FIGURE 4.

Seed development and embryonic cuticle integrity. Phenotype analyses of the independent transgenic events OsZOU-1#1, #4, and #5 and OsZOU-2#1, #3, and #5 were performed, but only images from OsZOU-1#4 and OsZOU-2#3, considered representative of all other events, are presented here. (A–D) The embryo of Col-0, Atzou-4, OsZOU-1 transgenic, and OsZOU-2 transgenic plants at ten DAP were observed under Differential Interference Contrast (DIC) microscopy. Scale bars = 0.1 mm. The embryo of Col-0 plant was bent, almost occupying the entire seed cavity with only one cell layer of endosperm surrounding the embryo (A). The Atzou-4 mutant embryo was arrested in the heart/torpedo stage and its endosperm remained intact (B). In OsZOU-1 transgenic seeds, the bent embryo occupied most of the seed cavity, and the endosperm showed breakdown (C). The embryo and endosperm of OsZOU-2 transgenic seeds were the same as those of the Atzou-4 mutant (D). Scale bars in (A–D) are 50 μm. (E–I) OsZOU-1 partially recovered the cuticle deficiency in Atzou-4. Scale bars in (E–I) are 1 mm. Seedlings of the Col-0 (E), Atzou-4 (F), OsZOU-1 (G), and OsZOU-2 (H) transgenic lines on the seventh day after germination were stained with TB. Results show that OsZOU-1 transgenic seedlings were weakly stained (G), while those of the OsZOU-2 seedlings showed strong staining similar to that in the Atzou-4 mutants (H). TB uptake in these lines was quantified spectrophotometrically and revealed similar results to microscopy analysis. The mean values of OsZOU-1#1, #4, and #5 represent the results for OsZOU-1 transgenic lines, while the mean values of OsZOU-2#1, #3, and #5 represent the results for OsZOU-2 transgenic lines. Error bars denote the SDs of three biological replicates, each containing 20 seedlings (I). Student’s t-test value: ^∗p < 0.05, ^∗∗p < 0.01.