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Our study provides the rst evidence that transient

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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 described previously29. 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. Analysis...
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