P2K1 Receptor, Heterotrimeric Gα Protein and CNGC2/4 Are Involved in Extracellular ATP-Promoted Ion Influx in the Pollen of Arabidopsis thaliana

As an apoplastic signal, extracellular ATP (eATP) is involved in plant growth and development. eATP promotes tobacco pollen germination (PG) and pollen tube growth (PTG) by stimulating Ca2+ or K+ absorption. Nevertheless, the mechanisms underlying eATP-stimulated ion uptake and their role in PG and PTG are still unclear. Here, ATP addition was found to modulate PG and PTG in 34 plant species and showed a promoting effect in most of these species. Furthermore, by using Arabidopsis thaliana as a model, the role of several signaling components involved in eATP-promoted ion (Ca2+, K+) uptake, PG, and PTG were investigated. ATP stimulated while apyrase inhibited PG and PTG. Patch-clamping results showed that ATP promoted K+ and Ca2+ influx into pollen protoplasts. In loss-of-function mutants of P2K1 (dorn1-1 and dorn1-3), heterotrimeric G protein α subunit (gpa1-1, gpa1-2), or cyclic nucleotide gated ion channel (cngc2, cngc4), eATP-stimulated PG, PTG, and ion influx were all impaired. Our results suggest that these signaling components may be involved in eATP-promoted PG and PTG by regulating Ca2+ or K+ influx in Arabidopsis pollen grains.


Introduction
The apoplast, including the cell wall and intercellular space, plays essential roles in modulating plant cell growth and development due to the existence of numerous signaling molecules within the apoplastic matrix. In recent years, growing evidence has shown that adenosine triphosphate (ATP) is present in the apoplast and participates in physiological processes such as vegetative growth, development, and stress response [1][2][3][4]. eATP is involved in maintaining cell viability [5] and the growth rate of cultured cells [6], regulating the rate or direction of the growth of roots [7], hypocotyls [8,9], and cotton fibers [10]. It is also involved in modulating stomatal movement [11][12][13]. Some stresses induce eATP release as a "danger signal", which in turn induces resistance responses to disease, hypotonic conditions, high salinity, and cold stress [14][15][16][17][18].

ATP Addition Impacts PG and PTG in Pollens from 34 Species
To verify the universality of eATP-regulated PG and PTG, we examined the effects of 0.1 mM ATP on PG and PTG of pollen grains from 34 species. Pollen germination rates increased significantly in 28 species (p < 0.05) and decreased significantly in 6 species (p < 0.05). Pollen tube length measurement results showed that tube growth was significantly promoted in 19 species (p < 0.05) and significantly inhibited in 5 species, while no effect was observed in 10 species (Figure 1).  Table 1. PG (A) and PTG (B) were noted. In each experiment, at least 300 pollen grains or 150 pollen tubes were counted or measured. Data from 3 replicates were calculated to obtain mean ± SD. Student's t-test p values: * p < 0.05, ** p < 0.01.  Table 1. PG (A) and PTG (B) were noted. In each experiment, at least 300 pollen grains or 150 pollen tubes were counted or measured. Data from 3 replicates were calculated to obtain mean ± SD. Student's t-test p values: * p < 0.05, ** p < 0.01. 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 Ba 2+ , 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).
To verify the role of Ca 2+ in eATP-promoted PG and PTG, 0.1 mM ATP was added to medium containing EGTA, series of concentrations of Ca 2+ , or Ca 2+ 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 CaCl 2 , PG and PTG were both stimulated by 0.1 mM ATP. In medium containing 1 mM CaCl 2 , pollen tube growth was markedly stimulated, while pollen germination was suppressed by 0.1 mM ATP. In medium containing 0.1 mM Ca 2+ -permeable channel blockers (Gd 3+ or La 3+ ), 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 Ca 2+ intake. 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 Ca 2+ in eATP-promoted PG and PTG, 0.1 mM ATP was added to medium containing EGTA, series of concentrations of Ca 2+ , or Ca 2+ 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 Ca 2+ -permeable channel blockers (Gd 3+ or La 3+ ), 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 Ca 2+ intake.
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). 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.  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.

