Extracellular ATP acts as a damage-associated molecular pattern (DAMP) signal in plants

1Multicellular organisms have assembled complex signaling networks that mediate specific and dynamic responses following various environmental stimuli. Among these are mechanisms that recognize a potentially life-threatening event as a danger signal. Danger signals include exogenous, enemy-derived signal molecules, e.g., pathogen-associated molecular patterns (PAMPs), which are recognized by pattern recognition receptors to activate immune responses. In addition, endogenous molecules and fragments from damaged cells and tissues can also be recognized as danger signals, referred to as damage-associated molecular patterns (DAMPs). Multicellular organisms use DAMPs for damage-self recognition to evoke immune inflammatory responses and damage healing independent of but in cooperation with exogenous danger signals.

2Because of their sessile nature, plants are continuously exposed to various stresses caused by changes in the environment and attacks by other organisms, i.e., abiotic and biotic stresses. Therefore, plants require sophisticated surveillance systems to detect a variety of danger signals. Indeed, plants have evolved a large number of receptor kinases (e.g., >600 genes in Arabidopsis), most of which are likely involved in the response to different stresses, based on the observation that many duplication events have occurred in the genes involved in defense responses and disease resistance. These plant receptors recognize not only exogenous danger signals such as PAMPs and herbivore-associated molecule patterns (HAMPs) but also endogenous danger signals such as DAMPs. A number of PAMPs have been identified, and their recognition systems and downstream signaling events are well studied and understood. In contrast, only a few DAMPs have been extensively studied (Table 1) and the details regarding their recognition and signaling mechanisms remain unclear. To date, few receptors have been identified that specifically recognize DAMP signals (Table 1). Further studies are needed to clarify each of their signaling mechanisms.

3Extracellular ATP is one of the well studied DAMP signals in both animals and plants. Although ATP is well recognized as a source of high energy phosphate bonds to support cellular metabolism, once ATP is released from cells following cellular damage, it acts as a DAMP signal (Figure 1). ATP is a good choice for such a role since cells contain a high concentration of ATP (1–10 mM), which is highly reactive and involved in more chemical reactions than any other compound except H2O. In animals, extracellular ATP has been studied for over 60 years. The released ATP is recognized by plasma membrane-localized purinergic receptors (P2X and P2Y) that involve a wide range of animal physiology. In plants, a number of earlier studies reported that extracellular ATP plays essential roles in plant growth and development. The recent discovery of the plant P2K1 (DORN1) receptor by demonstrated that extracellular ATP also serves as a DAMP signal in plants. For example, most ATP-responsive genes were also regulated in response to a wound treatment. As would be expected, dorn1 mutant plants showed a reduced transcriptional response to both ATP treatment and wounding. Similarly, overexpression of DORN1 resulted in an elevated response to both ATP and wounding. These data strongly suggest that ATP plays an important role in response to wounding, which is mediated by ATP recognition by the P2K1 (DORN1) receptor.

4Detailed analysis of ATP-induced genes by suggested that extracellular ATP signaling, mediated by P2K1, was also involved in plant responses to a variety of stresses. The suppression of ATP-hydrolyzing enzymes, apyrases (AtAPY1 and AtAPY2), which results in an elevation of extracellular ATP levels, induced the expression of genes involved in stress responses. Therefore, extracellular ATP likely plays a role as a central danger signaling during a variety of plant stress responses.

5The presence of extracellular ATP in plants was detected under various conditions (Table 2), which suggests the existence of mechanisms by which this energy molecule is released into the apoplast. Several mechanisms have been proposed for mediating ATP release (Figure 2). For example, similar to the mechanism of ATP release at animal neuronal synapses, ATP appears to be released at sites of active growth via vesicular exocytosis. AtPGP1, an ABC transporter, and PM-ANT1, a plasma membrane-localized nucleotide transporter were shown to export intracellular ATP into the extracellular milieu (ECM;; ). However, it remains unclear how these ATP-releasing systems are controlled by environmental stimuli.

