wild type upon application of oxidative and osmotic stress.
Mots clés
plant
Arabidopsis thaliana
vitamin B9
folate
tetrahydrofolate biosynthesis
enzyme
bifunctional enzyme
6-hydroxymethyl-7
8-dihydropterin pyrophosphokinase
dihydropteroate synthase
subcellular localization
cytosol
gene expression
stress
salt stress
oxidative stress
osmotic stress
seed development
seed germination
Domaines
Biochimie, Biologie Moléculaire
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2007_Storozhenko_Journal of Biological Chemistry_1.pdf (905.8 Ko)
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