Role of a nonselective de novo DNA methyltransferase in maternal inheritance of chloroplast genes in the green alga, Chlamydomonas reinhardtii

In the green alga, Chlamydomonas, chloroplast DNA is maternally transmitted to the offspring. We previously hypothesized that the underlying molecular mechanism involves specific methylation of maternal gamete DNA before mating, protecting against degradation. To obtain direct evidence for this, we focused on a DNA methyltransferase, DMT1, which was previously shown to be localized in chloroplasts. The full-length DMT1 protein with a molecular mass of 150 kD was expressed in insect cells, and its catalytic activity was determined. In vitro assays using synthetic DNA indicated methylation of all cytosine residues, with no clear selectivity in terms of the neighboring nucleotides. Subsequently, transgenic paternal cells constitutively expressing DMT1 were constructed and direct methylation mapping assays of their DNA showed a clear nonselective methylation of chloroplast DNA. When transgenic paternal cells were crossed with wild-type maternal cells, the frequency of biparental and paternal offspring of chloroplasts increased up to 23% while between wild-type strains it was ∼3%. The results indicate that DMT1 is a novel type of DNA methyltransferase with a nonselective cytosine methylation activity, and that chloroplast DNA methylation by DMT1 is one of factors influencing maternal inheritance of chloroplast genes.