Arabidopsis thaliana VTC4 encodes L-galactose-1-P phosphatase, a plant ascorbic acid biosynthetic enzyme

J Biol Chem. 2006 Jun 9;281(23):15662-70. doi: 10.1074/jbc.M601409200. Epub 2006 Apr 4.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Arabidopsis / enzymology
  • Arabidopsis / genetics*
  • Ascorbic Acid / biosynthesis*
  • Base Sequence
  • DNA Primers
  • Galactose / metabolism
  • Genes, Plant*
  • Humans
  • Mannose / metabolism
  • Molecular Sequence Data
  • Mutation
  • Phosphoric Monoester Hydrolases / chemistry
  • Phosphoric Monoester Hydrolases / genetics*
  • Phosphoric Monoester Hydrolases / metabolism
  • Phylogeny
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sequence Homology, Amino Acid

Substances

  • DNA Primers
  • VTC4 protein, Arabidopsis
  • Phosphoric Monoester Hydrolases
  • Mannose
  • Ascorbic Acid
  • Galactose