Significant evidence supporting the hypothesis that extracellular nucleotides act as signaling molecules regulating plant growth and development has accumulated over the last decade. As reviewed in Clark and Roux (2018) and Pietrowska-Borek et al. (2020), extracellular nucleotides lead to an increase [Ca²⁺]_cyt and reactive oxygen species (ROS), and these secondary messengers lead to diverse downstream changes that affect plant defense, development, and growth.

The first eATP receptor to be identified in plants was discovered in Arabidopsis. In contrast to eATP receptors characterized in animal systems, the plant receptor, DORN1 (DOes not Respond to Nucleotides 1), is identical to a previously characterized lectin receptor kinase (LecRK-I.9/P2K1; At5g60300). It contains an extracellular lectin domain that, upon ATP binding, activates an intracellular kinase domain (Choi et al., 2014). In addition to P2K1, an additional eATP receptor was recently identified and named P2K2 (LecRK-I.5; At3g45430) (Pham et al., 2020). Although the P2K2 extracellular domain binds eATP with a higher affinity than P2K1, these two receptors, P2K1 and P2K2, interact and cross phosphorylate when eATP is applied to plants (Pham et al., 2020).

Studies of extracellular nucleotides and their role in plant growth and development have recently expanded to include a more primitive system, spores from the aquatic fern, Ceratopteris richardii. This single-cell, model system has been used to study gravity-directed polarization for decades. Its value as a model system is high because its development is well characterized (Chatterjee and Roux, 2000), it can be genetically transformed (Plackett et al., 2014; Bui et al., 2015; Plackett et al., 2015), and the genome has been sequenced (Marchant et al., 2022). Spore germination is initiated by water and red light. Within hours of initiation, calcium enters through channels at the bottom of spores and exits through pumps at the top (Ul Haque et al., 2007; Salmi et al., 2011). The calcium efflux peaks between 7 and 12 h after light-initiated germination begins (Chatterjee et al., 2000). Polarity of development is set by gravity after 24-30 h of growth and can be clearly visualized by the emergence of a downward-growing rhizoid 72 h after germination begins (Edwards and Roux, 1994).

The calcium differential described above is gravity-responsive and highly dynamic. When a spore is turned 180°, the calcium current re-orients within 24 seconds (Ul Haque et al., 2007; Salmi et al., 2011). The calcium differential is also sensitive to the magnitude of the g- force (Ul Haque et al., 2007; Salmi et al., 2011). During parabolic flight, when spores were exposed to 2g, the magnitude of the calcium differential increased. When spores are exposed to micro-g on the same flight, the calcium differential decreased to baseline levels. Additionally, blocking calcium channels with nifedipine decreased the number of downward growing rhizoids (Chatterjee et al., 2000). All of these data point to the significance of the calcium current and its role in gravity-directed polarization.

To further understand the molecular mechanisms associated with gravity- directed polarization, Bushart et al. (2013) used an RNA-SEQ analysis to identify components of the transcriptome that influence or respond to the calcium current. In this study, a key sequence found was one that, when translated, had apyrase conserved domains and a 59% identity with an Arabidopsis apyrase that is postulated to help control the concentration of extracellular nucleotides (Lim et al., 2014). Additionally, a western blot using polyclonal antibodies raised to Arabidopsis apyrase confirmed the presence of an immunoreactive protein band of the predicted apyrase size in extracts of Ceratopteris spores (Bushart et al., 2013). Because apyrase expression can be induced by an increase in eATP (Wu et al., 2007), this discovery suggested the possibility that extracellular nucleotides could be playing a role in the gravity-directed polarization of spore cells.

As an initial test of this hypothesis, an assay of the ATP released by spores into the growth media was done, and it showed that extracellular ATP (eATP) accumulates during gravity-directed polarization of Ceratopteris spores (Bushart et al., 2013). Because eATP can promote the uptake of calcium into cells (Jeter et al., 2004; Demidchik et al., 2009; Demidchik et al., 2011), these results raised the question of whether the release of ATP from spores was polarized, and, if so, whether this eATP could contribute to the gravity-induced polarized entry of calcium along the bottom of the spores. These questions are addressed in this study, using an amperometric ATP biosensor (Vanegas et al., 2015) to measure the [eATP]. The novel results presented here are consistent with the hypothesis that gravity induces the polarized release of ATP from cells, and that the consequent asymmetric accumulation of eATP could potentially help induce one of the earliest steps in gravity signaling, the polarized entry of calcium into cells, which polarizes cell development.

