Fluorescent ratiometric imaging of Lilium longiflorum pollen tubes loaded with the Ca{dollar}sp{lcub}2+{rcub}{dollar} indicator fura-2 dextran has revealed a tip-focused gradient of free intracellular calcium ions in actively growing pollen tubes which extends from above 3.0 {dollar}mu{dollar}M at the apex to a uniform basal level of {dollar}sim{dollar}0.2 {dollar}mu{dollar}M within 20 {dollar}mu{dollar}m from the tip. Application of the Ca{dollar}sp{lcub}2+{rcub}{dollar} specific vibrating electrode revealed a tip-directed extracellular Ca{dollar}sp{lcub}2+{rcub}{dollar} influx between 1.4 and 14 pmol cm{dollar}sp{lcub}-2{rcub}{dollar} sec{dollar}sp{lcub}-1{rcub}{dollar}. The relationship between these phenomena and their role in tube growth was examined using different 1,2-bis (o-aminophenoxy)ethane {dollar}N,N,Nsp1,Nsp1{dollar}-tetraacetic acid (BAPTA)-type buffers, and hypertonic media. Injection of active BAPTA-type buffers or application of elevated sucrose levels reversibly inhibited growth, destroyed tip zonation of organelles, and modified normal patterns of cytoplasmic streaming. Simultaneously, these treatments dissipated both the intracellular tip-focused gradient and the extracellular Ca{dollar}sp{lcub}2+{rcub}{dollar} flux providing evidence that growing pollen tubes have open Ca{dollar}sp{lcub}2+{rcub}{dollar} channels in their tip which become inactivated in nongrowing tubes. The elevated sucrose studies support the view that stretching of the apical plasma membrane contributes to the maintenance of the Ca{dollar}sp{lcub}2+{rcub}{dollar} signal.; The presence and localization of actin and myosin have been examined in Lilium longiflorum and Nicotiana alata pollen tubes in order to determine their roles in pollen tube growth. Immunoblot analysis of pollen tube extracts with antibodies to actin, myosins IA and IB, myosin II, and myosin V reveals the presence of these contractile proteins. Immunofluorescence microscopy reaffirmed longitudinal actin localization. Myosin I was localized to the plasma membrane, larger organelles, the surface of the generative cell and the vegetative nucleus. Myosin V and Myosin II were found in the vegetative cytoplasm in a punctate fashion representing smaller and larger organelles, respectively. These results suggest the presence of three classes of myosins in pollen and lead to the following hypothesis: Myosin I may move the generative cell and vegetative nucleus unidirectionally toward the tip, while myosin V moves the smaller organelles and myosins I and II move the larger organelles (bidirectionally).
展开▼
