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.

Figure 1

Schematic representation of zygote-maturation time course. Cells are pictured with the nucleus as a blue circle and the chloroplast as a green cup-shaped structure containing nucleoids that are represented by small blue dots. cpDNA is not replicated in gametes or maturing zygotes. Replication resumes in germinating zygotes. Zygotic maturation is diagrammed as a time course with cell fusion occurring at 0 h. Nucleoids from the mt⁻ parent are shown disappearing ∼3–4 h after mating. In the next several hours, the flagella are withdrawn, the parental chloroplasts and nuclei fuse, and the zygotic cell wall begins forming. After 5 d of dark incubation, zygotes that are returned to light and nutrients will undergo meiosis and hatch within 12 to 24 h. Rare vegetative diploids can form prior to mt⁻ cpDNA destruction.

Figure 2

Bar graph showing the effect of 5adc on chloroplast DNA inheritance. (Horizontal axis) Parental strain that was grown on 5adc (200 μM); (vertical axis) number of exceptional progeny produced (those carrying the mt⁻ parental chloroplast marker). A cross with a relatively high frequency of exceptional progeny (∼5%) in the control mating (no 5adc treatment) was used to illustrate the ability of 5adc to increase or decrease the frequency of exceptional progeny depending on which parent is treated with the drug.

Figure 3

Dose-dependent responses of methylation levels, chloroplast DNA levels, and exceptional progeny frequencies to 5adc concentration. (A) mt⁺ cells were grown on varying concentrations of 5adc, and gametic DNA was prepared and cut with either MboI (methylcytosine insensitive) or its isoschizomer Sau3AI (methylcytosine sensitive). The DNA was used to prepare a Southern blot that was probed with atpB sequences. (Arrows) Positions of fully cut DNA fragments; (asterisks) methylated bands. (B) Gametes used in A were mated, and the frequency of exceptional progeny and level of gametic cpDNA methylation (vertical axes) were plotted against 5adc dosage (horizontal axis). Methylation was quantitated from data in A. (C) Same experiment as in B except the total amount of chloroplast DNA in gametes rather than methylation levels is plotted on the right vertical axis.

Figure 4

Effect of 5adc treatment on zygotic chloroplast DNA methylation. (A) Control mating. DNA from zygotes was prepared at the indicated times after mating, cut with either MboI (M) or Sau3AI (S), Southern blotted, and probed with atpB sequences. (B) Same experiment as in A except the mt⁺ parent was grown in the presence of 200 μM 5adc before mating.

Figure 5

Molecularly tagging the atpB locus. (A) Schematic of the recombination event that generated the tagged atpB locus in mt⁻ cpDNA. (Top line) Wild-type atpB locus with the coding region boxed. The locations of MboI/Sau3AI recognition sites are marked m/s. The atpB probe used in all these experiments is indicated by the thick shaded line above the locus. The recombining plasmid sequence is shown below the atpB locus with aadA (spectinomycin resistance) and atpB coding sequences boxed. (Bottom) Recombination product. (B) DNA from wild-type mt⁺ or tagged mt⁻ gametes was cut with MboI, Southern blotted, and probed with the atpB fragment indicated in A. (C) Gametes from B were mated, and DNA prepared from zygotes at the indicated time points was cut with MboI, and blotted as in B. Residual unmated gamete DNA was not removed from the zygote DNA preparations in this experiment.

Figure 6

Effect of 5adc on the fate of cpDNA. (A) DNA from gametes or zygotes formed from a wild-type mt⁺ strain mated to a tagged mt⁻ strain was cut with MboI, Southern blotted, and probed with chloroplast atpB sequences (top) or nuclear ezy1 sequences (bottom). The time course of zygote maturation extends to the end of the dark period (5 d). The last lane contains DNA from progeny that have germinated and grown vegetatively for several generations (4–5 d). Lanes from 24-h and 5-d samples contain less total DNA than the other lanes. (B) Same experiment as in A except the mt⁺ parent was grown on 200 μM 5adc before mating. (C) Same experiment as in A except that the DNA was cut with the methylcytosine-insensitive enzyme EcoRV, which releases a single ∼4.2-kb band containing the aadA transgene, and was probed with aadA sequences. The dots indicate the locations of weakly cross-hybridizing bands that are unrelated to the presence of the aadA transgene and serve as useful internal controls for levels of the authentic aadA band (marked as mt⁻). (D) Same experiment as in C except that the mt⁺ parent was grown on 200 μM 5adc before mating. (E) cpDNA signals from A and C were normalized to the nuclear signal and plotted on a logarithmic scale against the same time course of zygote maturation and germination. The symbols indicate control cross mt⁺ cpDNA (□), control cross mt⁻ cpDNA (○), 5adc-treated cross mt⁺ cpDNA (▪), and 5adc-treated cross mt⁻ cpDNA (●). Starting cpDNA for all samples was set at 100. The light and dark periods during which zygotes mature and germinate are also indicated.

Figure 7

Nuclear and chloroplast DNA levels during germination. (A, top) Graph of nuclear DNA levels during germination from a control (▵) and 5adc-treated (mt⁺) (▴) cross. (y axis) Relative nuclear DNA levels normalized to the starting concentration (0 h.); (x axis) time course of germination, starting with the transfer to light and liquid growth media at 0 hrs. (Bottom) PCR-amplified fragments from the nuclear gene fus1 used for the quantitation. (B, top) Graph of mt⁻ cpDNA levels from control (○) and 5adc-treated (mt⁺) (●) samples. (y axis) Relative amounts of mt⁻ cpDNA normalized to the starting nuclear DNA content. The x axis is labeled as in A. (Bottom) PCR-amplified fragments from the aadA gene (mt⁻ specific) used for the quantitation. (C, top) Graph of mt⁺ cpDNA levels from control (□) and 5adc-treated (mt⁺) (▪) samples. (y axis) Relative amounts of mt⁺ cpDNA normalized to the starting nuclear DNA content. The x axis is labeled as in A. (Bottom) PCR-amplified fragments from the 3′ region of the chloroplast atpB gene (mt⁺ specific) used for the quantitation.

Figure 8

Models for cpDNA replication during germination. (Top) Schematic of cpDNA methylation and replication during germination of a normal cross. Mature zygotes contain ∼100 times more mt⁺ than mt⁻ cpDNA. During germination, the heavily methylated mt⁺ cpDNA replicates better than the mt⁻ cpDNA, generating a final difference of ∼1000-fold in mt⁺ versus mt⁻ cpDNA levels. (Bottom) Schematic of cpDNA methylation and replication during germination when the mt⁺ parental cpDNA has been hypomethylated by 5adc. Mature zygotes contain a similar low level of mt⁻ cpDNA as the normal cross (∼1%), but the residual mt⁻ cpDNA outcompetes the hypomethylated mt⁺ cpDNA for replication during germination, increasing its representation by 10-fold in the resulting progeny.