ATP Stimulates K + and Ca 2+ 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 + Figure 3. eATP regulates PG and PTG of Arabidopsis thaliana by facilitating Ca 2+ uptake. ATP (0.1 mM) regulates PG (A) and PTG (B) in medium containing a Ca 2+ chelator, serial concentrations of Ca 2+ , 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 CaCl 2 . PG and PTG were not affected by these reagents in either KCl or CaCl 2 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 CaCl 2 . PG and PTG were significantly promoted by the three ATP salts (p < 0.05) in both the KCl and CaCl 2 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 CaCl 2 . PG and PTG were significantly promoted by ATPγS (p < 0.05) in the KCl and CaCl 2 media (Figure S1B-D).

ATP Stimulates K + and Ca 2+ 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). 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.

ATP Stimulates K + and Ca 2+ 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).  To further confirm the promotion of eATP on the Ca 2+ uptake of pollen grains, the effect of 0.1 mM ATP on inward Ca 2+ conductance was investigated. The addition of 0.1 mM ATP strongly promoted Ca 2+ 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). GdCl 3 (1 mM) significantly suppressed Ca 2+ conductance ( Figure 5A,C, n = 7, p < 0.05). In Gd 3+ -pretreated pollen protoplasts, ATP did not stimulate Ca 2+ inward conductance ( Figure 5A,C, n = 7, p > 0.05). effect of 0.1 mM ATP on inward Ca 2+ conductance was investigated. The addition of 0.1 mM ATP strongly promoted Ca 2+ 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 Ca 2+ conductance ( Figure  5A,C, n = 7, p < 0.05). In Gd 3+ -pretreated pollen protoplasts, ATP did not stimulate Ca 2+ inward conductance ( Figure 5A,C, n = 7, p > 0.05). . In each experiment, data from 7 protoplasts were recorded and combined to obtain the mean ± SD.

P2K1 Receptor, Heterotrimeric G Protein α Subunit and Two CNGCs Are Involved in eATP-Regulated PG and PTG by Modulating K + and Ca 2+ Influx
To verify the role of P2K1 receptor in eATP-regulated PG and PTG, pollen grains of two P2K1 receptor (DORN1) null mutants, dorn1-1 and dorn1-3, were germinated in vitro. In 0.1 mM KCl-containing medium, 0.1 mM ATP addition did not affect the PG or PTG of dorn1-1 nor dorn1-3 mutants ( Figure 6A,B). In 0.1 mM CaCl2-containing medium, ATP addition inhibited the PG of the two mutants but did not affect PTG in either ( Figure  6E,F). In the pollen protoplasts of dorn1-1 and dorn1-3 mutants, K + influx conductance was similar and exhibited weaker current intensity than wildtype pollen protoplasts. The addition of 0.1 mM ATP did not stimulate K + conductance ( Figure 6C,D). Ca 2+ current intensity in pollen protoplasts of both mutants was weaker than that in wildtype, and the addition of 0.1 mM ATP did not stimulate Ca 2+ conductance in either mutant ( Figure  6G,H). . In each experiment, data from 7 protoplasts were recorded and combined to obtain the mean ± SD.

