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First published online January 20, 2006; 10.1104/pp.105.073072 Plant Physiology 140:1222-1232 (2006) © 2006 American Society of Plant Biologists
Extracellular ATP Induces the Accumulation of Superoxide via NADPH Oxidases in Arabidopsis1Section of Molecular Cell and Developmental Biology, University of Texas, Austin, Texas 78712
Extracellular ATP can serve as a signaling agent in animal cells, and, as suggested by recent reports, may also do so in plant cells. In animal cells it induces the production of reactive oxygen species through the mediation of NADPH oxidase. Similarly, here we report that in leaves of Arabidopsis (Arabidopsis thaliana), applied ATP, but not AMP or phosphate, induces the accumulation of superoxide (O2–) in a biphasic, dose-dependent manner, with a threshold at 500 nM ATP. This effect did not require ATP hydrolysis for it was mimicked by ATP  S. ATP also induced increased levels of Arabidopsis respiratory burst oxidase homolog D (AtrbohD) mRNA, but ATP-treated plants that had disrupted AtrbohD and AtrbohF genes did not accumulate O2–, indicating that NADPH oxidases are responsible for the induced O2– accumulation. Inhibitors of mammalian P2-type ATP receptors abolished ATP-induced O2– production, suggesting that the ATP effects may be mediated through P2-like receptors in plants. Cytosolic Ca2+ and calmodulin are likely to help transduce the ATP responses, as they do in animal cells, because a Ca2+ channel blocker, a Ca2+ chelator, and calmodulin antagonist all reduced ATP-induced O2– accumulation. Furthermore, ATP treatment enhanced the expression of genes that are induced by wounds and other stresses. The ATP measured at wound sites averaged 40 µM, well above the level needed to induce O2– accumulation and gene expression changes. Transgenic plants overexpressing an apyrase gene had reduced O2– production in response to applied ATP and wounding. Together, these data suggest a possible role for extracellular ATP as a signal potentially in wound and stress responses.
Extracellular ATP (eATP) is a well-characterized signaling agent in mammals. It induces the respiratory burst in phagocytes, and it exerts this effect through P2 receptors (Ralevic and Burnstock, 1998  ; Di Virgilio et al., 2001) and a signaling chain that typically includes increased cytosolic Ca2+ concentration ([Ca2+]cyt) as an early step (Kuroki and Minakami, 1989; Dichmann et al., 2000). These receptors mediate diverse responses in animals including platelet aggregation, the inflammatory response, neurotransmission, and apoptosis (Ralevic and Burnstock, 1998; Di Virgilio et al., 2001). Recent reports indicate that there may be eATP signaling in plants. Low microgram concentrations of eATP induce increases in [Ca2+]cyt (Demidchik et al., 2003) and membrane depolarization (Lew and Dearnaley, 2000) in Arabidopsis (Arabidopsis thaliana) roots. At higher concentrations, eATP induces increases in [Ca2+]cyt and downstream gene expression changes associated with stress and wounding in intact seedling tissues (Jeter et al., 2004), and it inhibits auxin transport, root gravitropism (Tang et al., 2003), and pollen germination (Steinebrunner et al., 2003) in Arabidopsis. The destruction of eATP appears to induce programmed cell death in leaves of several plant species (Chivasa et al., 2005).
Among the signaling changes induced by eATP in animal cells is enhanced production of reactive oxygen species (ROS; Dichmann et al., 2000
The two best-characterized ROS are superoxide (O2–) and hydrogen peroxide (H2O2), but also included in this chemical category are hydroxyl radical, singlet oxygen, and hypochlorous acid (Henderson and Chappell, 1996
NADPH oxidase homologs have been implicated as key players in the production of O2– in plants (Mittler et al., 2004
Plants with reduced or disrupted gp91phox homologs have compromised responses to biotic and abiotic stress and have a reduced capability to accumulate ROS. Antisense lines of tomato Lerboh1 had a reduced ROS accumulation and compromised wound response, failing to produce wild-type levels of protease inhibitor II after being wounded (Sagi et al., 2004
Physical injury to plants can occur as a result of herbivory or environmental stresses such as wind, rain, or hail (Leon et al., 2001
Many aspects of the plant defense response are analogous to the animal immune response (Bergey et al., 1996
ATP and ADP Induce O2– Accumulation
In response to the range of ATP concentrations tested, there were two distinct peaks of increased O2– accumulation, one at 1 µM ATP and the other at 50 µM ATP (Fig. 1A
). All concentrations of ATP tested induced significantly higher O2– accumulation than the phosphate buffer (PB) control. The positive control of OGA, which is known to induce an oxidative burst in Arabidopsis (Hu et al., 2004
The O2– accumulation in response to infiltration of 50 µM ATP peaked at two distinct time points, 3 min and 4 h (Fig. 1C). At all of the time points measured, there was a significant accumulation of O2– in response to ATP treatment as compared to the PB control (P  0.0009). To determine whether the O2– assay was in fact detecting O2– in the leaves, 2 mM xanthine was added together with 5 units of xanthine oxidase to evolve O2–, and the O2– was detected using the same method. All times that were tested with xanthine and xanthine oxidase had significant increases in O2– compared to a buffer control (P 0.0002; data not shown), and the pattern of Nitroblue tetrazolium staining was similar to that observed after ATP treatment. A test of the threshold of the response to eATP revealed that 250 nM ATP induces no response, but 500 nM ATP induces a significant production of O2– that is at least equal to that induced by 1 µM ATP (data not shown). The kinetics of the response to 500 nM ATP for the first 60 min closely parallels the kinetic pattern induced by 50 µM ATP (Fig. 1C).
