Ca²⁺ imaging at a high spatiotemporal resolution in fast-growing Arabidopsis pollen tubes reveals fast and regular cytosolic Ca²⁺ oscillations and a unique pattern of Ca²⁺ influx on the apical PM. A and B, Time-lapse images (A) and kymograph along the central axis (B) of a fast-growing pollen tube expressing the cytosolic Ca²⁺ sensor CamelliA_CytR. C and D, Time course of the fluorescence intensity of the CamelliA_CytR in the tip region (6 µm from apex to the shank) of the pollen tube (C) and spectral analysis of the Ca²⁺ oscillations (D). E, Oscillation periods of the cytosolic Ca²⁺ (Cyt) and PM Ca²⁺ influx (PM) are plotted in the scatter plot. The mean ± 95% confidence interval is also shown. F, Time-lapse images of a fast-growing pollen tube expressing the PM-anchored Ca²⁺ sensor CamelliA_PmG. G, Kymograph of the CamelliA_PmG fluorescence along the growing PM region of 10 µm on each side of the apex (illustrated in the upper panel) by constantly tracking and analyzing the growing tip using the TipQAD algorithm. H and I, Time course of the fluorescence intensity of the CamelliA_PmG in the apical PM region (10 µm on each side of the apex) of the pollen tube (H) and spectral analysis of the Ca²⁺ oscillations (I). J, Kymographs of CamelliA_PmG (upper left), CamelliA_CytR (middle left), and the merged kymograph (lower left) were acquired from a simultaneous observation of both Ca²⁺ sensors in the same pollen tube. ROIs for generating the kymographs are indicated on the right side of each kymograph. K and L, Time course of the fluorescence intensity (K) and cross-correlation analyses (L) of the CamelliA_CytR and CamelliA_PmG observed simultaneously in the same pollen tube. The green and purple dotted lines and arrows highlight two events of sub-PM Ca²⁺ reaching the peak ahead of the cytosolic Ca²⁺. M, Distribution of the time delay between cytosolic and sub-PM Ca²⁺ reaching the peak within each oscillation cycle (data from five independent pollen tubes). N, Model of the Ca²⁺ influx across the apical PM, where the Ca²⁺ influx was initiated at the apex then expanded exclusively to the whole apical PM with stronger activity in the region surrounding the apex. Increases in fluorescence intensity indicate increases in Ca²⁺ concentration. Representative data from at least eight independent biological repeats were shown. a.u., arbitrary unit. Scale bars are 10 µm in A, F.