P2K1 Receptor, Heterotrimeric G Protein α Subunit and Two CNGCs Are Involved in eATP-Regulated PG and PTG by Modulating K + and Ca 2+ Influx
To verify the role of P2K1 receptor in eATP-regulated PG and PTG, pollen grains of two P2K1 receptor (DORN1) null mutants, dorn1-1 and dorn1-3, were germinated in vitro. In 0.1 mM KCl-containing medium, 0.1 mM ATP addition did not affect the PG or PTG of dorn1-1 nor dorn1-3 mutants ( Figure 6A,B). In 0.1 mM CaCl 2 -containing medium, ATP addition inhibited the PG of the two mutants but did not affect PTG in either ( Figure 6E,F). In the pollen protoplasts of dorn1-1 and dorn1-3 mutants, K + influx conductance was similar and exhibited weaker current intensity than wildtype pollen protoplasts. The addition of 0.1 mM ATP did not stimulate K + conductance ( Figure 6C,D). Ca 2+ current intensity in pollen protoplasts of both mutants was weaker than that in wildtype, and the addition of 0.1 mM ATP did not stimulate Ca 2+ conductance in either mutant ( Figure 6G,H). . In the pollen germination experiment, at least 300 pollen grains or 150 pollen were counted or measured; data from 3 replicates were combined to obtain the mean ± SD. Student's t test p values: * p < 0.05, ** p < 0.01. In the patch clamp experiment, data from 7 pollen protoplasts were recorded and combined to obtain the mean ± SD.
To verify the role of heterotrimeric G protein in eATP-regulated PG and PTG, pollen grains of two heterotrimeric Gα subunit null mutants, gpa1-1 and gpa1-2, were germinated in vitro. In 0.1 mM KCl or CaCl2 containing medium, the addition of 0.1 mM ATP did not promote PG or PTG in gpa1-1 and gpa1-2 mutants ( Figure 7A-F). In pollen protoplasts of gpa1-1 and gpa1-2 mutants, K + influx conductance was similar to that of wildtype pollen protoplasts. The addition of 0.1 mM ATP did not stimulate K + conductance. Ca 2+ current intensity in the pollen protoplasts of both mutants was weaker than that in wildtype, and the addition of 0.1 mM ATP did not stimulate Ca 2+ conductance in either mutant ( Figure 7C-H). . In the pollen germination experiment, at least 300 pollen grains or 150 pollen were counted or measured; data from 3 replicates were combined to obtain the mean ± SD. Student's t test p values: * p < 0.05, ** p < 0.01. In the patch clamp experiment, data from 7 pollen protoplasts were recorded and combined to obtain the mean ± SD.
To verify the role of heterotrimeric G protein in eATP-regulated PG and PTG, pollen grains of two heterotrimeric Gα subunit null mutants, gpa1-1 and gpa1-2, were germinated in vitro. In 0.1 mM KCl or CaCl 2 containing medium, the addition of 0.1 mM ATP did not promote PG or PTG in gpa1-1 and gpa1-2 mutants ( Figure 7A-F). In pollen protoplasts of gpa1-1 and gpa1-2 mutants, K + influx conductance was similar to that of wildtype pollen protoplasts. The addition of 0.1 mM ATP did not stimulate K + conductance. Ca 2+ current intensity in the pollen protoplasts of both mutants was weaker than that in wildtype, and the addition of 0.1 mM ATP did not stimulate Ca 2+ conductance in either mutant ( Figure 7C-H . In the pollen germination experiment, at least 300 pollen grains or 150 pollen were counted or measured; data from 3 replicates were combined to obtain the mean ± SD. Student's t test p values: * p < 0.05. In the patch clamp experiment, data from at least 7 pollen protoplasts were recorded and combined to obtain the mean ± SD. To verify the role of CNGC in eATP-regulated PG and PTG, pollen grains of two CNGC null mutants, cngc2 and cngc4, were germinated in vitro. In 0.1 mM KCl-or CaCl2-containing medium, the addition of 0.1 mM ATP did not stimulate PG or PTG in cngc2 or cngc4 mutants, while in 0.1 mM CaCl2-containing medium, PTG in cngc2 mutants was suppressed by ATP ( Figure 8A-F). In the pollen protoplasts of cngc2 and cngc4 mutants, K + influx conductance was weaker than in wildtype pollen protoplasts, and the addition of 0.1 mM ATP did not stimulate K + conductance. Ca 2+ current intensity in pollen protoplasts of both mutants was weaker than in wildtype, and the addition of 0.1 mM ATP did not stimulate Ca 2+ conductance in either mutant ( Figure 8C-H). . In the pollen germination experiment, at least 300 pollen grains or 150 pollen were counted or measured; data from 3 replicates were combined to obtain the mean ± SD. Student's t test p values: * p < 0.05. In the patch clamp experiment, data from at least 7 pollen protoplasts were recorded and combined to obtain the mean ± SD.
To verify the role of CNGC in eATP-regulated PG and PTG, pollen grains of two CNGC null mutants, cngc2 and cngc4, were germinated in vitro. In 0.1 mM KCl-or CaCl 2containing medium, the addition of 0.1 mM ATP did not stimulate PG or PTG in cngc2 or cngc4 mutants, while in 0.1 mM CaCl 2 -containing medium, PTG in cngc2 mutants was suppressed by ATP ( Figure 8A-F). In the pollen protoplasts of cngc2 and cngc4 mutants, K + influx conductance was weaker than in wildtype pollen protoplasts, and the addition of 0.1 mM ATP did not stimulate K + conductance. Ca 2+ current intensity in pollen protoplasts of both mutants was weaker than in wildtype, and the addition of 0.1 mM ATP did not stimulate Ca 2+ conductance in either mutant ( Figure 8C-H . In the pollen germination experiment, at least 300 pollen grains or 150 pollen were counted or measured; data from 3 replicates were combined to obtain the mean ± SD. Student's t test p values: * p < 0.05, ** p < 0.01. In the patch clamp experiment, data from at least 7 pollen protoplasts were recorded and combined to obtain the mean ± SD.