Several authors have shown that homologs of mammalian NADPH oxidase subunits are responsible for stress-induced production of O2– in plants (Torres et al., 2002
Presence of eATP at Arabidopsis Wound Sites To determine a possible physiological source of eATP that could induce ROS accumulation in plants, we measured the concentration of ATP in the extracellular fluid present at Arabidopsis wound sites. The sampling and measuring procedure used here was linear over a range from 100 nM to 10 mM ATP and closely matched the actual values of the ATP standards over that range (Fig. 2 ). Using the same procedure, we measured a mean value of 40 ± 22 µM [ATP] from seven different pooled samples collected from Arabidopsis rosette leaf wound sites (Fig. 2). The values for these seven samples ranged between 25 and 45 µM.
Plants Overexpressing Apyrase Have Reduced O2– Production in Response to eATP and Wounding
Plants, like animals, have ectoapyrase enzymes that regulate the concentration of eATP (Thomas et al., 2000
Transgenic line 4-4 was tested for its response to ATP S, a P2 receptor agonist that, like ATP, induces increase in [Ca2+]cyt, but, unlike ATP, cannot be hydrolyzed by apyrase or by phosphatases (Jeter et al., 2004). At the concentrations tested, the peak response of O2– production in both wild-type and apyrase-OE plants was at 1 µM, and the threshold response of both was at or below 250 nM (Fig. 3C). Wild-type and OE plants did not differ significantly in their O2– production response to ATPS. Both wounding and eATP application to unwounded leaves induced a biphasic response of O2– production (Fig. 3D), but the O2– response of transgenic lines 4-4 and 2-2 to wounding was muted relative to that of wild-type plants (Fig. 3D).
Many animal P2 receptors have broad nucleotide specificity (Ralevic and Burnstock, 1998
P2 Receptor Inhibitors and Adenosine Reduce eATP-Induced O2– Accumulation
Two inhibitors of the P2 receptors that mediate eATP signaling in animals, pyridoxalphosphate-6-azophenyl-2', 4'-disulphonic acid (PPADS) and reactive blue 2 (RB2; Ralevic and Burnstock, 1998
In neutrophils, adenosine, a product of ATP turnover, inhibits O2– production through the activation of A2 adenosine receptors (Cronstein et al., 1985 ; Bengtsson et al., 1996). To determine whether or not adenosine can inhibit eATP-induced O2– accumulation, we cotreated plants with 10 µM adenosine and 50 µM ATP and measured O2– accumulation (Fig. 4). Cotreatment with adenosine reduced eATP-induced O2– accumulation to levels similar to the buffer-only control (Fig. 4; P > 0.05). Adenosine treatment alone was not significantly different from the PB control (P > 0.05; data not shown).
Canonical P2 receptor signaling and wound signaling involve downstream Ca2+ signaling. When they were applied in addition to ATP, both 1,2-Bis(2-amino-5-bromophenoxy)ethane-N,N,N',N'-tetraacetic acid, which chelates extracellular Ca2+, and LaCl3, which blocks Ca2+ channels, reduced ATP-induced O2– accumulation to levels similar to control (Fig. 4; P To further study the involvement of Ca2+ mediation of ATP-induced O2– accumulation, we used a CaM antagonist, N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7), to block the action of CaM. Addition of W7 to the ATP treatment reduced O2– accumulation compared to ATP treatment in the absence of W7 (P < 0.0001; Fig. 4). N-(6-Aminohexyl)-1-naphthalenesulfonamide (W5), which is a much less potent CaM antagonist, was ineffective in blocking ATP-induced O2– accumulation (P > 0.05; data not shown). Responses to W5 and W7 alone also were not significantly different from the PB control (P > 0.05; data not shown).
