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

Abstract

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.

Graphical Abstract

The increase in ascorbate levels under continuous light and the decrease under continuous dark were enhanced and suppressed respectively by the transient expression of GGP/VTC2.

Key words:

• ascorbate
• biosynthesis
• GDP-l-galactose phosphorylase
• light and dark regulation
• VTC2

Acknowledgment

We are grateful to Dr. Nam-Hai Chua (Rockefeller University) for donating the pMDC7 vector. We thank Ms Mami Kondo, Mr Tsuyoshi Hirata, Ms Kanako Misaki, Mr Shigeyoshi Ishimoto, and Mr Ren Goto for technical assistance.

Funding

This work was supported by JSPS KAKENHI [grant number 24770046] (to K.Y.); Grant-in-Aid for Young Scientists (B), and by MEXT KAKENHI [grant number 22248042] (to S.S.); Grant-in-Aid for Scientific Research (A).

Notes

Abbreviations: DHA, dehydroascorbate; d-Man, d-mannose; GME, GDP-Man-3′,5′-epimerase; GMP, GDP-Man pyrophosphorylase; GGP, GDP-l-Gal phosphorylase/l-Gal guanylyltransferase; GPP, l-Gal-1-phosphate phosphatase; l-Gal, l-galactose; l-GalDH, l-Gal dehydrogenase; l-GalL, l-galactono-1,4-lactone; l-GalLDH, l-GalL dehydrogenase; PMI, phosphomannose isomerase; PMM, phosphomannose mutase.