Organization of the actin and microtubule cytoskeleton preceding pollen germination : An analysis using cultured pollen protoplasts of Lilium longiflorum.

Cultured pollen protoplasts of Lilium longiflorum Thunb. were used to study, with the aid of rhodamine-phalloidin staining and immunofluorescence, changes in the organization of actin filaments and microtubules (MTs) during cell-wall regeneration, transition of cell shape from spherical to ellipsoid, growth in cell size, and pollen-tube development. In freshly isolated protoplasts, an extensive network of minute actin filaments and disorganized MTs was observed in the central cytoplasm of the vegetative cell. After regeneration of the cell wall, cortical actin filaments and MTs gradually became organized but their orientation remained random. At 5-6 d of culture, when the protoplasts changed from a spherical to an ellipsoid shape, the cortical actin filaments were aligned almost in parallel, extending between two opposite foci, and in the growing ellipsoidal cell thick actin bundles became oriented transversely to the long axis of the cell. Following the ordering of the cortical actin filaments the cortical MTs also became arranged parallel to the actin filaments. After 8-10 d of culture, when the protoplasts germinated and formed a pollen tube, both actin filaments and MTs entered the tube. Cytochalasin B and colchicine added to the medium from the beginning of the protoplast culture inhibited the organization of the actin and MT cytoskeleton, and addition of the drugs from day 5 of culture onwards destroyed the organization and arrangement of the cytoskeleton. Disruption of the actin filaments by cytochalasin B resulted also in a disruption of the MT organization and arrangement, whereas disruption of the MTs by colchicine had little effect on the organization and arrangement of the actin filaments. Both drugs blocked pollen germination in the cultured protoplasts. From these observations it is assumed that the arrangement of actin filaments plays a predominant role in pollen germination, but that the arrangement of the MTs is also essential for pollen germination through maintaining cell polarity established by the actin arrangement.