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is to be improved much further, then methods to isolate embryo sac cells using
methods such as florescence-activated cell sorting, targeted genetic ablation by
expression of a cell-autonomous cytotoxin, or laser-assisted microdissection must
be developed [51,60-62].
The Embryo Sac Expressed Candidate Genes May Be Essential
for Female Gametophyte and Seed Development
Once we had validated the expression of the embryo sac expressed genes, we
considered whether these genes could play essential roles during embryo sac and
seed development. It is apparent from our work on five mutants, and mutant
data from the literature, that the embryo sac expressed genes that we report here
may play a crucial role during the embryo sac development or later during seed
formation. HOG1 is of special interest because we have provided evidence for
allelic phenotypic complementation by two mutant alleles. HOG1 is proposed
to act upstream of METHYL TRANSFERASE 1 (MET1) and CMT3 among
other methylases, and mutants for HOG1 have high levels of global hypom-
ethylation [54]. It has become clear that DNA hypomethylation plays a crucial
role during gametogenesis, and that mutations affecting the genes in this path-
way such as HOG1, MET1, and CMT3 affect embryo and endosperm develop-
ment [55,63,64]. It is interesting to note that we identified CMT3, MEA, and
FIS2 that are associated with pathways involving DNA and histone methylation
[63,65-68].
We have shown that our dataset will be a resource for targeted reverse ge-
netic approaches. The extensive reverse genetic tools available for Arabidopsis re-
searchers make such a large-scale functional study possible [69]. While screening
for female gametophytic mutants through T-DNA mutagenesis, we unexpect-
edly observed a number of female gametophytic mutants that had a very similar
phenotype: a complete arrest of female gametogenesis at the one-nuclear stage.
These, however, were not linked to the gene disruption. Agrobacterium-mediated
Arabidopsis T-DNA mutagenesis has been facilitated by floral dipping, which
involves integration of the T-DNA through the ovule, and the chromosomes of
the female gametophyte are the main target for T-DNA insertion [70]. Based on
our results from this study, and other independent observations (Johnston AJ,
Grossniklaus U, unpublished data), we believe that these unlinked gametophytic
lethal events arose because of translocations and other rearrangements of maternal
chromosomes during the integration of the T-DNA, and we advise due caution
in mutant screening.
