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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.
- Fall '09