eATP-mediated Cellular Events in Plants

To explore eATP signaling in plants, eATP concentrations [(eATP)] in the ECM are usually artificially altered by the applications of commercial hydrolysable and non-hydrolysable ATP, ATP-hydrolyzing enzymes (apyrase) and ATP-sequestering traps (hexokinase-glucose system).¹⁶ Exogenous application of ATP to plant cells caused numerous physiological responses, including alteration of membrane potential,¹⁷ induction of second messengers [e.g., cytosolic Ca²⁺, (Ca²⁺)_cyt; nitric oxide, NO; reactive oxygen species, ROS; and phosphatidic acid, PA],^6,18-24 inhibition of cotton fiber elongation, polar auxin transport, root hair and pollen tube growth,^25-28 initiation of programmed cell death,¹³ modification of gene expression,^8,19 stimulation of stomatal opening and closure,^14,15 and modulation of ion fluxes across the plasma membrane and tonoplast.^13,19,24 In addition to artificially increasing eATP concentrations in the ECM, a few studies focused on the effects of eATP depletion on plant and cellular responses. Slabas and colleagues reported that the removal of eATP caused expression of pathogenesis-related (PR) genes and cell death.^29-31

It has shown that the plant response to eATP is dose-dependent. In Arabidopsis, low concentrations of eATP (15 to 35 μM) triggered the opening of stomata while high concentrations (greater than 250 μM) caused stomatal closure.¹⁵ The application of 100 to 200 μM ATP increased hypocotyl elongation by 10–20% in etiolated seedlings but higher doses led to a 10–55% reduction of hypocotyl elongation.³² In addition, low doses of eATP (3 to 100 μM) induced an obvious Ca²⁺ influx in elongation zone of Arabidopsis roots while higher doses of eATP (1000 μM) caused a significant increase of Ca²⁺ efflux.²⁴ In Populus euphratica cells, ATP-induced cell death was seen when the applied dose of eATP greater than 500 μM, while low doses (10–200 μM) did not cause PCD over the observation period.¹³ These contrasting responses suggest that plant ATP sensors were discriminatively activated in response to different strength of ATP stimulus. The assumption is consistent to the observations in animal systems that P2X receptors are usually activated at higher eATP concentrations (micromoles), as compared with P2Y that activated by nanomoles ATP.¹ Therefore, identification of plant ATP receptors is undoubted to provide vital insight into how plant cells sense the ATP concentrations in the ECM.

Substantial evidence indicates that eATP signaling is mediated through a variety of second messengers, such as cytosolic Ca²⁺ gradient, NO, PA and ROS.^6,18-24 The inhibition effects of ATP_λS (a non-hydrolysable ATP homolog) on pollen germination and root hair growth were absent in Arabidopsis mutants with diminished NO and H₂O₂ production, e.g., nia1/nia2 and atrbohD/F.^26,28 Similarly, genetic evidence also suggests that NO and H₂O₂ contributed to the eATP-induced stomatal closure (> 150–250 μM).¹⁵ Moreover, intracellular Ca²⁺ mobilizations (including Ca²⁺ influx, Ca²⁺ elevation in the cytosol, Ca²⁺ release from the vacuole and mitochondrial Ca²⁺ uptake), and mitochondrial H₂O₂ burst were implicated in the signaling pathway of eATP-induced PCD in P. euphratica cells.¹³ Under salt shock, the release of intracellular ATP from P. euphratica cells and the activation of putative plasma membrane ATP receptors were found able to trigger the downstream signaling components, H₂O₂ and Ca²⁺ (Sun J, Deng S and Chen S, unpublished data). Taking together, Ca²⁺, NO and H₂O₂ are likely downstream signaling components of eATP in plants. However, it should be noted that the effects of ROS and NO on intracellular ATP release or eATP levels were less investigated in plants. Thus, it is hard to discriminate the cross talk of these signals in a specific biological background that can induce both intracellular ATP release and the production of other messengers.