To determine if eATP is released from germinating spores during gravity-directed polarization, an ATP-selective micro-biosensor was used to directly measure ATP release from germinating spores. Calibrations were conducted before/after each experiment, and in the presence of amino acids, organic acids, BFA, and GdCl₃ to ensure no calibration drift caused bias in the data (see Supplemental Section for data). This assay showed that ATP was released from spores during their gravity-directed polarization, and that this release occurs as early as 30 min after light exposure, nearly coincident with the earliest detection of a gravity-induced calcium differential. The data presented here favor the hypothesis that the asymmetric accumulation of eATP preferentially along the bottom of germinating spores helps promote the gravity-directed polarization of these cells. Because eATP promotes the uptake of calcium in both animal and plant cells, we postulate that one potential role of eATP in spore cell polarization is to enhance the entry of calcium into the spores, and thus promote the trans-cell calcium current that orients cell polarization. Although eATP does induce calcium entry into Arabidopsis cells (Jeter et al., 2004; Demidchik et al., 2009), whether it can do so also in Ceratopteris cells remains to be tested.

In our previous work, we showed that the accumulation of ATP outside of spore cells was temporally dynamic (Vanegas et al., 2015), meaning that although there was a net efflux of ATP, this signaling molecule cyclically peaks in concentration every 10-12 min followed by a decrease in its concentration at the surface of the spores for 6-9 min. The temporally dynamic nature of ATP accumulation is evidence of modulation of the [eATP], presumably by ecto-phosphatases. The best characterized of these enzymes are ecto-apyrases, the phosphatases with the lowest Km for ATP thus far reported (Knowles, 2011). Maintaining precise spatial and temporal control of eATP during development ensures that a pivotal cellular decision dependent on the presence of this signal, such as polarization, is tightly regulated. Although the [eATP] dynamically changes, there is also a net increase in the accumulation of this molecule over time. This increase in accumulation may point to the necessity to surpass a threshold level in order to activate calcium channels. The opening of additional calcium channels would lead to an increase in the uptake of calcium and an increase in the accumulation of intracellular calcium along the bottom of the spore, promoting polarization.

Since a gradient of eATP is present as early as 30 min after light exposure and persists throughout polarization, the physiological relevance of this gradient was explored. That eATP could play a role in the spore gravity response was first shown in Bushart et al. (2013), who found that adding to the growth medium either a high concentration of extracellular nucleotides or an extracellular ATP receptor antagonist caused a statistically significant decrease in gravity directed polarization. Based on these results, Bushart et al. (2013) postulated that if gravity induced an asymmetric release of ATP this could help polarize the spore cells. The data presented here provide support for that postulate.

Blocking eATP responses with 350 μM PPADS significantly reduced the effect of gravity on spore polarization (Bushart et al., 2013). In the experiments shown in Figure 2A , both 150 μM and 250 μM PPADS were applied to spores in order to determine if a lower concentration would also disrupt the gravity response in Ceratopteris spores. Based on the results shown in Figure 2A , the eATP receptor antagonist threshold is 250 μM or between the two concentrations tested because 150 μM had no effect on polarization.

Hydrolyzing the eATP gradient using acid phosphatase ( Figure 3A ) or apyrase caused a small but statistically significant decrease in the gravity response in this system. This result suggests two alternative hypotheses that could explain the subtleness of the physiological response to this treatment. First, given the relatively large size of acid phosphatase (58 kD) and the denseness of the thick spore coat, only very low levels of these enzymes would be likely to penetrate through to the plasma membrane. This would limit the access of the enzyme to the site of eATP action and thus limit its effect on the eATP gradient and on gravity-directed polarization. In addition, since the Km of acid phosphatase is in the µM range, this treatment may not have a substantial effect because the concentration of ATP released into the external medium by spores is in the nM range. The alternative hypothesis is that eATP is promoting gravity-directed polarization rather than initiating it. According to this second hypothesis, in the absence of an extracellular nucleotide gradient, most spores would still polarize in response to gravity. However, if more eATP accumulated at the bottom pole of the cell, additional calcium channels would be opened there, causing an increase in the uptake of calcium and an increase in the percent of spores polarizing in response to the increased calcium channel activity.

In order to begin investigating the source of ATP release during polarity fixation, spores were treated with BFA and GdCl₃. In Figures 4A , 5 μM BFA treatment caused a statistically significant decrease in gravity-directed polarization. Since ATP can be released by both MS channels and as a result of secretory vesicles fusing with the membrane, this treatment does not differentiate between the two mechanisms, but the result in Figure 4A could be showing the necessity of ATP release by one or both of them. The BFA also caused a statistically significant decrease in the [eATP] measured on the top and bottom of Ceratopteris spores during gravity-directed polarization ( Figure 4C ). Additionally, the fold-change between the bottom and the top of spores decreases significantly, perhaps enough to fall below a hypothetical threshold. A decrease in ATP release as a result of BFA treatment has previously been reported in growing root hairs of Arabidopsis thaliana (Kim et al., 2006).

The release of ATP could also be mediated by MS channels during gravity-directed polarization in Ceratopteris spores. Treating spores with a MS channel antagonist, GdCl₃, caused a statistically significant decrease in the gravity response ( Figure 5A ). In addition, treating spores with 1 mM GdCl₃ decreased the [eATP] measured on the top and bottom of spores during gravity-directed polarization. Unlike BFA, GdCl₃ treatment disrupted the ATP gradient ( Figure 5C ). Treatment of Ceratopteris spores with GdCl₃ will also cause a decrease in the uptake of Ca²⁺ through MS channels, and, as noted by Kim et al. (2006), a decrease in Ca²⁺ uptake would decrease the [ATP] released. These results identify two potential mechanisms of ATP release during the gravity response in Ceratopteris spores, secretion and MS channels.

