Hypomethylation and transcriptional reactivation of retrotransposon-like sequences in ddm1 transgenic plants of Brassica rapa

DNA methylation and histone modification play important roles in regulating gene expression. The DDM1 gene in Arabidopsis thaliana (AtDDM1) is required for the maintenance of DNA methylation level and histone H3 methylation pattern. We isolated DDM1 homologs of Brassica rapa, BrDDM1a and BrDDM1b, which have 84.4% and 84.1% deduced amino acid sequence identities with AtDDM1, respectively. Both the BrDDM1a and BrDDM1b genes were found to be expressed in vegetative and reproductive tissues. B. rapa ddm1-RNAi transgenic plants with reduced levels of BrDDM1a/BrDDM1b expression showed genome-wide and non-tissue-specific demethylation. These results suggest that the BrDDM1a and BrDDM1b genes are orthologs of AtDDM1 and are required for the maintenance of DNA methylation as is AtDDM1. Despite genome-wide demethylation, developmental abnormalities were not found in the ddm1-RNAi transgenic plants. Dominance relationships of SP11/SCR alleles, the determinant of pollen recognition specificity in Brassica self-incompatibility, in S heterozygotes in B. rapa were not influenced by the low level of the BrDDM1 expression. Transcriptional reactivation of retrotransposon-like sequences observed in the ddm1-RNAi transgenic plants indicates that BrDDM1a and BrDDM1b participate in silencing of retrotransposons. Hypomethylation states of the ddm1-RNAi transgenic plants were inherited by plants of the next generation even by plants which had lost the RNAi construct by segregation. Remethylation was observed in a few progenies. Efficiencies of remethylation in the progenies without the RNAi construct were different between 18S rDNA, BoSTF12a/15a, and BrTto1 sequences.