John Dowdle

Shimane University, Matsu, Shimane, Japan

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Publications (7)29.82 Total impact

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    ABSTRACT: Plants synthesize ascorbate from guanosine diphosphate (GDP)-mannose via L-galactose/L-gulose, although uronic acids have also been proposed as precursors. Genes encoding all the enzymes of the GDP-mannose pathway have previously been identified, with the exception of the step that converts GDP-L-galactose to L-galactose 1-P. We show that a GDP-L-galactose phosphorylase, encoded by the Arabidopsis thaliana VTC2 gene, catalyses this step in the ascorbate biosynthetic pathway. Furthermore, a homologue of VTC2, At5g55120, encodes a second GDP-L-galactose phosphorylase with similar properties to VTC2. Two At5g55120 T-DNA insertion mutants (vtc5-1 and vtc5-2) have 80% of the wild-type ascorbate level. Double mutants were produced by crossing the loss-of-function vtc2-1 mutant with each of the two vtc5 alleles. These show growth arrest immediately upon germination and the cotyledons subsequently bleach. Normal growth was restored by supplementation with ascorbate or L-galactose, indicating that both enzymes are necessary for ascorbate generation. vtc2-1 leaves contain more mannose 6-P than wild-type. We conclude that the GDP-mannose pathway is the only significant source of ascorbate in A. thaliana seedlings, and that ascorbate is essential for seedling growth. A. thaliana leaves accumulate more ascorbate after acclimatization to high light intensity. VTC2 expression and GDP-L-galactose phosphorylase activity rapidly increase on transfer to high light, but the activity of other enzymes in the GDP-mannose pathway is little affected. VTC2 and At5g55120 (VTC5) expression also peak in at the beginning of the light cycle and are controlled by the circadian clock. The GDP-L-galactose phosphorylase step may therefore play an important role in controlling ascorbate biosynthesis.
    Preview · Article · Dec 2007 · The Plant Journal
  • N. Smirnoff · J. Dowdle · T. Ishikawa

    No preview · Article · Apr 2007 · Comparative Biochemistry and Physiology - Part A Molecular & Integrative Physiology
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    ABSTRACT: The mechanisms of metal hyperaccumulation are still not understood, so we conducted a quantitative trait locus (QTL) analysis of zinc (Zn) hyperaccumulation in Arabidopsis halleri, in a cross between this and its sister species, A. petraea, in order to determine the number and approximate location of the genomic regions significantly contributing to this adaptation. An F2 cross between the two species was made, and the leaf Zn concentration of 92 individuals was measured at both low (10 microm) and high (100 microm) Zn concentrations. Twenty-five markers were established that were distributed on all of the eight chromosomes. Mapping of the markers established that they were essentially collinear with previous studies. QTLs exceeding a logarithm to the base 10 of the odds (LOD) value of 3 were found on chromosomes 4 (low Zn), 6 (high Zn) and 7 (both high and low Zn). Evidence for a QTL on chromosome 3 (low Zn) was also found. This analysis validates a previously used method of QTL analysis, based on microarray analysis of segregating families. Genes that have altered during the evolution of this character should also be QTL: this analysis calls into question a number of candidate genes from consideration as such primary genes because they do not appear to be associated with QTLs.
    Full-text · Article · Feb 2007 · New Phytologist
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    ABSTRACT: One of the challenges of comparative genomics is to identify specific genetic changes associated with the evolution of a novel adaptation or trait. We need to be able to disassociate the genes involved with a particular character from all the other genetic changes that take place as lineages diverge. Here we show that by comparing the transcriptional profile of segregating families with that of parent species differing in a novel trait, it is possible to narrow down substantially the list of potential target genes. In addition, by assuming synteny with a related model organism for which the complete genome sequence is available, it is possible to use the cosegregation of markers differing in transcription level to identify regions of the genome which probably contain quantitative trait loci (QTLs) for the character. This novel combination of genomics and classical genetics provides a very powerful tool to identify candidate genes. We use this methodology to investigate zinc hyperaccumulation in Arabidopsis halleri, the sister species to the model plant, Arabidopsis thaliana. We compare the transcriptional profile of A. halleri with that of its sister nonaccumulator species, Arabidopsis petraea, and between accumulator and nonaccumulator F(3)s derived from the cross between the two species. We identify eight genes which consistently show greater expression in accumulator phenotypes in both roots and shoots, including two metal transporter genes (NRAMP3 and ZIP6), and cytoplasmic aconitase, a gene involved in iron homeostasis in mammals. We also show that there appear to be two QTLs for zinc accumulation, on chromosomes 3 and 7.
    No preview · Article · Oct 2006 · Molecular Ecology
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    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.
    Full-text · Article · Jul 2006 · Journal of Biological Chemistry
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    ABSTRACT: l-Ascorbic acid (vitamin C) is synthesized from hexose sugars. It is an antioxidant and redox buffer, as well as an enzyme cofactor, so it has multiple roles in metabolism and in plant responses to abiotic stresses and pathogens. Plant-derived ascorbate also provides the major source of vitamin C in the human diet. An understanding of how ascorbate metabolism is controlled should provide a basis for engineering or otherwise manipulating its accumulation. Biochemical and molecular genetic evidence supports synthesis from GDP-d-mannose via l-galactose (d-Man/l-Gal pathway) as a significant source of ascorbate. More recently, evidence for pathways via uronic acids has been obtained: overexpression of myo-inositol oxygenase, d-galacturonate reductase and l-gulono-1,4-lactone oxidase all increase leaf ascorbate concentration. Interestingly, this has proved more effective in pathway engineering than overexpressing various d-Man/l-Gal pathway genes. Ascorbate oxidation generates the potentially unstable dehydroascorbate, and the overexpression of glutathione-dependent dehydroascorbate reductase has resulted in increased ascorbate. Ascorbate is catabolized to products such as oxalate, l-threonate and l-tartrate. The enzymes involved have not been identified, so catabolism is not yet amenable to manipulation. In the examples of pathway engineering so far, the increase in ascorbate has been modest on an absolute or proportional basis. Therefore, a deeper understanding of ascorbate metabolism is needed to achieve larger increases. Identifying genes that control ascorbate accumulation by techniques such as analysis of quantitative trait loci (QTL) or activation tagging may hold promise, particularly if regulatory genes can be identified.
    Preview · Article · Feb 2006 · Physiologia Plantarum
  • T. Ishikawa · M. Tanimoto · J. Dowdle · N. Smirnoff

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