Fig 1.
Loading of fluo-4/AM into pollen tubes under different conditions.
(a, b) Control pollen tube that was not loaded with fluo-4/AM. (c-h) Pollen tubes loaded with fluo-4/AM along with cell lysis solution for 5 min (c, d), 15 min (e, f) and 30 min (g, h). (i, j) Pollen tubes loaded for 30 min without cell lysis solution.
Fig 2.
Comparison of the influence of the cell lysis solution and pluronic F-127 on fluo-4/AM dyeing.
Fluorescence intensity using cell lysis solution as an auxiliary reagent was significantly higher (P < 0.05, Student’s t-test) compared with the values obtained using pluronic F-127. Vertical bars indicate ± SE. Each data point represents the mean of three replicates with more than 10 pollen tubes each. CLS: cell lysis solution.
Fig 3.
Effect of the cell lysis solution on the pollen tube membranes.
(a) Pollen tube dyed with FM4-64 without cell lysis solution (control). (b and d) Pollen tubes after dyeing with FM4-64 for 15 min with 1:500 (v/v) cell lysis solution in the medium (b), with 1:5 (v/v) cell lysis solution in the medium (c) and with 1:1 (v/v) cell lysis solution in the medium (d). (e) Fluorescence value comparison. The difference was not significant. Scale bars: 50 μm (a and b) and 60 μm (c and d). Vertical bars indicate ±SE. Each data point represents the mean of three replicates with more than 10 pollen tubes each.
Fig 4.
Effect of Cd2+ on the Ca2+ gradient in pollen tube apical regions.
(a, b) Results obtained using a loading time of 15 min and the cell lysis solution (control). (c, d) Results after inclusion of 50 μM Cd2+ during loading. (e) Statistical analysis of fluorescence values at the pollen tube tips (n = 15). ** corresponds to a significant difference (P < 0.01, Student’s t-test). Vertical bars indicate ± SE. Each data point represents the mean of three replicates with more than 10 pollen tubes each. CLS: cell lysis solution.
Fig 5.
Effect of La3+ on the Ca2+ gradient in the pollen tube apical regions.
(a, b) Results obtained using a loading time of 15 min and the cell lysis solution (control). (c, d) Results after inclusion of 10 μM La3+ during loading. (e) Statistical analysis of fluorescence values at the pollen tube tips (n = 10). * corresponds to a significant difference (P < 0.05, Student’s t-test). Vertical bars indicate ±SE. Each data point represents the mean of three replicates with more than 10 pollen tubes each.
Fig 6.
Effect of different concentrations of La3+ on the calcium gradient in the pollen tube apical regions.
(a) Pollen tube loaded with fluo-4/AM without La3+ (control). Pollen tubes after loading with fluo-4/AM followed by the addition of 10 μM La3+ (b), 100 μM La3+ (c), and 1 mM La3+ (d). (e) Pollen tube after loading with fluo-4/AM in the presence of 1 mM La3+. Scale bars: 100 μm (a), 90 μm (b and c), 60 μm (d) and 70 μm (e).
Fig 7.
Effect of EGTA on the Ca2+ gradient in the pollen tube apical regions.
(a) Pollen tube loaded with fluo-4/AM without EGTA (control). (b) Pollen tube after addition of 1 mM EGTA following fluo-4/AM loading. (c) Pollen tube after addition of 1 mM EGTA during fluo-4/AM loading. Scale bars: 100 μm (a), 60 μm (b) and 30 μm (c).
Fig 8.
Loading fluo-4/AM into the root hair and pollen tube of Arabidopsis.
(a) Control root hair that was not loaded with fluo-4/AM. (b) Root hair loaded with fluo-4/AM along with cell lysis solution for 15 min. (c-d) Pollen tubes loaded with fluo-4/AM along with cell lysis solution for 15 min. Note: The root hairs were barely observable in the fluorescence microscopy image because the autofluorescence of the root hairs was very weak. To see the root hairs clearly in the image, we improved the overall image brightness with Photoshop software. Therefore, the background of image (a) is different from the background of the other images (b, c and d).