Biology Reference
In-Depth Information
a simple genetic subtraction strategy, which successfully identified a large num-
ber of candidate genes that are expressed in the cell types of the embryo sac.
The wealth of data reported here lays the foundation to elucidate the regulatory
networks of transcriptional regulation, signaling, transport, and metabolism that
operate in these unique cell types of the haploid phase of the life cycle. Given that
many of the genes in our expression dataset are essential to female gametophyte
and seed development, targeted functional studies with further candidate genes
promise to yield novel insights into the development and function of the embryo
sac. Another major finding of this work is the identification of 108 genes that are
enriched for embryo sac expression and thus probably play important roles for the
differentiation and function of these specific cell types. The surprising finding that
many genes are deregulated in sporophytic tissues in the absence of an embryo sac
suggests a much more complex interplay of the haploid gametophytic with the
diploid sporophytic tissues than was previously anticipated. Understanding the
sporophytic regulatory network governed by the embryo sac will be of key interest
for future studies.
Materials and Methods
Plant Material and Growth Conditions
The coatlique (coa) mutant was identified in Arabidopsis var Landsberg (erecta
mutant; Ler) background and Ler was used as a wild-type control in the microar-
ray and in situ hybridization experiments. Before transplanting, seeds were sown
on Murashige and Skoog media (1% sucrose and 0.9% agar; pH 5.7) supple-
mented with appropriate selection markers and stratified for two days at 4°C (see
Table 1 for description of mutants plants and selection markers). The seeds were
germinated and grown for up to 15 days under 16-hour light/8-hour dark cycles
at 22°C. Plants were then transplanted into ED73 soil (Einheitserde, Schopfheim,
Germany) and grown in greenhouse conditions under a 16-hour photo-period at
22°C and 60% to 70% relative humidity.
Histological Analysis
For phenotypic characterization, the gynoecia of Arabidopsis wild-type, coa
and gametophytic mutants, and siliques of the zygotic mutants were cleared
in accordance with a protocol described in the report by Yadegari and co-
workers [78]. Samples were observed using a Leica DMR microscope (Leica
Microsystems, Mannheim, Germany) under differential interference contrast
(DIC) optics.