eATP Regulates PG and PTG in Dozens of Plant Species
eATP plays a role in vegetative and reproductive tissue cells in plants. The addition of mM levels of ATP inhibited PG and PTG [64,65], while low concentrations of eATP stimulated PG and PTG [63,66]. Here, the responses of 34 plant species in terms of PG and PTG pointed to a dual effect of eATP: eATP had a positive effect on the pollen of most plant species, while had an inhibitory effect on the pollen of a few other species. Such a dual regulatory effect of eATP had been noticed in various plant systems [2,15,67]. The dual effect might result from the sensitivity of pollen to eATP. In most species, PG and PTG were promoted or inhibited simultaneously. Nevertheless, the responses of PG and PTG to ATP were not consistent. PG in Paeonia lactiflora, Magnolia denudata, Robinia pseudoacacia, Clivia miniata, and Pinus tabuliformis was promoted by eATP, but PTG did not change markedly. PG in Jasminum nudiflorum, Malus halliana, and Orychophragmus violaceus was promoted by ATP, whereas PTG was inhibited. For Kolkwitzia amabilis and Cotoneaster horizontalis, PG was inhibited by ATP, but PTG was not affected. These results revealed the universality of eATP's regulatory effect on PG and PTG. Further research into the mechanism of eATP-regulated PG or PTG will shed light on the mechanisms that regulate plant sexual reproduction. . In the pollen germination experiment, at least 300 pollen grains or 150 pollen were counted or measured; data from 3 replicates were combined to obtain the mean ± SD. Student's t test p values: * p < 0.05, ** p < 0.01. In the patch clamp experiment, data from at least 7 pollen protoplasts were recorded and combined to obtain the mean ± SD.

eATP Regulates PG and PTG in Dozens of Plant Species
eATP plays a role in vegetative and reproductive tissue cells in plants. The addition of mM levels of ATP inhibited PG and PTG [64,65], while low concentrations of eATP stimulated PG and PTG [63,66]. Here, the responses of 34 plant species in terms of PG and PTG pointed to a dual effect of eATP: eATP had a positive effect on the pollen of most plant species, while had an inhibitory effect on the pollen of a few other species. Such a dual regulatory effect of eATP had been noticed in various plant systems [2,15,67]. The dual effect might result from the sensitivity of pollen to eATP. In most species, PG and PTG were promoted or inhibited simultaneously. Nevertheless, the responses of PG and PTG to ATP were not consistent. PG in Paeonia lactiflora, Magnolia denudata, Robinia pseudoacacia, Clivia miniata, and Pinus tabuliformis was promoted by eATP, but PTG did not change markedly. PG in Jasminum nudiflorum, Malus halliana, and Orychophragmus violaceus was promoted by ATP, whereas PTG was inhibited. For Kolkwitzia amabilis and Cotoneaster horizontalis, PG was inhibited by ATP, but PTG was not affected. These results revealed the universality of eATP's regulatory effect on PG and PTG. Further research into the mechanism of eATP-regulated PG or PTG will shed light on the mechanisms that regulate plant sexual reproduction.

