Figure 4.

ALA reduces cytosolic Ca²⁺ concentration and tip-focused Ca²⁺ gradient in pollens. Pollens were evenly placed in petri dishes containing basal medium alone (A₁), or with 10 mg/L ALA (A₂), 10 mg/L ALA + 10⁻³ M Ca²⁺ (A₃), 10 mg/L ALA + 10⁻³ M Ca²⁺ + 10⁻³ M EGTA (A₄), and incubated at 25°C at 100% humidity, in the dark for 1 h. Then pollen solutions were loaded with 1 μM Fluo-3 AM (dissolved in DMSO) for 2 h in darkness at 4°C, then excess dye was removed. The fluorescence of the above-treated pollens (A) was observed using a laser scanning confocal microscope (Carl Zesis 780, LSCM) and Time-course and Photoshop software. For each treatment, the first picture is bright field image and the following are fluorescence images corresponding to the bright field image at 5 min intervals. Scale bar: 30 μm. (B) Time course changes of the relative fluorescence in the middle (B₁) and the tip (B₂) of pollen tubes. (C) Time course changes of the fluorescence intensity gradient between the tip and the middle pollen tube. Values are the means of 15 measurements ± SE from three independent experiments.