An interesting observation is that eATP modulates ion fluxes across the plasma and vacuolar membranes in plant cells. The application of ATP outside root epidermis of Arabidopsis induced a rapid influx of extracellular Ca²⁺ into the cytosol.¹⁹ Ca²⁺-channel inhibitors blocked the ATP-induced gene expression, indicating that Ca²⁺ signal is critical for mediating eATP effects in plants.⁸ Using protoplasts derived from root epidermis, Davies and colleagues show that the perception site of eATP is at the PM.¹⁹ In a more recent report, high concentrations (300 and 1000 μM) of ATP caused a net Ca²⁺ efflux from Arabidopsis roots.²⁴ This is likely a defense response since overloading of Ca²⁺ in the cytosol would trigger programmed cell death in plant cells.¹³ ATP application into intact root vacuoles of beet plants was found to induce an obvious Ca²⁺ release, which is possibly mediated by the activation of depolarization-activated slow vacuolar (SV) channels.³³ In P. euphratica cell cultures, eATP (1.0 mM) caused a release of vacuolar Ca²⁺ into cytosol and this process was mediated by cADPR-dependent activation of vacuolar Ca²⁺ channels.¹³ Suramin, an antagonist of animal P2 receptor, abolished the eATP induction of vacuolar Ca²⁺ release.¹³ The pharmacological results show that the vacuolar Ca²⁺ efflux induced by eATP is a downstream event of ATP perception at the PM. eATP also caused an obvious K⁺ efflux in Arabidopsis roots;²⁴ however, the physiological significance of the eATP-induced K⁺ efflux needs further studies to clarify.

Disruption of eATP Homeostasis Initiates Cell Death in Plants

Like other signaling agents such as ROS and cytosolic Ca²⁺, extracellular ATP has dual functions in plant cell signaling. A lethal level of eATP (extremely low or high) causes cell death, whereas a moderate level of eATP benefits plant growth and development.^2,13,29 Therefore, the concentration of eATP should be strictly controlled under normal conditions. Recent work about eATP signaling underlines the significance of eATP homeostasis in plant cell death regulations. Using apyrase and hexokinase-glucose system as ATP-traps, Chivasa et al. showed that continuous removal of eATP led to cell death in different plant species and cell types.²⁹ Their result indicates that a moderate level of eATP is required for normal growth of plant cells. In this report, FB1, a mycotoxin that triggers programmed cell death in plant cells, caused an eATP depletion and occurrence of cell death.²⁹ Supplement with millimole range of ATP rescued Arabidopsis root and suspension cells from FB-1-induced cell death.²⁹ These results suggest the involvement of eATP degradation in pathogen-induced hypersensitive cell death and biotic stress response. The correlation between plant pathogen resistance and eATP depletion was consequently established in a following report. In tobacco leaves, enzymatic depletion of eATP triggered salicylic acid (SA)-dependent PR gene expression and enhanced resistance to tobacco mosaic virus and Pseudomonas syringae pv tabaci.³⁰ Artificially addition of ATP reversed the effects of eATP depletion on gene expression and pathogen resistance.³⁰ The above mentioned results show that the eATP degradation during pathogen infecting is an important signaling to induce downstream defense responses and hypersensitive cell death.

In cell suspension cultures of Arabidopsis thaliana, proteomic analysis showed that FB-1-induced depletion of eATP altered abundance of various proteins, including molecular chaperones, glycolysis-related enzymes, antioxidant enzymes, cytoskeleton-related proteins and ATP synthesis machinery.³⁴ It is noting that mitochondrial ATP synthase β-subunit was significantly downregulated after the application of FB1.³⁴ Thus it can be speculated that the cell death, caused by FB1-triggered degradation of eATP, is due to the exhaust of intracellular energy since ATP is a critical factor for the survival of plant cells.³⁵ The supplement of ATP accompanied with FB-1 reversed the inhibitory effect of FB-1 on mitochondrial ATP synthase β-subunit.³⁴ Chivasa et al. conclude that the inhibition of FB-1 on protein abundance is mediated by eATP and not due to a direct toxicity of FB-1. In addition, knockout of mitochondrial ATP synthase β-subunit enhanced the capacity of Arabidopsis to tolerate FB-1.³⁴ Therefore, mitochondrial ATP synthase β-subunit is a main molecular target mediating the physiological process of eATP in plants. Accordingly, it would be interesting to explore the regulatory mechanisms of eATP on mitochondrial ATP synthesis machinery.

Besides the signaling effects of extremely low [eATP] in plant cells, excess [eATP] was found to trigger programmed cell death in P. euphratica cells.¹³ Chen and colleagues put forward a speculated model to elucidating the pathway of eATP-induced PCD.¹³ In this model, eATP induction of PCD was intermediated by the activation of mitochondrial ATP synthesis machinery. This is consistent to the result of Chivasa et al. who reported that mitochondrial ATP synthase β-subunit is a main molecular target of eATP signaling.³⁴ The activation of mitochondrial ATP synthesis by excess eATP was mediated by a series of signaling events, including purinergic receptor activation, extracellular Ca²⁺ influx, Ca²⁺ elevation in the cytosol, Ca²⁺ release from the vacuole, mitochondrial Ca²⁺ uptake, mitochondrial hyperpolarization and H₂O₂ bursts in the mitochondria.¹³ The elevation of mitochondrial ATP synthesis contributed to the activation of caspase-like proteases, the main enzymes executing the cell suicide program.³⁵

Taken together, these new findings indicate that both eATP depletion and eATP overproduction induce programmed cell death in plants. Therefore, it can be inferred that the adaptation of plant cells to environmental stimuli are dependent on the fine-regulated ATP homeostasis in the ECM. Further investigations about eATP homeostasis control would give an insight to the understanding of eATP signaling in specific processes related to PCD, such as leaf senescence and abscission, self-incompatibility induced programmed cell death and root architecture remodeling under salinity stress.