ATP Regulates PG and PTG of Arabidopsis thaliana
It had been reported that eATP regulates PG and PTG in Picea meyeri [66] and Nicotiana tabacum [63]. However, null mutants of eATP signaling components are difficult to generate in these species. Here, Arabidopsis pollen grains were used to verify the role of eATP in PG and PTG, and several mutants of eATP signaling components were used to clarify the role of these molecules in PG, PTG, and ion influx.
The addition of apyrase inhibited PG and PTG (Figure 2), indicating that endogenous eATP may be involved in maintaining PG and PTG. Endogenous eATP may be secreted during pollen development. The addition of ATP stimulated PG and PTG, indicating that endogenous eATP might be not sufficient and that PG and PTG can be promoted by increasing eATP level. Our finding that the addition of high levels of ATP inhibited the PG and PTG of Arabidopsis thaliana is in agreement with previous reports [64,65]. These reports used a high concentration of ATP (>1 mM), compared to the lower concentration of 0.1 mM ATP added here, which stimulated PG and PTG. These contrasting results indicate that, in a certain concentration range, eATP acts as a positive regulator. ATP hydrolytes, including ADP, AMP, and adenosine, did not affect PG or PTG, indicating that ATP hydrolysis is unnecessary for this regulatory effect. The addition of ADP stimulated some reactions that are evoked by eATP [3,23,25,68,69]. In our previous work, ADP was found to promote the PG and PTG of tobacco [63]. The results presented here indicate that Arabidopsis thaliana pollen may respond to ATP differently than tobacco pollen. A weakly hydrolyzable ATP analog, ATPγS, promoted PG and PTG of Arabidopsis thaliana similarly to ATP, confirming that ATP may function in its intact form as a signaling molecule. Various ATP salts showed similar positive effects on PG and PTG ( Figure S1), indicating that the cations in these salts did not affect PG or PTG.

K + /Ca 2+ Influx Mediates ATP Regulation of PG and PTG
In our previous work, we demonstrated the existence of eATP-regulated K + and Ca 2+ uptake in tobacco pollen [63]. Here, we found that K + and Ca 2+ uptake is necessary for resting or eATP-stimulated PG and PTG in Arabidopsis thaliana. Similar to the results in tobacco pollen, we found that ATP promoted PG and PTG in medium containing low concentrations of K + or Ca 2+ and inhibited PG and PTG in medium containing high concentrations of K + or Ca 2+ . In medium containing 1 mM CaCl 2 , the pollen tube growth of tobacco was inhibited, while the pollen tube growth of Arabidopsis was strongly promoted ( Figure 3B). In tobacco pollen, 0.1 mM Ca 2+ channel blockers weakly suppressed eATPpromoted ion influx and PG and PTG, while in Arabidopsis thaliana, 0.1 mM Ca 2+ channel blockers almost entirely blocked ATP-induced Ca 2+ influx and PG and PTG. These results indicate that K + and Ca 2+ influx through ion channels may be a central event in PG and PTG, that endogenous eATP is necessary for maintaining resting ion uptake, that apyrase decreases eATP levels, and that decreased ion influx might explain apyrase-inhibited PG and PTG. The positive effects of ATP-supplementation on PG and PTG may partially result from increased ion absorption. The proper influx and accumulation of ions is essential for PG and PTG, while the overload of ions suppresses PG and PTG [32,33,37,40,45]. The data presented here suggest that, when eATP or ion concentrations are above normal levels, the overload of K + or/and Ca 2+ influx may inhibit PG and PTG.

