Fig. 3. Epidermal phenotypes of zou mutant seedlings. (A,B) Seedlings stained with Toluidine Blue. Arrowheads indicate leaves, arrows indicate cotyledons. Unlike wild type (A), in zou-4 mutant (B) cotyledons and hypocotyls are stained strongly. (C-I) Cryo-SEM images of seedling epidermis. (C)zou-4 mutant seedling. Leaves (arrowhead) are normal, whereas cotyledons (arrow) have large tears in the epidermis. (D) Epidermis of wild-type cotyledon, showing jigsaw-shaped pavement cells and stomata. (E) Epidermis of zou-4 cotyledons. Normal cell types are present, although stomatal spacing is abnormal. (F)zou-2 mutant cotyledon showing large tear, with protruding mesophyll cells. (G)zou-4 mutant cotyledon showing gaps between pavement cells (arrows). The rough cell surfaces are due to adhering endosperm. (H)zou-4 acr4-2 double mutant, showing severely defective cotyledons (arrows). Adhering seed coat is indicated by arrowhead. (I)ale2-1 zou-4 double mutant cotyledons are minute, mis-shapen and partially fused. (J) TEM image of wild-type adaxial cotyledon surface showing cell wall (cw) cuticularized layer (cl) and cuticle proper (cp). (K) TEM image of zou-2 adaxial cotyledon surface showing thickness variability and discontinuity (arrow) of the cp. (L)zou-2 cotyledon surface showing adhering endosperm tissue (arrow) on surface of embryo (e). Scale bars: 1 cm in A,B; 1 mm in C; 300µm in D; 400µm in E; 500µm in F; 50µm in G; 500µm in H; 400µm in I; 100 nm in J,K; 2µm in L.