Insensitivity of chloroplast gene expression to DNA methylation

Presence and possible functions of DNA methylation in plastid genomes of higher plants have been highly controversial. While a number of studies presented evidence for the occurrence of both cytosine and adenine methylation in plastid genomes and proposed a role of cytosine methylation in the transcriptional regulation of plastid genes, several recent studies suggested that at least cytosine methylation may be absent from higher plant plastid genomes. To test if either adenine or cytosine methylation can play a regulatory role in plastid gene expression, we have introduced cyanobacterial genes for adenine and cytosine DNA methyltransferases (methylases) into the tobacco plastid genome by chloroplast transformation. Using DNA cleavage with methylation-sensitive and methylation-dependent restriction endonucleases, we show that the plastid genomes in the transplastomic plants are efficiently methylated. All transplastomic lines are phenotypically indistinguishable from wild-type plants and, moreover, show no alterations in plastid gene expression. Our data indicate that the expression of plastid genes is not sensitive to DNA methylation and, hence, suggest that DNA methylation is unlikely to be involved in the transcriptional regulation of plastid gene expression.