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J. Biol. Chem., Vol. 281, Issue 23, 15662-15670, June 9, 2006

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Arabidopsis thaliana VTC4 Encodes L-Galactose-1-P Phosphatase, a Plant Ascorbic Acid Biosynthetic Enzyme^*

Patricia L. Conklin¹, Stephan Gatzek², Glen L. Wheeler³, John Dowdle, Marjorie J. Raymond, Susanne Rolinski, Mikhail Isupov, Jennifer A. Littlechild, and Nicholas Smirnoff⁴

From the Department of Biological Sciences, State University of New York, Cortland, New York 13045 and School of Biosciences, University of Exeter, Exeter EX4 4QD, United Kingdom

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-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 50% of wild-type plants. In addition, vtc4-1 plants incorporate significantly more radiolabel from [2-³H]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.

Received for publication, February 14, 2006 , and in revised form, March 28, 2006.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by the National Research Initiative of the United States Department of Agriculture Cooperative State Research, Education, and Extension Service Grant 1998-35100-12987 and a grant from the Dr. Nula McGann Drescher Affirmative Action/Diversity Leave Program.

² Present address: Novartis Pharma AG, Biomarker Development, WKL-136.1.15, CH-4002 Basel, Switzerland.

³ Present address: Plymouth Marine Laboratory, Prospect Place, The Hoe, Plymouth PL1 3DH, UK.

⁴ Supported by the Biotechnology and Biological Sciences Research Council (UK) and Bio-Technical Resources (Manitowoc, WI). To whom correspondence should be addressed: School of Biosciences, Geoffrey Pope Bldg., University of Exeter, Stocker Road, Exeter EX4 4QD, UK. Tel.: 44-1392-263756; Fax: 44-1392-263700; E-mail: N.Smirnoff{at}exeter.ac.uk.

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