Antisense suppression of <scp>l</scp>‐galactose dehydrogenase in <i>Arabidopsis thaliana</i> provides evidence for its role in ascorbate synthesis and reveals light modulated <scp>l</scp>‐galactose synthesis

Gatzek, Stephan; Wheeler, Glen; Smirnoff, Nicholas

doi:10.1046/j.1365-313x.2002.01315.x

Cited by 229 publications

(240 citation statements)

References 62 publications

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“…Additional evidence for feedback control of the pathway comes from analysis of A. thaliana plants with antisense suppression of L-Gal dehydrogenase. Not only does L-Gal accumulate as predicted (18), but gas chromatography-mass spectrometry analysis shows that Man (including Man-1-P and GDP-Man) also accumulates, 6 suggesting that accumulation of L-Gal feeds back to the GDP-Man-3,5-epimerase step (Fig. 1).…”

Section: Discussionmentioning

confidence: 80%

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

Conklin

Gatzek

Wheeler

et al. 2006

Journal of Biological Chemistry

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168

119

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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 ϳ50% 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.

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Section: Discussionmentioning

confidence: 80%

“…1) (21). Reduction of L-Gal dehydrogenase and L-galactono-1,4-lactone dehydrogenase activities in planta (via antisense suppression) leads to reduced levels of ascorbate (18,22). Recently, a gene encoding an enzyme with L-Gal-1-P phosphatase activity was identified in kiwi fruit and A. thaliana (23).…”

mentioning

confidence: 99%

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

Conklin

Gatzek

Wheeler

et al. 2006

Journal of Biological Chemistry

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168

119

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“…The Fe 31 to Fe 21 assay does not cross-react with free thiol groups (e.g. glutathione; data not shown) and has been successfully employed to detect ascorbate in plant material (Gatzek et al, 2002). The sum of ascorbate and dehydroascorbate was determined after reduction of dehydroascorbate with dithiothreitol and the redox state calculated as Asc/(Asc 1 DHA) (Kampfenkel et al, 1995).…”

Section: Quantification Of Tocopherol Ascorbate and Glutathionementioning

confidence: 99%

Alterations in Tocopherol Cyclase Activity in Transgenic and Mutant Plants of Arabidopsis Affect Tocopherol Content, Tocopherol Composition, and Oxidative Stress

et al. 2005

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Tocopherol belongs to the Vitamin E class of lipid soluble antioxidants that are essential for human nutrition. In plants, tocopherol is synthesized in plastids where it protects membranes from oxidative degradation by reactive oxygen species. Tocopherol cyclase (VTE1) catalyzes the penultimate step of tocopherol synthesis, and an Arabidopsis (Arabidopsis thaliana) mutant deficient in VTE1 (vte1) is totally devoid of tocopherol. Overexpression of VTE1 resulted in an increase in total tocopherol of at least 7-fold in leaves, and a dramatic shift from a-tocopherol to g-tocopherol. Expression studies demonstrated that indeed VTE1 is a major limiting factor of tocopherol synthesis in leaves. Tocopherol deficiency in vte1 resulted in the increase in ascorbate and glutathione, whereas accumulation of tocopherol in VTE1 overexpressing plants led to a decrease in ascorbate and glutathione. Deficiency in one antioxidant in vte1, vtc1 (ascorbate deficient), or cad2 (glutathione deficient) led to increased oxidative stress and to the concomitant increase in alternative antioxidants. Double mutants of vte1 were generated with vtc1 and cad2. Whereas growth, chlorophyll content, and photosynthetic quantum yield were very similar to wild type in vte1, vtc1, cad2, or vte1vtc1, they were reduced in vte1cad2, indicating that the simultaneous loss of tocopherol and glutathione results in moderate oxidative stress that affects the stability and the efficiency of the photosynthetic apparatus.Vitamin E encompasses a class of lipid antioxidants consisting of four forms each of tocopherol and tocotrienol. Because of its high economical value and importance for human nutrition, much effort has been invested to elucidate the tocopherol biosynthetic pathway in plants and Cyanobacteria and to identify limiting steps by overexpression of candidate genes in transgenic plants. The hydroquinone ring of tocopherol is derived from the shikimate pathway of aromatic amino acid synthesis. Homogentisate, the precursor for the synthesis of tocopherol, tocotrienol, and plastoquinone, is synthesized by p-hydroxyphenylpyruvate dioxygenase (HPPD; Fig. 1; Norris et al., 1998) (Collakova et al., 2003a). Final methylation by g-tocopherol methyltransferase (g-TMT, VTE4) results in the production of a-tocopherol. a-Tocopherol is the predominant form in leaves, whereas g-tocopherol is most abundant in seeds of Arabidopsis (Shintani and DellaPenna, 1998). Side reactions of VTE1, VTE3, and VTE4 result in the production of minor tocopherol forms in Arabidopsis seeds or leaves (d-tocopherol, b-tocopherol; Fig. 1).Under oxidative stress, the amounts of different antioxidants strongly increase in plants, and it is believed that this results in an increased capacity to scavenge reactive oxygen species (Noctor and Foyer, 1998;Collakova et al., 2003b). The accumulation of antioxidants under stress is at least in part mediated by induction of gene expression. For example, HPPD and HPT1, two steps of tocopherol synthesis, were shown to be induced under light stress (Coll...

