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Unformatted text preview: ricts their role to somatic growth.
Therefore, epigenetic regulatory circuits operating in somatic
and gametophytic phases of plant development might be functionally separated. Our study provides the ﬁrst evidence that
transient loss of DNA methylation in the two or three cell divisions during haploid stage, and the ensuing release of gene
silencing, cannot be rescued by the subsequent presence of wildtype MET1. In other words, chromatin modiﬁcations that may
have been partially retained during the postmeiotic divisions and
early embryonic development are not sufﬁcient to provide the
required information for rapid recruitment of DNA methylation
and establishment of TGS to a locus. Rather, CpG methylation
provides essential information for subsequent histone modiﬁcations as recently suggested (refs. 27,28 and M. Tariq, H.S., A.V.
Probst, J. Lichota, Y. Habu and J.P., manuscript submitted).
The observed epigenetic segregation suggests that hemimethylation provides a template for methylation on both strands in
heterozygotes directly after fertilization, which restores the previous epigenetic makeup. This would co-exist with other fully
demethylated loci, generating new epigenetic gene variants. Furthermore, considering the possibility of post-replicative sister
chromatid crossovers and random chromatid segregation in
postmeiotic divisions, met1 heterozygous progeny will be characterized by excessive epigenetic variability. Although the haploid
gametophytic phase has been reduced considerably during plant
evolution, it has not declined beyond two postmeiotic divisions,
the minimum required to obtain completely demethylated
copies of genetic information in the case of MET1 deﬁciency.
This design may secure appropriate maintenance as well as ﬂexibility of epigenetic determination in the gametophytic phase. Methods
Plant growth conditions. We grew wild-type A. thaliana and both
mutant lines (ecotype Columbia) at 16 h light/8 h dark cycles either
under aseptic condition at 22.5 °C or in soil at 21 °C during the day and
16 °C during the night.
Histochemical GUS assay. We selected backcrossed heterozygous MET1
plants for the GUS activation test based on the presence of the T-DNA
insertion by growth on germination medium containing 15 µg ml–1 phosphinotricin. We subjected 2-wk-old plants to histochemical X-gluc assay as
Screening for TGS mutants. We pooled a population of T-DNA insertion
mutants in ecotype Columbia into samples of 100 independent lines each
and grew them on soil. We isolated 5 µg total RNA from 2-wk-old
seedlings in pools and used it for RT–PCR to detect expression of TSI16.
Primer sequences for RT–PCR are available on request. We analyzed the
PCR products on a 1.5% agarose gel stained with ethidium bromide. We
divided positive pools into ten subpools (10 lines each) and analyzed individual lines of positive subpools for TSI expression by RNA gel-blot analysis as previously described16.
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- Fall '09