Signaling Underlying ATP-Regulated PG and PTG of Arabidopsis thaliana
P2K1 receptor DORN1 was the first reported eATP receptor and is involved in plant immune and stress responses [13,14,21]. However, the role of the P2K1 receptor in sexual reproduction has not been investigated, although DORN1 is expressed in flowers ( Figure S2). Results here showed that DORN1 is involved in PG and PTG. In 0.1 mM KCl-containing medium, the resting pollen germination rate and pollen tube length were similar between wildtype and both DORN1-null mutants, while in 0.1 mM CaCl 2 -containing medium, the pollen germination rate of both mutants was significantly lower than in wildtype, indicating that DORN1 is necessary for maintaining Ca 2+ uptake and resting pollen germination, possibly through interactions with endogenous eATP. Patch clamping showed that resting K + and Ca 2+ current intensities were weaker than those in wildtype, demonstrating a role of DORN1 in maintaining the resting ion influx. Added ATP did not promote PG, PTG, or ion influx in dorn1-1 and dorn1-3, indicating that DORN1 plays a key role in ATP-regulated PG and PTG through regulating K + /Ca 2+ influx in pollen cells. DORN1 is involved in eATP-regulated intracellular Ca 2+ concentration elevation in Arabidopsis root cells [19,26,70,71]. The results here provided new evidence for a role for DORN1 in eATP-evoked Ca 2+ signaling in reproductive cells as well. Heterotrimer G protein is involved in eATP-regulated root growth and stomatal movement and acts by regulating Ca 2+ influx [3,12,22,72,73]. Heterotrimeric G protein-regulated PG, PTG, and Ca 2+ influx in Arabidopsis thaliana was reported in our previous work [61]. Nevertheless, the upstream regulator that governs the heterotrimeric G protein is still unknown. The data presented here indicates that eATP may be an upstream regulator of heterotrimeric G protein in pollen cells, suggesting that eATP-stimulated G protein activation may play similar roles in vegetable and reproductive growth. The resting pollen germination rate and pollen tube length of gpa1-1 and gpa1-2 mutants were lower than those in wildtype, and the addition of ATP did not promote PG or PTG in either mutant, indicating that Gα may play a key role in eATP-regulated PG and PTG. In the protoplasts of both null mutants, resting K + or Ca 2+ current intensities were slightly weaker than those in wildtype, and the addition of ATP did not stimulate K + or Ca 2+ conductance, indicating that Gα may be involved in eATP-regulated ion influx. These data provide new evidence that heterotrimeric G protein is involved in eATP-regulated PG and PTG through regulating K + or Ca 2+ influx.
As channels permeable to divalent cations and K + , CNGCs had been reported to participate in key processes involved in plant growth, development, and stress responses, including apical growth, the vegetative development of plants, sexual reproduction, immunity, response to drought, and temperature stress [26,[74][75][76][77]. CNGC2 and CNGC4 form ion channels by heterodimerization in Arabidopsis thaliana, after which they mediate ion influx and function in plant growth and pathogen defense [78][79][80]. In our preliminary experiments, the responses of pollen from several CNGC null mutants to ATP supplementation were investigated. Most mutants responded to eATP similarly to wildtype (data not shown). Only mutants for CNGC2 and CNGC4 responded to eATP different from Col-0 pollen. The resting pollen germination rates of cngc2 or cngc4 mutants were lower than that of wildtype, indicating that ion channels containing CNGC2 and CNGC4 are essential for maintaining Ca 2+ and K + uptake. In pollen grains from both mutants, the addition of ATP did not promote PG or PTG, indicating that CNGC2 and CNGC4 may play key roles in eATP-stimulated PG and PTG. Patch clamping data showed that resting K + and Ca 2+ conductance in mutants was weaker than in wildtype, confirming that resting ion influx may be mediated by ion channels containing CNGC2 and CNGC4. The addition of ATP did not stimulate K + or Ca 2+ conductance in the pollen protoplasts of cngc2 or cngc4 mutants, indicating that eATP-stimulated ion influx may be mediated by CNGC2/CNGC4containing ion channels. Single null mutants of CNGC2 or CNGC4 showed impaired resting and eATP-stimulated ion influx, indicating that the heterodimerization of these two proteins is necessary for the construction of functional ion channels. It was reported that CNGC18 and CNGC16 are involved in the pollen development, germination and tube growth in Arabidopsis thaliana [59,81,82], whether these CNGCs are involved in eATP signaling need to be further investigated.
Our results confirmed which components are involved in eATP-stimulated PG, PTG, and ion flux and provided new clues to help elucidate the mechanisms of eATP-regulated reproduction. Based on these results, it is reasonable to speculate that eATP first binds the P2K1 receptor, then stimulates heterotrimer G protein, then activated Gα stimulates CNGC2/CNGC4, finally leading to increased Ca 2+ /K + influx and the promotion of PG and PTG. How these components are connected remains unclear. Some receptor-like kinases directly bind and phosphorylate heterotrimeric G protein or activate heterotrimeric G protein through RGS1 (regulator of G protein signaling 1) [83][84][85]. Hence, the interaction of DORN1 and RGS1 (or heterotrimeric Gα) need to be carefully investigated before we can draw a conclusion. Heterotrimeric G protein is involved in the regulation of ion flux in plant cells [86]; however, whether heterotrimeric G protein directly activates ion channels or does so indirectly through altering membrane potential or generating cytoplasmic messengers (e.g., ROS or NO) needs to be clarified in the future.