6Cell breakage is a simple mechanism for ATP release that could be caused by herbivore attack (i.e., wounding) or pathogen-induced cell lysis (i.e., necrosis). This released ATP would be expected to act as a DAMP. Interestingly, ∼60% of the genes induced by ATP treatment are also induced by wounding, 90% of which responded early to wounding. In previous studies, the ATP concentrations measured at the wound sites of Arabidopsis leaves and damaged roots were 40 μM and 80 nM, respectively. The effective concentration for ATP signaling at damaged sites may be lower than these measurements, considering that the ATP pool is competitively shared with other ectoenzymes, such as ectophosphatases, ectokinases, and other cell wall enzymes. Given the high affinity of P2K1 for ATP recognition (Kd ∼46 nM), these levels of extracellular ATP would be sufficient to trigger P2K1-mediated ATP signaling. This finding suggests that extracellular ATP released during wounding acts as a DAMP signal and is recognized by the P2K1 receptor (Figure 2).

7More subtle treatments of plants have also been shown to release sufficient ATP to potentially activate the P2K1 receptor. For example, nanomolar levels of ATP were released from Arabidopsis roots when a mild touch was applied with a force of 198 ± 178 mN (Table 2), which is a typical range for plants growing through soil. Plants can also experience mechanical stress when infected by microbes. reported that pathogenic fungi and oomycetes exert mechanical stress by turgor pressure from penetrating pegs and hyphae. Indeed, mechanical stimulation was shown to activate disease resistance. Interestingly, pathogen attachment causes nuclear repositioning and cytoplasmic streaming at the contact points on the plant cell. These cytoskeletal arrangements are general responses caused by mechanical stimulation. Considering that exogenous ATP can also cause cytoplasmic streaming in plant cells, it is conceivable that mechanical stress following pathogen infection likely stimulates plants to release ATP as a DAMP signal, which eventually evokes cellular responses against the invaders. ATP-induced actin filament formation and cytoskeletal rearrangements are widely observed in animal cells in which ATP modulates inflammation, wound healing and angiogenesis. Although such a role for ATP in plants is speculative at this point, it is clear that ATP has the potential to be a missing signal that mediates many of the responses previously attributed solely to mechanical stimulation.

8In addition to wounding and mechanical stress, ATP is also released, without visible cell damage, in response to treatment with biotic and abiotic stress agents (Table 2). Abiotic stimuli such as osmotic and salt stress, as well as abscisic acid (a stress-related hormone), and L-glutamate (known as a fast excitatory neurotransmitter in the mammalian nervous system) induce ATP release in plants (Table 2). Biotic stress agents such as pathogen-derived elicitors (i.e., chitin, yeast extract, and mycotoxin beauvericin) also trigger ATP release (Table 2).

9Taken together, these findings clearly demonstrate that plants release ATP outside cells in response to various stresses, and the released ATP likely acts as a DAMP signal to trigger plant defenses and environmental stress responses (Figure 2). Further studies are necessary to understand how the ATP-releasing machinery (e.g., transporters and exocytotic vesicles) are regulated by an array of different stimuli.

10Herbivore feeding, similar to physical wounding, would be expected to passively release high doses of ATP into the extracellular milieu. If this ATP triggers plant defense mechanisms, it would be expected that insects may have adapted to counter this defense. Indeed, some herbivorous caterpillars and whitefly larvae secrete saliva containing apyrases while feeding on plant leaves. Interestingly, the exogenous application of purified caterpillar apyrase at the wound site suppressed glandular trichome production, which is involved in the defense against herbivores, and the expression of defense-related genes regulated by jasmonic acid and ethylene. A clear mechanism for this response would be that the salivary apyrases dampen wound-released ATP to restrict the plant defense response. Furthermore, a higher catalytic activity of apyrases was detected in globose insect galls on Calliandra brevipes. Similar mechanisms for controlling extracellular ATP concentrations are observed in the interactions between animals and parasitic worms or blood sucking insects.

