Biology Reference
In-Depth Information
Functional Classification of the Candidate Genes
The genes identified as embryo sac expressed or over-expressed sporophytic can-
didates were grouped into eight functional categories based on a classification
system reported previously [53] (Figure 1). The gene annotations were improved
based on the Gene Ontology annotations available from 'The Arabidopsis Infor-
mation Resource' (TAIR). The largest group in both gene datasets consisted of
genes with unknown function (35% of embryo sac expressed genes and 37% of
over-expressed sporophytic candidate genes), and the next largest was the class of
metabolic genes (24% and 27%; Figure 1). Overall, both the gametophytic and
sporophytic datasets comprised similar percentages of genes within each func-
tional category (Figure 1). In both datasets, we found genes that are predicted to
be involved in transport facilitation and cell wall biogenesis (15% of embryo sac
expressed genes and 13% of over-expressed sporophytic candidate genes), tran-
scriptional regulation (10% and 9%), signaling (7% and 6%), translation and
protein fate (5% each), RNA synthesis and modification (3% and 1%), and cell
cycle and chromosome dynamics (1% each).
Validation of Expression for Known Embryo Sac-Expressed
genes
The efficacy of the comparative profiling approach used here was first confirmed
by the presence of 18 genes that were previously identified as being expressed
in the embryo sac. They included embryo sac expressed genes such as PROLIF-
ERA, PAB2 and PAB5 (which encode poly-A binding proteins) and MEDEA,
and genes with cell-specific expression such as central cell expressed FIS2 and
FWA, synergid cell expressed MYB98, and antipodal cell expressed AT1G36340.
Therefore, our comparative profiling approach potentially identified novel genes
that could be expressed either throughout the embryo sac or in an expression pat-
tern that is restricted to specific cell types.
In Situ Hybridization and Enhancer Detector Patterns Confirm
Embryo Sac Expression of Candidate Genes
In order to validate the spatial expression of candidate genes in the wild-type
embryo sac, the six following genes were chosen for mRNA in situ hybridiza-
tion on paraffin-embedded pistils: AT5G40260 (encoding nodulin; 1.99-fold)
and AT4G30590 (encoding plastocyanin; 1.88-fold); AT5G60270 (encoding
a receptor-like kinase; 1.56-fold) and AT3G61740 (encoding TRITHORAX-
LIKE 3 [ATX3]; 1.47-fold); and AT5G50915 (encoding a TCP transcription
factor; 1.36-fold) and AT1G78940 (encoding a protein kinase; 1.35-fold). Broad
