Light (A–D, F, and G) and immunoelectron (E) microscopy analysis of the localization of PAPhy in the developing wheat grain, approximately 18 DPA. A, Toluidine blue-stained semithin cross-section of endosperm, aleurone, and pericarp tissues. B, Differential interference contrast microscopy with indications of the aleurone vacuoles. C, Immunofluorescence detection of PAPhy in 1-μm-thick sections. The aleurone vacuoles are clearly labeled, while there is no fluorescence from any other compartment of the cell, the apoplast (arrowheads), or other cell types. D, Immunofluorescence of a 1-μm-thick section incubated with secondary antibody only. There is virtually no background from the secondary antibody. E, Immunoelectron microscopy analysis showing an aleurone vacuole with gold labeling of protein crystalloid. F, Transgenic wheat grain transformed with a TaPAPhy_a1-GUS construct and showing GUS activity in the embryo and the seed coat fraction (arrows). G, GUS activity is restricted to the embryo scutellum. al, Aleurone; EnvM, globoid crystal-enveloping membrane; GC, globoid crystal; n, nucleus; pb, protein body; PC, protein crystalloid; s, starch; v, vacuole.