title: Impact of oxidative stress on ascorbate biosynthesis in Chlamydomonas via regulation of the VTC2 gene encoding a GDP-L-galactose phosphorylase.
abstract: The L-galactose (Smirnoff-Wheeler) pathway represents the major route to L-ascorbic acid (vitamin C) biosynthesis in higher plants. Arabidopsis thaliana VTC2 and its paralogue VTC5 function as GDP-L-galactose phosphorylases converting GDP-L-galactose to L-galactose-1-P, thus catalyzing the first committed step in the biosynthesis of L-ascorbate. Here we report that the L-galactose pathway of ascorbate biosynthesis described in higher plants is conserved in green algae. The Chlamydomonas reinhardtii genome encodes all the enzymes required for vitamin C biosynthesis via the L-galactose pathway. We have characterized recombinant C. reinhardtii VTC2 as an active GDP-L-galactose phosphorylase. C. reinhardtii cells exposed to oxidative stress show increased VTC2 mRNA and L-ascorbate levels. Genes encoding enzymatic components of the ascorbate-glutathione system (e.g. ascorbate peroxidase, manganese superoxide dismutase, and dehydroascorbate reductase) are also up-regulated in response to increased oxidative stress. These results indicate that C. reinhardtii VTC2, like its plant homologs, is a highly regulated enzyme in ascorbate biosynthesis in green algae and that, together with the ascorbate recycling system, the L-galactose pathway represents the major route for providing protective levels of ascorbate in oxidatively stressed algal cells.
authors.affiliation: Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095, USA.
authors.forename: Eugen I
authors.initials: EI
authors.lastname: Urzica
authors.forename: Lital N
authors.initials: LN
authors.lastname: Adler
authors.forename: M Dudley
authors.initials: MD
authors.lastname: Page
authors.forename: Carole L
authors.initials: CL
authors.lastname: Linster
authors.forename: Mark A
authors.initials: MA
authors.lastname: Arbing
authors.forename: David
authors.initials: D
authors.lastname: Casero
authors.forename: Matteo
authors.initials: M
authors.lastname: Pellegrini
authors.forename: Sabeeha S
authors.initials: SS
authors.lastname: Merchant
authors.forename: Steven G
authors.initials: SG
authors.lastname: Clarke
mesh_terms: D000975:Antioxidants / Q000378:metabolism; D017360:Arabidopsis / Q000201:enzymology; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism*; D001205:Ascorbic Acid / Q000378:metabolism; D016825:Chlamydomonas reinhardtii / Q000378:metabolism*; D002736:Chloroplasts / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005786:Gene Expression Regulation; D008954:Models, Biological; D008969:Molecular Sequence Data; D018384:Oxidative Stress; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics* / Q000378:metabolism*; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D000975:Antioxidants; D029681:Arabidopsis Proteins; D011994:Recombinant Proteins; C522629:GDP-L-galactose phosphorylase, Arabidopsis; D010744:Phosphoric Monoester Hydrolases; D001205:Ascorbic Acid