FIGURE 5.

Evaluation of seed development and detection of the embryo cuticle. (A–E) The seeds at 12 DAP were cleared and observed by DIC microscopy. The embryo in Col-0 showed complete bending, occupying the entire seed cavity (A) and arrested in the heart/torpedo stage in the Atzou-4 mutant (B). In GmZOU-1 transgenic seeds, the semi-bent and larger embryo occupied the seed (C,D), and the embryo in the GmZOU-2 transgenic seeds was the same as that in the Atzou-4 mutant (E). Scale bars: 100 μm. (F–J) Embryonic and endosperm development was detected by paraffin sectioning at about 10 DAP. The wild-type seeds were in the mature stage, showing a completely bent embryo and only one cell layer of endosperm surrounding the embryo (F); the Atzou-4 mutant embryos were arrested in the heart stage and their cellularized endosperm remained intact (G). In GmZOU-1 transgenic seeds, the endosperm showed breakdown and the embryo expanded to later stages. Furthermore, the expanded embryo still exhibited non-uniform development based on the amount of residual endosperm (H,I). In the GmZOU-2 transgenic seeds, the embryo and endosperm remained arrested, similar to that in the Atzou-4 mutant (J). Scale bars: 100 μm in (F) and 50 μm in (G–J). (K,L) GmZOU-1 partially recovered the cuticle deficiency in Atzou-4. The seedlings of Col-0, Atzou-4, and the GmZOUs transgenic lines at about 7 days after germination were stained with TB. The GmZOU-1 transgenic seedlings were weakly stained, whereas the GmZOU-2 transgenic seedlings showed strong staining similar to that in the Atzou-4 mutants (K). TB uptake in these lines was quantified spectrophotometrically, showing similar results to that of microscopy analysis. Error bars represent s.d. among three biological replicates, each containing 20 seedlings (L). Scale bars: 5 mm.