To examine the mechanism of these effects we crossed

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Unformatted text preview: type, suggests dominant-like effects of both loss-of-function mutations. To examine the mechanism of these effects, we crossed T2 plants heterozygous with respect to either met1 allele with the transgenic line 6b5 (ref. 19), which contains an epigenetically silent and hypermethylated locus consisting of several copies of the β-glucuronidase gene (GUS; Fig. 3a). In sharp contrast to the rapid demethylation and reactivation of the GUS locus in F1 hybrids between 6b5 and AsMET1, we did not detect any change in DNA methylation at the GUS locus in any F1 hybrid heterozygous with respect to met1-3 or met1-4 and the GUS locus (Fig. 3b and data not shown). This was also true in the late developmental stages of F1 hybrids; the GUS locus remained methylated and silent (data not shown). Thus, the downregulation of MET1 by antisense expression, but not the met1 mutations, showed sporophytic dominance with respect to demethylation and reactivation of the GUS locus. This excludes the possibility that the effects of the met1 mutations are due to dominant-negative properties of the mutated alleles (for example, antisense MET1 RNA initiated from the TDNA inserts or interference of truncated MET1 protein) or to haploinsufficiency of MET1. Therefore, the discrepancy in demethylation and TGS release from endogenous targets, but not from the GUS locus in met1 heterozygotes, must originate from their different exposure to the mutation. Half of the endogenous sequences in the heterozygotes are inevitably inherited in conjunction with the mutated allele, whereas the GUS repeat entered the F1 hybrid as an ‘immaculate’ test locus. Ingression of a target sequence either together with or independent from the mutated allele might make a difference if MET1 function was required during transition from one to the next generation. Whereas in most animals the genomes of gametes are combined directly after or even before the end of meiosis, the haploid phase of the life cycle of higher plants is extended by postmeiotic divisions before zygote formation. In angiosperm female gametogenesis, the primary postmeiotic haploid cell undergoes three mitoses resulting in an embryo sac with eight nuclei, whereas the two male sperm nuclei fertilizing the egg and the central cell are formed by two postmeiotic nuclear divisions20. Division of haploid cells that inherited the mutated alleles could result in passive demethylation of their DNA. Consequently, the epigenetic state of the GUS locus is expected to change only when it is transmitted through a met1 gamete. This was tested in consecutive backcrosses of the hybrids (Fig. 3a). Indeed, we observed progeny expressing GUS from plants containing a met1 mutated allele (genotype MET1+/– GUS+/–; Fig. 3a,b). The different number of post-meiotic divisions during female and male gametogenesis provided an opportunity to further challenge the hypothesis of passive demethylation. Assuming fertilization with haploid gametes that are arrested in G1...
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This document was uploaded on 03/17/2014.

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