Chloroplast DNA methylation and inheritance in Chlamydomonas

Abstract

When Chlamydomonas reinhardtii cells mate, a zygotic maturation program is activated, part of which leads to destruction of chloroplast DNA (cpDNA) from the mating type minus (mt − ) parent, and, therefore, to uniparental inheritance of mating type plus (mt + ) cpDNA. A long-standing model that explains the selective destruction of mt − cpDNA in zygotes invokes a methylation-restriction system. We tested this model by using the potent methylation inhibitor 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 the parental mt + cpDNA is hypomethylated. Instead, loss of methylation affects the relative rates of replication of residual mt − cpDNA and mt + cpDNA in germinating zygotes. The mode of action for 5adc on cpDNA replication in germinating zygotes may be via hypomethylation of mt + cpDNA, but is also consistent with its action as a DNA-damaging agent. Interestingly, 5adc causes reduced cpDNA replication only in germinating zygotes, not in vegetatively grown cells, indicating that cpDNA replication is qualitatively different in these two stages of the life cycle. Our results demonstrate that methylation is not necessary for protection of the mt + cpDNA in early zygotes and uncover a novel stage of the Chlamydomonas life cycle when replication of cpDNA is highly susceptible to perturbation. Our data support a model in which differential cpDNA replication in germinating zygotes is used as a mechanism to selectively amplify intact and properly methylated cpDNA molecules.

Conservation and divergence of methylation patterning in plants and animals

“…Chlamydomonas was previously shown to exhibit heavy methylation of chloroplasts during mating, which contributes to uniparental inheritance of chloroplast DNA (12), but methylation of the nuclear genome has not been characterized. We used vegetative cells not containing gametes in our analysis and found only low levels of methylation in the chloroplast genome (Fig.…”

Gene silencing in microalgae: Mechanisms and biological roles

“…In C. reinhardtii, DNA cytosine methylation was initially observed in chloroplast DNA during gametogenesis and implicated in the uniparental inheritance of mating type plus chloroplast DNA (Umen and Goodenough, 2001;Nishiyama et al, 2004). Recent findings indicated that the nuclear genome of this alga is also methylated at low levels and, similarly to Volvox, CG methylation is preferentially enriched in transposon sequences and to a much lower degree in gene bodies (Feng et al, 2010).…”

Synergism between Inositol Polyphosphates and TOR Kinase Signaling in Nutrient Sensing, Growth Control, and Lipid Metabolism in Chlamydomonas

“…Amplification of the pHYG3 insertion junction was performed according to a previously described strategy (O'Malley et al, 2007) using genomic DNA prepared as described (Umen and Goodenough, 2001). Genomic DNA from 266F3 was digested with PvuII, SmaI, StuI, and PmlI and blunt-end ligated to annealed adaptor oligonucleotides (Supplemental Table 1).…”