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 μm in panels a to i; and 100 μm and 50 μm in the inserts of panels e and h, respectively.