11Pathogen attacks cause cell damage that allows cytoplasmic ATP to leak into the extracellular space. The reduction of extracellular ATP levels was observed following plant infection by the bacterial pathogen Pseudomonas syringae. Interestingly, this reduction was not observed using a P. syringae strain lacking a canonical protein secretion system, suggesting that some effector proteins may contribute to ATP depletion. A recent study showed that adenylate kinases (which phosphorylates AMP using ATP as a phosphate donor) are secreted by Xanthomonas oryzae. These enzymes could play a role in quenching DAMP-utilized ATP during colonization. Adenylate cyclase were also shown to be important for the virulence of animal pathogens. Given that extracellular ATP was shown to be involved in cytolysis, cell adhesion, and growth of various microbes, these pathogenic microbes may be merely protecting themselves from ATP, a very reactive molecule. Correspondingly, a recent report by demonstrated that conidial germination, appressorium formation and pathogenicity of rice blast fungus Magnaporthe oryzae were significantly reduced by pretreatment with nucleotidase-specific inhibitors.

12The above results suggest that pathogens and insects directly manipulate extracellular ATP levels to weaken or block plant immunity; thereby promoting colonization of the plant host (Figure 1).

13Plants use hormone-mediated pathways, either salicylic acid, jasmonic acid and/or ethylene, to activate defense against pathogens or herbivores. The application of ATP induces the expression of genes encoding biosynthetic enzymes for jasmonic acid and ethylene. Interestingly, observed that ATP addition to slightly wounded tissue induced the secretion of extrafloral nectar in the lima bean. The secretion of extrafloral nectar is a jasmonic acid-dependent defense response, known to attract ants and other predatory insects. These results suggest that ATP released following wound damage activates signaling by stress hormones that eventually activates additional defense systems.

14It is expected that more than two DAMPs would be present together in the plant apoplastic area at the wound site, where the signals would function together to activate defense response signaling. Oligogalacturonic acid (OGA), another DAMP generated by cell wall degradation (Table 1), is known to trigger cytosolic Ca2+ elevation, reactive oxygen species (ROS) production, and other downstream defense responses. These same responses are also triggered by ATP. Co-treatment with both OGA and ATP resulted in an enhancement of cytosolic Ca2+ elevation. Given that similar defense signaling and gene expression are triggered by both ATP and OGA, it is likely that these two signals may act synergistically to insure a robust response to wounding.

15Wound healing is an essential biological process composed of complex, sequential steps to restore function of damaged cells and surrounding tissues. In animals, this process starts with conserved cellular damage signals (e.g., Ca2+, ROS, and ATP) that diffuse into ambient tissue, surround the wounded area, and initiate immediate cellular events including cell shape changes, actomyosin formation, and immune cell recruitment.

16Plants lack cellular mobility because of the cell wall and must restore tissue through cellular regeneration at the damaged site. A recent report by using RNAi silencing of ectoapyrases suggested that accumulated levels of extracellular ATP caused significant changes in the expression of genes regulating wall composition and extensibility. The silenced lines showed changes in wall lignification and decreased methyl ester bonds. These results suggest that extracellular ATP induced by wounding could play an important role in wall reorganization during tissue healing.

17The molecular mechanisms of the early stage, plant healing process remain largely unknown, in contrast to those of later-stage wound healing, which has been well studied. noted that auxin plays an important role during communication between the repair site and other parts of the plant. Auxin accumulates on the acropetal side of the wound while lower levels are found on the basipetal side. Given that extracellular ATP strongly inhibits polar auxin transport, ATP released by wounding could contribute to the auxin distribution at the wound site. Jasmonic acid and ethylene has been postulated to activate specific wound responsive genes that further promote wound healing. The addition of ATP induces the expression of genes involved in the biosynthesis of jasmonic acid and ethylene. Although definitive experimental evidence is lacking, the literature certainly contains clues that extracellular ATP could be playing a very important signaling role during the wound healing process.

18The role of extracellular ATP in symbiosis has been suggested by studies of ectoapyrases, which control the level of extracellular nucleotides. For example, overexpression of soybean ectoapyrase in Lotus japonicus, Medicago truncatula, and soybean resulted in enhanced infection by rhizobia and an increased number of nodules. In contrast, RNAi silencing and antisense suppression of ectoapyrases in soybean and L. japonicus blocked symbiont infection, resulted in markedly reduced nodulation. These results are corroborated by earlier studies in which nodule formation on soybean and Dolichos biflorus was inhibited by treatment with specific antibodies against ectoapyrases. A recent study by also demonstrated that the suppression of ectoapyrase blocked infection by arbuscular mycorrhizal fungi. Taken together, these data suggest that ectoapyrases are essential for both rhizobial and mycorrhizal symbiosis, presumably due to their ability to modulate extracellular ATP levels.

