To verify the role of ion uptake in eATP-promoted PG and PTG, Arabidopsis thaliana pollen was germinated in vitro, and the effects of apyrase or ATP addition on PG and PTG were investigated. In the basic medium, which contained 0.1 mM KCl, apyrase inhibited PG and PTG significantly. After 100 units/mL apyrase treatment, the pollen germination rate decreased from 39.2 ± 2.5% to 26.7 ± 3.4%, and the pollen tube length decreased from 16.9 ± 2.4 μm to 10.3 ± 1.8 μm (p < 0.05). Control treatment with heat-denatured apyrase or bovine serum albumin (BSA) did not affect PG or PTG (p > 0.05) (Figure 2A,D). At low K⁺ concentrations (0.02–0.1 mM), ATP supplementation (0.1 mM) promoted PG and PTG significantly, while at high K⁺ concentration (1.0 mM), ATP supplementation slightly inhibited PG but did not affect PTG (Figure 2B,E). To confirm that eATP supplementation promotes PG and PTG by increasing K⁺ uptake, 1 mM of the K⁺ channel blockers Ba²⁺, Cs⁺, or TEA (tetraethylammonium) was added into the basic medium containing 0.1 mM KCl. In these conditions, the effects of eATP on PG or PTG were significantly suppressed (p < 0.01) (Figure 2C,F).

Figure 2. eATP regulates the PG and PTG of Arabidopsis thaliana by facilitating K⁺ uptake. Apyrase-inhibited PG (A) and PTG (D), ATP-regulated PG (B), and PTG (E) in medium containing serial concentrations of KCl. K⁺ channel blockers (1 mM) inhibited ATP-promoted PG (C) and PTG (F). In (A–F), the K⁺ concentration in the medium is 0.1 mM. In each experiment, at least 300 pollen grains were counted to obtain PG, and at least 150 pollen tubes were measured to obtain the pollen tube length. Data from 3 replicates were combined to obtain the mean ± SD. Student’s t test p values: * p < 0.05, ** p < 0.01.

To verify the role of Ca²⁺ in eATP-promoted PG and PTG, 0.1 mM ATP was added to medium containing EGTA, series of concentrations of Ca²⁺, or Ca²⁺ channel blockers. In 0.5 mM EGTA-containing medium, PG and PTG were significantly suppressed, and ATP supplementation did not have any effect. In medium containing 0.1 mM CaCl₂, PG and PTG were both stimulated by 0.1 mM ATP. In medium containing 1 mM CaCl₂, pollen tube growth was markedly stimulated, while pollen germination was suppressed by 0.1 mM ATP. In medium containing 0.1 mM Ca²⁺-permeable channel blockers (Gd³⁺ or La³⁺), resting PG and PTG were both suppressed, and added ATP did not have any effect on PG and PTG (Figure 3). These data indicate that eATP may promote PG and PTG by facilitating Ca²⁺ intake.

Figure 3. eATP regulates PG and PTG of Arabidopsis thaliana by facilitating Ca²⁺ uptake. ATP (0.1 mM) regulates PG (A) and PTG (B) in medium containing a Ca²⁺ chelator, serial concentrations of Ca²⁺, and channel blockers (0.1 mM). In each experiment, at least 300 pollen grains were counted to obtain PG, and at least 150 pollen tubes were measured to obtain the pollen tube length. Data from 3 replicates were combined to obtain the mean ± SD. Student’s t test p values: * p < 0.05, ** p < 0.01.

To verify the role of ATP hydrolytes in PG and PTG, 0.1 mM ADP, AMP, adenosine, adenine, or phosphate (Pi) was added to the medium containing 0.1 mM KCl or CaCl₂. PG and PTG were not affected by these reagents in either KCl or CaCl₂ medium (Figure S1A,C). To verify the effect of various ATP salts on PG and PTG, 0.1 mM ATP sodium, Tris, or magnesium salt was added to the medium containing 0.1 mM KCl or CaCl₂. PG and PTG were significantly promoted by the three ATP salts (p < 0.05) in both the KCl and CaCl₂ media (Figure S1B–D). To ensure that ATP acts as a signal rather than an energy carrier, 0.1 mM ATPγS, a weakly-hydrolyzable ATP analog, was added to the medium containing either 0.1 mM KCl or CaCl₂. PG and PTG were significantly promoted by ATPγS (p < 0.05) in the KCl and CaCl₂ media (Figure S1B–D).

4. ATP Stimulates K⁺ and Ca²⁺ Influx in Arabidopsis thaliana Pollen Protoplast

To confirm that eATP stimulates K⁺ uptake in pollen grains, whole-cell patch clamping was performed to detect the effect of eATP on K⁺ conductance in the PM. In pollen protoplasts, a time-dependent inward-rectifying K⁺ current was recorded at a negative voltage of more than −140 mV. The addition of 1 mM CsCl significantly suppressed the current intensity, confirming that the inward currents may be carried by K⁺ influx (Figure 4A). The addition of 0.1 mM ATP stimulated the inward K⁺ current: the maximum current intensity increased from −129 ± 32 pA to −254 ± 34 pA at −200 mV (n = 7, p < 0.05), and the open potential shifted to more positive (Figure 4B). In CsCl-pretreated protoplasts, the effect of ATP on the inward K⁺ currents was markedly suppressed. The maximum current intensity decreased from −270 ± 35 pA to −83 ± 18 pA at −200 mV (n = 7, p < 0.05) (Figure 4C).

Figure 4. CsCl inhibits eATP-stimulated K⁺ influx in the protoplasts of Arabidopsis pollen grains. (A) CsCl (1 mM) inhibited resting and 0.1 mM ATP-stimulated inward K⁺ currents. Representative current traces from step-voltage clamping are shown. (B,C) I-V relationship curves of inward K⁺ currents (n = 7). In each experiment, data from 7 protoplasts were recorded and combined to obtain the mean ± SD.

To further confirm the promotion of eATP on the Ca²⁺ uptake of pollen grains, the effect of 0.1 mM ATP on inward Ca²⁺ conductance was investigated. The addition of 0.1 mM ATP strongly promoted Ca²⁺ influx in pollen protoplasts: the maximum inward current intensity at −200 mV increased from −147.6 ± 21.9 pA to −243.1 ± 22.8 pA (n = 7, p < 0.05) (Figure 5A,B). GdCl₃ (1 mM) significantly suppressed Ca²⁺ conductance (Figure 5A,C, n = 7, p < 0.05). In Gd³⁺-pretreated pollen protoplasts, ATP did not stimulate Ca²⁺ inward conductance (Figure 5A,C, n = 7, p > 0.05).

Figure 5. GdCl₃ inhibits eATP-stimulated Ca²⁺ influx in the protoplasts of Arabidopsis pollen grain. (A) GdCl₃ (0.1 mM) inhibited resting and 0.1 mM ATP-stimulated inward Ca²⁺ currents. Representative current traces from step-voltage clamping are shown. (B,C) I-V relationship curves of the inward Ca²⁺ currents (n = 7). In each experiment, data from 7 protoplasts were recorded and combined to obtain the mean ± SD.