Methylation Of Chloroplast DNA Research Articles

Chloroplast DNA methylation in the kelp Saccharina latissima is determined by origin and possibly influenced by cultivation.

DNA cytosine methylation is an important epigenetic mechanism in genomic DNA. In most land plants, it is absent in the chloroplast DNA. We detected methylation in the chloroplast DNA of the kelp Saccharina latissima, a non-model macroalgal species of high ecological and economic importance. Since the functional role of the chloroplast methylome is yet largely unknown, this fundamental research assessed the chloroplast DNA cytosine methylation in wild and laboratory raised kelp from different climatic origins (High-Arctic at 79° N, and temperate at 54° N), and in laboratory samples from these origins raised at different temperatures (5, 10 and 15°C). Results suggest genome-wide differences in methylated sites and methylation level between the origins, while rearing temperature had only weak effects on the chloroplast methylome. Our findings point at the importance of matching conditions to origin in restoration and cultivation processes to be valid even on plastid level.

Methylation of chloroplast DNA does not affect viability and maternal inheritance in tobacco and may provide a strategy towards transgene containment

We report the integration of a type II restriction-methylase, mFokI, into the tobacco chloroplast genome and we demonstrate that the introduced enzyme effectively directs the methylation of its target sequence in vivo and does not affect maternal inheritance. We further report the transformation of tobacco with an E. coli dcm methylase targeted to plastids and we demonstrate efficient cytosine methylation of the plastid genome. Both adenosine methylation of FokI sites and cytosine methylation of dcm sites appeared phenotypically neutral. The ability to tolerate such plastid genome methylation is a pre-requisite for a proposed plant transgene containment system. In such a system, a chloroplast located, maternally inherited restriction methylase would provide protection from a nuclear-encoded, plastid targeted restriction endonuclease. As plastids are not paternally inherited in most crop species, pollen from such plants would carry the endonuclease transgene but not the corresponding methylase; the consequence of this should be containment of all nuclear transgenes, as pollination will only be viable in crosses to the appropriate transplastomic maternal background.

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 approximately 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.

Methylation of chloroplast DNA in Chlamydomonas.

Methylation of chloroplast DNA in Chlamydomonas.

The mat-1 gene in chlamydomonas regulates DNA methylation during gametogenesis

The inheritance of chloroplast genes in Chlamydomonas is regulated by methylation of chloroplast DNA during gametogenesis. The wild-type pattern of maternal inheritance results from the methylation of chloroplast DNA in female ( mt +) but not in male ( mt −) gametes, leading to preferential degradation of chloroplast DNA of male origin in zygotes. This paper describes the distribution of 5-methyl cytosine residues in restriction fragments of chloroplast DNA sampled during gametogenesis by two methods: ethidium bromide staining of agarose gels, and binding of antibody directed against 5-methyl cytosine onto restriction fragments blotted to nitrocellulose paper. Methylated cytosines are located in most if not all Eco RI and Msp I fragments, but the extent of methylation is not proportional to fragment size. The mat-1 mutation carried by males converts maternal inheritance of chloroplast genes to biparental inheritance. Chloroplast DNA of male gametes carrying the mat-1 mutation becomes methylated during gametogenesis. This methylation protects against restriction enzyme-promoted degradation in zygotes, as shown by physical data demonstrating the transmission to progeny of chloroplast genes carried on chloroplast DNA of the mat-1 male parent. Thus the mat-1 gene, which is linked to the mating-type locus, determines whether or not methylation of chloroplast DNA will occur in males during gametogenesis.