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“…It is formally possible that the vtc2 is also deficient in GDP-Man, as the AsA deficiency in this mutant is the result of a mutation in a gene of unknown function. Further confirmation of the above phenotypes by experimentation with additional AsA-deficient lines with defects downstream of GDP-Man synthesis (such as the antisense L-Gal dehydrogenase lines generated by N. Smirnoff;Gatzek et al, 2002) will be necessary. Additionally, possible reversion of the premature senescence and pathogen resistance of vtc1 and vtc2 by artificial elevation of AsA requires further investigations.…”

Section: Ascorbate Deficiency In Vtc1 Is Associated With Promotion Ofmentioning

confidence: 99%

The Timing of Senescence and Response to Pathogens Is Altered in the Ascorbate-Deficient Arabidopsis Mutant vitamin c-1

et al. 2004

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The ozone-sensitive Arabidopsis mutant vitamin c-1 (vtc1) is deficient in l-ascorbic acid (AsA) due to a mutation in GDP-Man pyrophosphorylase (Conklin et al., 1999), an enzyme involved in the AsA biosynthetic pathway (Smirnoff et al., 2001). In this study, the physiology of this AsA deficiency was initially investigated in response to biotic (virulent pathogens) stress and subsequently with regards to the onset of senescence. Infection with either virulent Pseudomonas syringae or Peronospora parasitica resulted in largely reduced bacterial and hyphal growth in the vtc1 mutant in comparison to the wild type. When vitamin c-2 (vtc2), another AsA-deficient mutant, was challenged with P. parasitica, growth of the fungus was also reduced, indicating that the two AsA-deficient mutants are more resistant to these pathogens. Induction of pathogenesis-related proteins PR-1 and PR-5 is significantly higher in vtc1 than in the wild type when challenged with virulent P. syringae. In addition, the vtc1 mutant exhibits elevated levels of some senescence-associated gene (SAG) transcripts as well as heightened salicylic acid levels. Presumably, therefore, low AsA is causing vtc1 to enter at least some stage(s) of senescence prematurely with an accompanying increase in salicylic acid levels that results in a faster induction of defense responses.

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Antisense suppression of l‐galactose dehydrogenase in Arabidopsis thaliana provides evidence for its role in ascorbate synthesis and reveals light modulated l‐galactose synthesis

Cited by 229 publications

References 62 publications

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

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

Alterations in Tocopherol Cyclase Activity in Transgenic and Mutant Plants of Arabidopsis Affect Tocopherol Content, Tocopherol Composition, and Oxidative Stress

The Timing of Senescence and Response to Pathogens Is Altered in the Ascorbate-Deficient Arabidopsis Mutant vitamin c-1

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