Chloroplast DNA methylation and inheritance in Chlamydomonas

When Chlamydomonas reinhardtii cells mate, a zygotic maturation program is activated, part of which leads to destruction of chloroplast DNA (cpDNA) fr om the mating type minus (mt(-)) parent, and, therefore, to uniparental inh eritance of mating type plus (mt(+)) cpDNA. A long-standing model that expl ains the selective destruction of mr cpDNA in zygotes invokes a methylation -restriction system. We tested this model by using the potent methylation i nhibitor 5-aza-2'-deoxycytidine (5adc) to hypomethylate parental cpDNA and found that the pattern of cpDNA inheritance is altered by 5adc in a manner that is consistent with the model. Surprisingly, however, hypomethylated mt (+) cpDNA is not destroyed in zygotes as the methylation-restriction model predicts it should be. Destruction of mt(-) cpDNA is also unaffected when t he parental mt(+) cpDNA is hypomethylated. Instead, loss of methylation aff ects the relative rates of replication of residual mt(-) cpDNA and mt(+) cp DNA in germinating zygotes. The mode of action for 5adc on cpDNA replicatio n in germinating zygotes may be via hypomethylation of mt(+) cpDNA, but is also consistent with its action as a DNA-damaging agent. Interestingly, 5ad c causes reduced cpDNA replication only in germinating zygotes, not in vege tatively grown cells, indicating that cpDNA replication is qualitatively di fferent in these two stages of the life cycle. Our results demonstrate that methylation is not necessary for protection of the mt(+) cpDNA in early zy gotes and uncover a novel stage of the Chlamydomonas life cycle when replic ation of cpDNA is highly susceptible to perturbation. Our data support a mo del in which differential cpDNA replication in germinating zygotes is used as a mechanism to selectively amplify intact and properly methylated cpDNA molecules.