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expression in all cells of the mature embryo sac was observed for genes AT5G40260,
AT4G30590, AT5G60270, and AT4G01970 (Figure 2). The trithorax group gene
ATX3 and AT5G50915 were predominantly expressed in the egg and the central
cell, and the expression of the receptor-like kinase gene AT5G60270 was found to
be restricted to the egg cell alone (Figure 2). In addition to the in situ hybridiza-
tion experiments, we examined the expression of transgenes where specific pro-
moters drive the expression of the bacterial uidA gene encoding
β
-galacturonidase
(GUS) or in enhancer detector lines. We show that CYCLIN A2;4 (1.28-fold)
and AT4G01970 (encoding a galactosyl-transferase; about 1.51-fold) were broad-
ly expressed in the embryo sac, and that PUP3 (encoding a purine permease; 1.3-
fold) was specifically expressed in the synergids (Figure 2). CYCLIN A2;4 appears
to be expressed also in the endothelial layer surrounding the embryo sac (Figure
2e). Diffusion of GUS activity did not permit us to distinguish unambiguously
embryo sac expression from endothelial expression. In short, both broader and
cell type specific expression patterns in the embryo sac were observed for the nine
candidate genes. Hence, we could validate the minimal fold change cut-off of
1.28 and the statistical methods employed in this study.
Figure 2.
Confirmation of embryo sac expression for selected genes.
Embryo sac
expression of nine candidate genes is shown by in situ hybridization (panels a, c, d, f, g, and i) or
histochemical reporter gene (GUS) analysis (b, e, and h). Illustrated is the in situ expression of broadly expressed
genes: (a) AT1G78940 (encoding a protein kinase that is involved in regulation of cell cycle progression), (c)
AT5G40260 (encoding a nodulin), and (d) AT4G30590 (encoding a plastocyanin). Also shown is the restricted
expression of (f ) AT3G61740 (encoding the trithorax-like protein ATX3), (g) AT5G50915 (encoding a TCP
transcription factor), and (i) AT5G60270 (encoding a protein kinase). The corresponding sense control for panels
a, b, c, d, f, g, and i did not show any detectable signal (data not shown). GUS staining: (b) an enhancer-trap line
for AT4G01970 (encoding a galactosyltransferase) shows embryo sac expression, (e) a promoter-GUS line for
AT1G80370 (encoding CYCLIN A2;4) shows a strong and specific expression in the embryo sac and endothelium
(insert: shows several ovules at lower magnification), and (h) a promoter-GUS line for AT1G28220 (encoding the
purine permease PUP3) shows synergid specific expression (insert; note the pollen-specific expression of PUP3-
GUS when used as a pollen donor on a wild-type pistil). CC, central cell; EC, egg cell; SC, synergids. Scale bars:
50
µ
m in panels a to i; and 100
µ
m and 50
µ
m in the inserts of panels e and h, respectively.