19Lipochitooligosaccharides (LCOs), which are known as Nod factors and Myc factors, are key symbiotic signals essential for rhizobial and mycorrhizal symbioses, respectively. Treatment of soybean seedlings with LCO was shown to increase ectoapyrase gene expression. LCO treatment was also shown to result in a re-localization of the apyrase to the root hair tip, which is the site of rhizobial infection. Indeed, LCO treatment was shown to increase the enzymatic activity of the ectoapyrase isolated from D. biflorus demonstrating that this ectoapyrase has the ability to bind directly to LCO. Therefore, one can hypothesize that, in order to enhance infection, the symbionts use LCO to increase ectoapyrase expression and/or activity; thereby quenching DAMP, extracellular ATP, either released due to mechanical stress during infection or as a result of a response to LCO. Studies have shown that ATP, but not ADP, induces ROS production in the root hairs of Medicago truncatula and Arabidopsis, as well as Arabidopsis epidermal cells. ROS are well-known signals in the plant defense response. Therefore, symbionts may control ectoapyrase activity using LCOs, in which the ATP function as a DAMP is suppressed so that plant defenses are not triggered.

20The products of ectoapyrase activity are ADP and/or AMP, which may also have biological activity. For example, ADP was shown to change plasma membrane conductance in root hairs and induce calcium influx in root epidermal cells. Interestingly, while RNAi silencing of ectoapyrase expression in soybean reduced nodulation, this effect was partially reversed by the addition of exogenous ADP, suggesting a role for extracellular ADP in the nodulation process. used electrophysiological methods to verify the presence of an ADP signaling pathway, independent of ATP signaling, with ionotrophic receptor-like activity at the plasma membrane. P2K1 (DORN1) shows slightly less affinity for ADP over ATP. It is possible that a second, distinct receptor could exist in plants for ADP, as suggested by. Alternatively, it is also possible that ADP recognition by P2K1 could activate distinct downstream signaling events, which would also account for the apparent distinct response of ADP vs. ATP. Further research on ADP/ATP signaling pathways, possible additional purinoceptors and P2K1 function should provide answers to these and other questions relevant to nucleotide signaling in plants.

21The release and response to extracellular ATP is conserved across many taxa, including lower vertebrates, insects, protozoa, and prokaryotes, indicating that a role for ATP as an extracellular signal is an ancient trait. Therefore, it is not surprising that ATP also plays such a role in plants. However, the P2K purinoceptor family is quite distinct from the P2X and P2Y receptors found in animal systems. Hence, although ATP signaling is widely conserved, the means by which plants and animals recognize this signal appears to have evolved independently. However, caution should be used in making such broad conclusions since we are clearly at a very early stage of our understanding of ATP recognition and action in plants. Additional comparative studies are needed to fully elucidate the similarities and differences between the mechanism of action of extracellular ATP in plants and animals.

22Plant enemies such as pathogens and insects usually interfere with one or more defense signals to promote colonization and infection. It is now clear that among these mechanisms are strategies for modulating the levels of extracellular ATP. This underlines the important role that ATP plays in general plant stress response (Figure 1).

23Although a number of molecular components need to be identified for a complete understanding of extracellular ATP signaling, the recent discovery of the P2K1 (DORN1) purinoceptor creates the possibility for further genetic and biochemical studies. As shown in Figure 2, the repertoire of the molecular components identified to date covers a variety of responses induced by extracellular ATP. The following research avenues are of particular interest for further investigation: (1) understanding how these molecular components are controlling extracellular ATP levels following various environmental stimulations and (2) learning the molecular mechanisms of how the ATP signal influences the immune system and subsequent wound healing processes. We propose that extracellular ATP is a key missing signal in many plant stress responses that were previously believed to be due to a direct effect of the stress stimulus itself. Therefore, further knowledge of the role of ATP in plants is crucial to understanding how plants adapt to environmental changes, a crucial question in lieu of a rapidly changing climate.

24The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.