Figure 4

Confocal images of Zinnia suspension cultured cells, representing the cell morphology during TEs differentiation. A. FDA stained living cell - visible are diffuse nuclei, intact cytoplasm and intact vacuole; B. Double FDA-PI stained cells showing different stages of vacuole rupture: living TE with intact vacuole, preserved cytoplasm and single SCW, lacking a "dead" nucleus (the lower cell), dead cell - FDA is partially penetrated in the vacuole, but the cellular organelles are still preserved (the upper larger cell); dead cell with ruptured vacuole and lacking FDA stained cytoplasm (right), dead TEs with "dead" nuclei (underneath the living TE) - PI fluorescence is visualised in pink; C. Fully differentiated TE - autofluorescence from the lignified secondary cell walls; D. Field image of living, dead and differentiated cells after specific CFW staining of the cellulose micro fibrils in the cell walls; E. Differentiating cells representing intermediate state - combined staining with FDA (visible ruptured vacuole) and PI staining of a nucleus in dead cells; F. PI staining of a hollow differentiating cell with preserved nuclei and lysed cell content; G. CFW staining of the secondary cell walls in a cluster of differentiated cells; H. Cell cluster consisting of a differentiated and differentiating vacuolated cells. Scale bars (A, B, C, E, G, H) = 20 μm; D = 50 μm; F = 10 μm. Images were collected by using a TCS SP2 AOBS confocal laser scanning microscopy system (Leica-Microsystems GmbH, Mannheim, Germany) mounted on an inverted Leica DM IRE2 microscope. Three different lasers (405, 488 and 561 nm) were employed for excitation and three emission channels for fluorescence imaging and one separate channel for non-confocal transmission imaging. Overlays and orthogonal projections were made using the Leica Confocal software. The images were taken 120 h after administration of 1 mg/L NAA and 1 mg/L BA and stained with FDA (fluorescence visible in the cytoplasm of living cells and as faded green in the ruptured vacuole), PI (stains nuclei in dead cells) and CFW (specific for cell wall visualization). Cyt, cytoplasm; cw, cell wall; dc, dead cell; difc, differentiated cell; lc, living cell; L-TE, living TE; nu, nucleus; scw, secondary cell wall; v, vacuole.