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Dynamics of the apical vesicle accumulation and the rate of growth are related in individual pollen tubes

Institute of Cell and Molecular Biology, University of Edinburgh, Mayfield Road, Edinburgh, EH9 3JH, UK
^* Author for correspondence (e-mail: rparton{at}srv0.bio.ed.ac.uk )

View larger version (64K): [in a new window]   Fig. 1. FM4-64 staining distribution in a growing pollen tube of L. longiflorum. (A) Typical median focal plane confocal optical section; the bright field image at 1/3 size appears as an insert. (B) Pixel values along a central transect through the fluorescence image in (A). (C) Representative diagram of FM4-64 staining in the L. longiflorum pollen tube apex. The apical V-shaped `clear zone' of secretory vesicles defined previously (Lancelle and Hepler, 1992) on the basis of electron microscopy analysis is shown in relation to the different `zones' defined by the FM4-64 signal (see text).

View larger version (80K): [in a new window]   Fig. 2. Plasmolysis of an FM4-64-loaded L. longiflorum pollen tube. (A) 5 minutes after applying standard medium with 0.3 M sorbitol and 2 µM FM4-64. The detector sensitivity was increased to show dye fluorescence associated with Hechtian strands at the apex (arrowhead). (B) Corresponding bright field image. Bar, 15 µm.

View larger version (33K): [in a new window]   Fig. 3. (A-E) Sequence of confocal optical sections (Z-series) taken at 1.25 µm stepped focus positions through a growing FM4-64-stained L. longiflorum pollen tube. Half of the ten-mage sequence is shown from the periphery to the median focal plane. (The other five images of the sequence (not shown) were very similar reflecting the radial symmetry of the pollen tube.) Pixel intensity values along a central transect through each image are shown. Bar, 15 µm.

View larger version (100K): [in a new window]   Fig. 4. FM4-64-uptake time course in a growing L. longiflorum pollen tube. (A-J) Median confocal fluorescence images at increasing times (minutes) after addition of FM4-64 (2 µM in 115% standard medium). To avoid osmotic perturbation, pollen tubes were pretreated with 115% medium before dye application. Inserts show bright field images at 1/3 size. Bar, 20 µm.

View larger version (83K): [in a new window]   Fig. 5. Median focal plane images of FM4-64-stained L. longiflorum pollen tubes (A) 15 minutes, (B) 30 minutes, (C) 24 hours and (D) 48 hours after staining. Inserts show bright field images at 1/3 size. Bar in A,B, 15 µm; bar in C,D, 15 µm.

View larger version (120K): [in a new window]   Fig. 6. Injection of aqueous FM4-64 into a L. longiflorum pollen tube. Insert shows the bright field image at 1/3 size. (A) Tip region; (B) subapical region. Bar, 15 µm.

View larger version (95K): [in a new window]   Fig. 7. Effects of sodium azide on FM4-64 uptake by L. longiflorum pollen tubes. (A) 30 minutes after applying 2 µM dye to a tube pre-treated for 2 minutes with 500 µM sodium azide. (B) Similar treatment as in A with 1 mM sodium azide pre-treatment. Dye was applied in the continued presence of inhibitor. Inserts show bright field images at 1/3 size. Bar, 15 µm.

View larger version (77K): [in a new window]   Fig. 8. FM4-64-washout time course from a growing pollen tube of L. longiflorum preloaded with dye for 3 hours. Washout was achieved by continuous perfusion with dye-free medium. (A) Selected images showing the pattern of decline in fluorescence; times relative to the start of perfusion are given in minutes. (B) Plots of normalised fluorescence intensity within four regions, defined in A by the areas a-d: (a) extreme apex, (b) subapical region, (c) plasma membrane (plotted as c minus d) and (d) periphery. Bar, 15 µm.

View larger version (79K): [in a new window]   Fig. 9. Time course of changes in growth rate and dye distribution of FM4-64-loaded L. longiflorum pollen tubes treated with BFA. (A) Confocal fluorescence images of a pollen tube at different times (in minutes) following treatment with 3.6 µM BFA. (B) Typical growth rates for tubes treated with 0.36, 3.6, 18 and 36 µM BFA. An arrow indicates addition of BFA. Bar, 15 µm.

View larger version (108K): [in a new window]   Fig. 10. The effects of Cytochalsin D treatment on the FM4-64 staining pattern of an L. longiflorum pollen tube; times are in seconds. Bar, 15 µm.

View larger version (87K): [in a new window]   Fig. 11. Movement of FM4-64-stained material in the apex of a L. longiflorum pollen tube (average growth rate 21.9 µm/minute). (A-C) Median section confocal images taken 2 seconds apart showing bulk movement of FM4-64-stained material from a region 5 µm behind the apex away from the tip along the path of cytoplasmic streaming (arrows). (D-I) Images from the same time series, taken 1 second apart, showing movement of FM4-64-stained material from a region 25 µm subapically back towards the apex (arrows). Diagrammatic interpretation of the `reverse fountain' path of cytoplasmic streaming in a growing L. longiflorum pollen tube (J) (see main text), and of the movement of FM4-64-stained material based upon time-sequence imaging (see A-I) and FRAP experiments (see text for details) (K). Bar, 20 µm.

View larger version (93K): [in a new window]   Fig. 12. Fluorescence recovery after photobleaching (FRAP) analysis showing the direction of movement of FM4-64-stained material around the pollen tube. (A,B) Movement from the apex down the centre of the cell to more subapical regions. (A) Time sequence of confocal fluorescence images; time relative to bleaching is in seconds. The original area of bleaching is shown as the boxed area. (B) Pixel intensity plots along a central transect through the images in A. The original bleached zone is indicated as the boxed area. The relative position of the original bleached zone to the apex is shown as the dashed boxed area; graphs have been aligned from the cell apex. (C,D) Movement from subapical peripheral regions into the apex. (C) Time sequence of confocal fluorescence images; the first image indicates the lateral bleaching areas (box a). (D) Graphs of fluorescence intensity against time for the areas indicated a-c. The relative position of the sample areas to the apex, shown in the first image of (C), was maintained throughout. The grey shaded area indicates time of bleaching. Bars, 15 µm.

View larger version (49K): [in a new window]   Fig. 13. Quantitative analysis of the relationship between FM4-64 staining and oscillatory growth of a L. longiflorum pollen tube. (A) Growth rate for a pollen tube derived from confocal images of FM4-64 fluorescence. (B-D) Corresponding plots of average pixel value against time for the regions A-C defined in diagram (E). Heavy dashed lines show peak growth rate; grey boxed areas show the times when the apical 3-5 µm region is most distinctly defined from the more subapical FM4-64 staining (see Results and F). (E) Sample areas A-C: apical 1-3 µm, 5 µm and 10 µm subapically, respectively. (F) Graphs of pixel values along a central transect of the pollen tube are shown (as in Fig. 1) for images covering the growth period outlined by the cross-hatched area in A. The maximum and minimum rate growth phases of the period are indicated.

View larger version (56K): [in a new window]   Fig. 14. FM4-64 staining pattern of a L. longiflorum pollen tube grown under restrictive growth conditions. Confocal images of FM4-64 fluorescence are shown with corresponding bright-field images. (A) 15% sucrose medium (growth 8.7 µm/minute). (B) 25 mM calcium medium (growth <1 µm/minute), note the thickened apical cell wall. (C) Growth on 15 mM calcium medium (4.6 µm/minute). Bars, 15 µm.

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