Molecular cloning of the monodehydroascorbate reductase gene from Brassica campestris and analysis of its mRNA level in response to oxidative stress

In a majority of living organisms, a fundamental protection mechanism from reactive oxygen species is by the ascorbate-glutathione cycle in which an important antioxidant, ascorbate (vitamin C), is utilized to convert harmful H₂O₂ to H₂O. Monodehydroascorbate reductase (MDHAR) maintains reduced pools of ascorbate by recycling the oxidized form of ascorbate. By screening a Brassica campestris cDNA library, we identified a B. campestris MDHAR cDNA (BcMdhar) which encodes a polypeptide of 434 amino acids possessing domains characteristic of FAD- and NAD(P)H-binding proteins. The predicted amino acid sequence of the open reading frame (ORF) shows a high level of identity to the cytosolic MDHAR of rice, pea and tomato, and does not possess N-terminal leader sequence suggesting that it encodes a cytosolic form of MDHAR. Genomic Southern blot analysis indicated that a single nuclear gene encodes this enzyme. Northern hybridization analysis detected BcMdhar transcripts in all plant tissues examined. The level of BcMdhar mRNA increased in response to oxidative stress invoked by hydrogen peroxide, salicylic acid, paraquat, and ozone.