title: Arabidopsis thaliana VTC4 encodes L-galactose-1-P phosphatase, a plant ascorbic acid biosynthetic enzyme.
abstract: In plants, a proposed ascorbate (vitamin C) biosynthesis pathway occurs via GDP-D-mannose (GDP-D-Man), GDP-L-galactose (GDP-L-Gal), and L-galactose. However, the steps involved in the synthesis of L-Gal from GDP-L-Gal in planta are not fully characterized. Here we present evidence for an in vivo role for L-Gal-1-P phosphatase in plant ascorbate biosynthesis. We have characterized a low ascorbate mutant (vtc4-1) of Arabidopsis thaliana, which exhibits decreased ascorbate biosynthesis. Genetic mapping and sequencing of the VTC4 locus identified a mutation (P92L) in a gene with predicted L-Gal-1-P phosphatase activity (At3g02870). Pro-92 is within a beta-bulge that is conserved in related myo-inositol monophosphatases. The mutation is predicted to disrupt the positioning of catalytic amino acid residues within the active site. Accordingly, L-Gal-1-P phosphatase activity in vtc4-1 was approximately 50% of wild-type plants. In addition, vtc4-1 plants incorporate significantly more radiolabel from [2-(3)H]Man into L-galactosyl residues suggesting that the mutation increases the availability of GDP-L-Gal for polysaccharide synthesis. Finally, a homozygous T-DNA insertion line, which lacks a functional At3g02870 gene product, is also ascorbate-deficient (50% of wild type) and deficient in L-Gal-1-P phosphatase activity. Genetic complementation tests revealed that the insertion mutant and VTC4-1 are alleles of the same genetic locus. The significantly lower ascorbate and perturbed L-Gal metabolism in vtc4-1 and the T-DNA insertion mutant indicate that L-Gal-1-P phosphatase plays a role in plant ascorbate biosynthesis. The presence of ascorbate in the T-DNA insertion mutant suggests there is a bypass to this enzyme or that other pathways also contribute to ascorbate biosynthesis.
authors.affiliation: Department of Biological Sciences, State University of New York, Cortland, New York 13045, USA.
authors.forename: Patricia L
authors.initials: PL
authors.lastname: Conklin
authors.forename: Stephan
authors.initials: S
authors.lastname: Gatzek
authors.forename: Glen L
authors.initials: GL
authors.lastname: Wheeler
authors.forename: John
authors.initials: J
authors.lastname: Dowdle
authors.forename: Marjorie J
authors.initials: MJ
authors.lastname: Raymond
authors.forename: Susanne
authors.initials: S
authors.lastname: Rolinski
authors.forename: Mikhail
authors.initials: M
authors.lastname: Isupov
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Littlechild
authors.forename: Nicholas
authors.initials: N
authors.lastname: Smirnoff
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics*; D001205:Ascorbic Acid / Q000096:biosynthesis*; D001483:Base Sequence; D017931:DNA Primers; D005690:Galactose / Q000378:metabolism; D017343:Genes, Plant; D006801:Humans; D008358:Mannose / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
chemical_list: D017931:DNA Primers; C512578:VTC4 protein, Arabidopsis; D010744:Phosphoric Monoester Hydrolases; D008358:Mannose; D001205:Ascorbic Acid; D005690:Galactose