Plant Apyrases and eATP Homeostasis

In animal and plant systems, a wide range of enzymes helps control the concentration of extracellular ATP in the ECM.³ Ecto-apyrases (Nucleoside Triphosphate-Diphosphohydrolase), which remove the terminal phosphate from extracellular ATP and ADP to form non-signaling agent AMP, are shown to be the most important in ATP hydrolyzing and have been well characterized in animals.³ Plant apyrases contain four apyrase conserved regions that are similar to the conserved regions of animal apyrases.^3,5 Two apyrase genes, termed APY1 and APY2, have been characterized from model plants Arabidopsis thaliana and other species. APY1 and APY2 use ATP as their preferred substrate and exhibit ecto-apyrases activity.^2-5 Substantial molecular evidence indicates that these genes are involved in mediating eATP signaling and plant behaviors. The expression level of APY1 and APY2 is usually highest in the cells exhibiting active growing, such as elongating cotton fibers and apical region of Arabidopsis primary roots.^25,36 Intracellular ATP is released into the ECM of growing cells, and apyrases function as an ATP-scavenger to maintain the ATP homeostasis in the ECM. Chemical inhibition of apyrase activity with NGXT 191 inhibited root hair growth in Arabidopsis and cotton fiber elongation,^25,26 suggesting a detrimental effect of excess eATP on plant growth. Artificially addition of non-hydrolysable ATP also inhibited polar growth of root hairs and pollen tube in Arabidopsis.^25,28 Arabidopsis double-knockout mutants, apy1apy2, are dwarf phenotypes that exhibited a complete inhibition of pollen germination and reduced cell elongation.^36,37 Overexpression of AtAPY1 in Arabidopsis increased growth rate of pollen tubes and etiolated hypocotyls, compared with wild-type plants.³⁶ Taken together, a reasonable degradation of ATP concentration in the ECM could stimulates growth in different plant cell types.

The transcript abundance of plant apyrases is usually upregulated under adverse conditions. In Arabidopsis, hypertonic stress induced an obvious elevation of [eATP] and is accompanied by an increased expression of APY1 and APY2.^8,9 Similarly, we found that the transcripts of PeAPY1 and PeAPY2 in cell cultures of P. euphratica were upregulated by a various abiotic stresses (Sun J, Deng S and Chen S, unpublished data). These data reveal that apyrases, which contribute to eATP homeostasis, play an important role in regulating the plant adaptations to environmental stress. In Arabidopsis, the ABA-induced stomatal closure was mediated by eATP and apyrase.¹⁵ Chemical inhibition of apyrase activities led to stomatal closure and ABA treatment induced an obvious elevation of eATP in guard cells.¹⁵ These results suggest that ABA-induced stomatal closure is partially caused by an inhibition of apyrase activity and a higher accumulation of eATP around guard cells. However, they also found that RNAi suppression of APY1 in apy2 single knockout mutant resulted in increased stomatal apertures compared with the wild type.¹⁵ Thus, the regulation of apyrases on eATP, and the role of apyrases and eATP in stomatal movement and drought tolerance are extremely complicated and need further investigations.

In AtAPY1-overexpressed Arabidopsis, the growth inhibition induced by paraquat, which triggers oxidative stress, was less remarkable.³⁸ This work highlights a regulatory function of apyrases on antioxidant capabilities in plants. Our data reveal that overexpression of PeAPY2, a AtAPY2 homologous gene isolated from P. euphratica, increased cold and salt tolerance in both transgenic Arabidopsis and tobacco BY-2 cells. We found that the PeAPY2-transgenic Arabidopsis and tobacco showed less ROS accumulation, compared with the wide type plants and cells (Deng S, Sun J and Chen S, unpublished data). These results suggest that apyrases act as antioxidant machinery in response to abiotic stress. It should be noted that the application of higher dose of eATP could trigger ROS burst in plant cells.^6,13,19 Thus, the regulatory mode of apyrase on eATP homeostasis and antioxidant capabilities should be established in the future.