Transient expression analysis revealed the importance of VTC2 expression level in light/dark regulation of ascorbate biosynthesis in Arabidopsis.

Ascorbate (AsA) is an important antioxidant and an enzyme cofactor involved in various metabolic pathways. In this study, we investigated the effects of estrogen (ES)-inducible transient expression of genes encoding enzymes involved in the d-mannose/l-galactose (d-Man/l-Gal) pathway for plant AsA biosynthesis on AsA levels under light and dark conditions. No significant difference was observed in AsA levels between Arabidopsis plants transiently expressing phosphomannose isomerase (PMI1), GDP-d-Man pyrophosphorylase (GMP/VTC1), GDP-Man-3',5'-epimerase (GME), and l-Gal 1-phosphate phosphatase (GPP/VTC4), but AsA levels in the plants transiently expressing GDP-l-Gal phosphorylase (GGP/VTC2) were 2.5-fold higher than those in control plants 7 d after ES treatment. The increase in AsA levels under continuous light conditions and the decrease in AsA levels under dark conditions were enhanced and suppressed, respectively, in the ES-treated plants. These results suggest that GGP/VTC2 acts as a rate-limiting step regulating AsA biosynthesis in response to light and dark conditions.