Plant Materials
Pollen grains of 34 plant species (Table 1) were collected from newly opened flowers and then stored (adding silica gel as a dehydrator) at −20 • C.
Arabidopsis thaliana plants were grown in a greenhouse running at 22 • C with illumination intensity of 120-130 µmol·m −2 ·s −1 , relative humidity of 70%, and a 16/8 h light/dark cycle.

In Vitro Pollen Germination
The basic medium contained 18% sucrose and had a pH of 7.0. Nutrient salts were added according to individual experimental aims. All reagents were dissolved in deionized water, and 0.5% agarose was added to solidify the medium for pollen germination.
Pollen grains were suspended in a basic medium and spread onto the surface of a solid medium. Then, pollen grains were incubated in the dark at 28 • C with a relative humidity of 100%. The incubation duration varied among different species since the time needed to reach maximum germination rate for each species is different (see detail in Table 1). After incubation, pollen grains were photographed with a DS-Fi1c imaging system equipped to a microscope (Nikon Eclipse 80i, Japan). Images were analyzed with NIS-Element BR 4.0 software to count germinated pollen grains and measure pollen tube length. In each experiment, no less than 300 pollen grains were counted to obtain germination rate (pollen grains with emerging tubes longer than 5 µm were considered as germinated pollen), and the lengths of no less than 150 pollen tubes were measured. Data from 3 replicates were calculated to obtain mean values. Data was analyzed with Statistica 8.0 software.

Patch-Clamp Recording
All patch-clamp solutions were adjusted to 1.5 Osm/kg with sorbitol. For K + current detection, the bath solution specifications were 10 mM KC 6 H 11 O 7 , 5 mM Mes, and pH 5.8., and the pipette solution specifications were 100 mM KC 6  Patch-clamp recording methods were manipulated according to Véry & Davies [87]. Glass pipettes (World Precision Instruments, FL, USA) were pulled with a vertical puller (PC-10, Narishige, Japan). A whole-cell voltage clamping configuration was used. Current signal was recorded with an AXON 200B amplifier (AXON Instruments, IL, USA) equipped with Digidata 1440A data acquisition system (AXON Instruments, IL, USA), which was controlled by Clampex 10.3 software (AXON Instruments, IL, USA). Data was sampled at 1 kHz and filtered at 200 Hz. Voltage-clamp protocols included a series of depolarizing and/or hyperpolarizing steps of 1.5 s from a holding potential in the range of 0 to −200 mV. Current-voltage relationships (I-V curves) were then constructed with total whole-cell currents measured after 1.5 s step-voltage clamping. Data were analyzed with Microcal Origin 6.0.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10 .3390/plants10081743/s1, Figure S1: The effect of ATP, ATP agonist or hydrolytes on PG and PTG of Arabidopsis thaliana. Figure  Data Availability Statement: The study did not report any data.