Figure 4

Calcofluor staining and callose inmunolocalisation in unpollinated and pollinated pistils of Malus x domestica at the stigma (A-C), mid- style (D-H), and at the style base (I-M). (A) Cellulose stained pollen grain intine, pollen tube walls, and stigmatoid tissue cell walls one day after pollination (1DAP). (B) Same section immunolocalising linear β-(1,3)-glucans (callose) along the inner side of the pollen tube wall, (C) overlapped images of both cellulose and callose. (D) Cellulose and other polysaccharides detected (arrows) in the cells of the style transmitting tissue of unpollinated pistils two days after anthesis (2DAA). (E) Callose immunolocalization (fluorescent green-arrows) in an unpollinated pistil (2DAA). (F) Pollen tubes (asterisks) in the transmitting tissue two days after pollination (2DAP). (G) Callose immunolocalized at pollen tube wall, but not in the transmitting tissue cells of pollinated pistils. (H) Overlapped image of callose and cellulose in the pollen tube wall two days after pollination. (I) Cellulose and other polysaccharides at the base of the stylar transmitting tissue (arrows) in unpollinated pistils accumulated later, by three days after anthesis (3DAA). (J) Callose co-localized with cellulose (arrows) in the transmitting tissue of unpollianted pistils. (K) While cellulose and other polysaccharides were no longer detected in pollinated pistils three days after pollination (3DAP), (L) pollen tube walls labelled for callose (asterisks) in cross sections. (M) Overlapped image of calcofluor white and callose at the stylar base. Calcofluor staining (A, D, F, I, K), linear β-(1–3)-glucans immunolocalization with FITC secondary Ab labelling in fluorescent green (B, E, G, J, L), and merged images callose-calcofluor (C, H, M) in 4μm longitudinal (A-C), and transversal (D-M) sections of upper (A-C), middle (D-H), and lower (I-M) style. Asterisks indicate pollen tubes. ct, cortical tissue; P, papillae; pg, pollen grain; tt, stylar transmitting tissue. Scale bars: 10 μm.