Identification of tobacco genes encoding proteins possessing removal activity of 5-methylcytosines from intact tobacco DNA

Cytosines in eukaryotic DNA is often methylated to yield 5-methylcytosines (m 5 C), which play an important role in controlling gene expression. This is referred as DNA methylation, and its status dynamically changes during plant growth by active methylation and demethylation. DNA glycosylases have been known to possess base excision DNA repair activity, and an Arabidopsis enzyme, ROS1 (Repressor of Silencing 1), was recently shown to exhibit m 5 C DNA glycosylase activity. We isolated and characterized four genes encoding ROS1-like proteins from tobacco plants (Nicotiana tabacum), and designated as NtROS1, NtROS2a, NtROS2b and NtROS3, each respectively encoding a polypeptide with 1796, 1673, 1673 and 1662 amino acids. Purified NtROS1 and NtROS2a proteins expressed in Sf9 insect cells clearly exhibited activity of m 5 C removal from tobacco genomic DNA in vitro. GFP fusion assay showed that NtROS1 and NtROS2a were localized in nucleus. Transcripts of NtROS1, NtROS2a and NtROS3 were induced by abiotic stresses, including aluminium, salt and reactive oxygen species. These observations suggested that NtROS proteins function in demethylating process of genomic DNA during plant stress response, thereby maintaining the balance of gene expression in combination with DNA methyltransferases.