Overall, the significant decrease in gravity-directed polarization after BFA and GdCl₃ treatment, the lower [ATP] released from spores as a result of BFA and GdCl₃ treatments, and the significant decrease in the average fold difference as a result of BFA and GdCl₃ treatments are consistent with a hypothetical model that predicts the release of ATP by MS channels and targeted secretion will promote gravity-directed polarization in Ceratopteris spores ( Figure 6 ). The transcript encoding a putative MS channel that has been identified in the transcriptome of Ceratopteris spores ( Supplementary T able S1 ) is to date the only one confirmed to be expressed during the polarization period of development. Stronger support for the predictions of Figure 6 would require identifying the MS channel(s) that are localized on the plasma membrane in Ceratopteris and then showing that the knockout of this channel (or these channels) blocks graviresponsiveness of the spore.

Multiple experiments using Ceratopteris spores support the idea that an electrical gradient is generated by the asymmetrical distribution of Ca²⁺ during gravity-directed polarization (Chatterjee et al., 2000; Ul Haque et al., 2007; Salmi et al., 2011). This electrical gradient is essential for gravity-directed polarization in these spores (Chatterjee and Roux, 2000). The hypothetical model generated and supported by the experiments outlined in this paper support the hypothesis that ATP released from MS channels, secretory vesicles, or both is promoting the uptake of Ca²⁺ necessary to generate an intracellular electrical gradient. This hypothetical intracellular electrical gradient could be the initiating signal that sets up that polarization of early growth and developmental steps in Ceratopteris spores, including nuclear migration, asymmetric cell division, and emergence and growth of a rhizoid in the downward direction in response to gravity. Previous studies in zygotes of Fucus and Pelvetia, brown algae, have shown that the formation of an intracellular Ca²⁺ gradient is essential for directed polarization (Robinson, 1996). These studies support the hypothesis that the extracellular Ca²⁺ gradient previously measured in Ceratopteris spores (Chatterjee et al., 2000; Ul Haque et al., 2007; Salmi et al., 2011) is suggestive of an intracellular gradient that is necessary for gravity-directed polarization.

The idea of a differential distribution of a signaling molecule leading to morphological polarity is not unique. In this regard, gradients of auxin are among the best documented to be inductive of cell polarity (Dhonukshe, 2009). The differential distribution of eATP during gravity-directed polarization could be an early step in a series of asymmetrical molecular mechanisms that ultimately lead to the downstream cellular and morphological changes needed to generate a gravity-directed, downward growing rhizoid in Ceratopteris spores.

Precedent data relevant to this hypothesis are those of Tang et al. (2003), who showed that a high [eATP] inhibits the gravity response in Arabidopsis roots. They interpreted their data as evidence that an eATP gradient could be one of the first steps involved in the gravity response in plant cells, and, in support of that interpretation, they showed that high levels of eATP decreased shoot-ward auxin transport in a dose-dependent manner, leading to an inhibition in the gravity-response.

Like applied ATP, applied brefeldin and gadolinium also block gravity responses in roots (Abas et al., 2006; Soga, 2013), and all three of these agents would also be expected to affect, directly or indirectly, tip-focused calcium gradients that are characteristic of polarly growing cells and required for polarized growth (Campanoni and Blatt, 2007; Rincon-Zachary et al., 2010). Taken together these data underscore the likelihood that signaling components of the gravity response have been conserved in evolution from ferns through flowering plants.

These results show a clear role for eATP in gravity-directed polarization of Ceratopteris spores. However, there are still questions remaining about the molecular components and downstream mechanisms involved in this response. Although data support the idea that eATP could open Ca2+ channels leading to a higher influx during polarization, additional experiments are required to show a clear link between eATP and Ca2+ influx. Nonetheless, our data reveal that quantifying and evaluating gradients of eATP can lead to a more thorough understanding of how plants use ATP release to direct growth and development.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/genbank/, KX961683.

AC: Conceptualization, Investigation, Methodology, Supervision, Validation, Writing – original draft, Writing – review & editing. DV: Formal Analysis, Investigation, Methodology, Validation, Writing – review & editing. TS: Investigation, Writing – review & editing. MS: Funding acquisition, Investigation, Methodology, Supervision, Writing – original draft, Writing – review & editing. JW: Formal Analysis, Investigation, Writing – review & editing. GC: Conceptualization, Formal Analysis, Funding acquisition, Investigation, Methodology, Resources, Supervision, Writing – original draft, Writing – review & editing. EM: Conceptualization, Formal Analysis, Investigation, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing. SR: Conceptualization, Funding acquisition, Methodology, Project administration, Supervision, Writing – original draft, Writing – review & editing.