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Vitamin C biosynthesis and vitamin C-deficient mutants of Arabidopsis

Plants synthesize vitamin C (L-ascorbic acid) via several pathways. The major AA biosynthetic pathway uses the precursor glucose-6-phosphate and includes the intermediates GDP-mannose and L-galactose.


Evidence for this pathway is provided by biochemical and genetic studies (Wheeler et al. 1998, Conklin et al. 2000, Wolucka et al. 2001, Gatzek et. 2002, Jander et al. 2002, Laing et al. 2004, Conklin et al. 2006). The isolation of Arabidopsis vitamin C-deficient vtc mutants (Conklin et al. 1999, 2000) facilitated the identification of the VTC1 gene, encoding a GDP-mannose pyrophosphorylase (Conklin et al. 1999), and the VTC4 gene, which encodes an L-galactose-1-phosphate phosphatase (Laing et al. 2004, Conklin et al. 2006). In addition to vtc1 and vtc4, two other vtc mutants, vtc2 and vtc3, have been isolated. The VTC2 gene encodes a GDP-L-galactose phosphorylase (Linster et al. 2007). The VTC3 gene has not yet been identified. Other enzymes in the major AA biosynthetic pathway include: 1, phosphoglucose isomerase; 2, phosphomannose isomerase; 3, phosphomannose-mutase; 5, GDP-mannose 3,5-epimerase; 8, L-galactose-1-dehydrogenase; 9, L-galactono-1,4-lactone dehydrogenase.

The vtc mutants we are using in our studies were generated by ethyl methane sulfonate mutagenesis (which causes point mutations). They have been isolated by their virtue of sensitivity to ozone, an air pollutant and reactive oxygen species generator. All mutants are characterized by a defect in vitamin C biosynthesis (see above), thus resulting in low endogenous levels of this antioxidant (ranging between 30% and 50% of the wild-type leaf vitamin C content; Conklin 2001).

Research on the role of vitamin C in regulating plant pathogen defense responses

Another interesting discovery about the vtc mutants is the fact that they are more tolerant to infection by the virulent (i.e. disease-causing) pathogens Pseudomonas syringae pv. maculicola (and P. syringae pv. tomato), the cause of leaf spot disease, and the fungus Peronospora parasitica pv. Noco, the cause of downy mildew (Barth et al. 2004, Pavet et al. 2005).

 
Conidiophore production in wild type and vtc1 mutants inoculated with P. parasitica pv. Noco. A, Pronounced conidiophore production (arrow) and massive hyphae spread (arrowhead) in the wild type. B, In vtc1, conidiophore production and hyphal development is much lower than in the wild type after infection with Noco. Scale bar = 100 µm
  
The vtc1 mutant contains elevated levels of the plant hormone salicylic acid and pathogenesis-related proteins, suggesting an up-regulation of salicylic acid-dependent defense pathways. The up-regulation of defense response genes occurs independently of the growth photoperiod (Barth et al. 2004, Pavet et al. 2005). Our lab is using a genetic approach in order to illuminate how vitamin C affects phytohormone-mediated defense responses.