Applied ATP induced increased abundance of transcripts for the Phe ammonia-lyase gene 1 (PAL1), which is also induced by ROS (Levine et al., 1994
P2 Receptor Inhibitors Block eATP-Induced Changes in Gene Expression We also examined the effect of P2 receptor inhibitors and adenosine on eATP-induced changes in gene expression (Fig. 6 ). Pretreatment of seedlings with 250 µM PPADS, 30 µM RB2, or 10 µM adenosine reduced the eATP-induced expression of AtrbohD and of PAL1, a gene whose expression can be induced by ROS (Fig. 6, A and B).
Parallels in Animal and Plant Stress Signaling
When activated by a pathogen, phagocytic blood cells have a respiratory burst that results in the production of ROS (Vignais, 2002
The low (submicromolar) concentrations at which ATP elicits O2– accumulations in leaves suggests that eATP is most likely acting as a signal in leaf wound responses, just as when it induces increased [Ca2+]cyt in root cells (Demidchik et al., 2003 The samples for directly measuring the [ATP] in the extracellular fluid at wound sites were typically collected <3 min after wounding, coincident with the first peak in O2– accumulation. Immediately after wounding, we would expect the initial [ATP] present in the ECM to be higher than the low micromolar level reported here. However, our measurement of an average of 40 µM ATP remaining in the extracellular fluid within 3 min after wounding suggests that levels of ATP within the range that induces O2– accumulation persist for some period of time after wounding.
There are additional mechanisms for ATP release into the ECM. Plasma membrane proteins from the ATP-binding cassette transporter family can release ATP into the ECM, and the overexpression of an ATP-binding cassette transporter family member MDR1 in Arabidopsis resulted in increased levels of ATP available on the surface of leaves compared to wild-type plants (Thomas et al., 2000
Our observation that transgenic plants OE apyrase show muted O2– production responses to applied ATP and to wounding suggests that increased apyrase expression lowers the effective [ATP] in the vicinity of the postulated receptors that respond to this agonist. Related studies in animal cells suggest that P2 nucleotide receptors and ectoapyrases occur together in a cell surface microenvironment, and that they compete there for the nucleotides that are released from cells during stress or mechanical stimulation (Joseph et al., 2003
As observed in both animal systems and Arabidopsis (Jeter et al., 2004 Our results showing the effects of apyrase overexpression on nucleotide-induced O2– production beg the question whether enhanced apyrase expression will also affect the ability of wound-released ATP to induce O2– production. Figure 3D answers this question in the affirmative, providing further support for the hypothesis that the ATP released at wound sites participates importantly in early steps of the wound-signaling cascade.
The fact that adenosine inhibits ATP-induced O2– production and changes in gene expression, suggests that it may act as a negative regulator of eATP signaling in plants as it does in animals. Ectophosphatases such as apyrase can hydrolyze ATP and ADP to AMP, and 5' nucleotidase hydrolyzes AMP to adenosine (Zimmerman, 1996
In the absence of any pathogens, elicitors, wounding, ozone, or mechanical stress, two temporally distinct peaks of eATP-induced O2– accumulation were observed (Fig. 1C). Biphasic peaks have been described in response to avirulent pathogens, ozone, wounding, and mechanical stress (Lamb and Dixon, 1997
Our finding that the atrbohD/F mutant did not accumulate O2– in response to eATP treatment (Table I) indicates that the NADPH oxidase subunits, AtrbohD and AtrbohF, are required for the production of O2– induced by eATP. Also supporting this conclusion is our finding that the suicide substrate inhibitor of mammalian NADPH oxidase, DPI, suppressed the ATP-induced O2– accumulation. The AtrbohD and AtrbohF subunits and NADPH oxidase activity also appear to be needed for ROS-dependent ABA signaling in Arabidopsis (Kwak et al., 2003
In animal systems, nucleotides fulfill their roles as signals through binding to P2 receptors. Our observation that nucleotides induce O2– production and that two P2 receptor inhibitors, PPADS and RB2, prevent ATP induction of O2– and ATP-induced changes in gene expression is consistent with the hypothesis that in plants, eATP can act as a signal through interaction with a receptor at least functionally similar to the P2 receptors in animals. Moreover, animal P2 receptors are typically activated by a rather broad range of nucleotides (Ralevic and Burnstock, 1998
P2 receptor signaling is mediated through [Ca2+]cyt increases in animals, and Ca2+ is an intermediate signal leading to the ATP-induced respiratory burst (Kuroki and Minakami, 1989
Twelve mammalian P2 receptors with diverse affinities for different nucleotides have been characterized (Di Virgilio et al., 2001
Applied ATP induces the accumulation of transcripts of genes that are also induced by wounding or pathogen infection. Given the release of ATP into the ECM during wounding and the potential for ATP release in response to pathogen attack, our findings suggest that eATP could be an early signaling agent in the stimulus-response pathway leading from wounding and pathogen attack to increased mRNA levels for wound- and defense-response genes such as PAL1, LOX2, and ACS6. PAL1 is a gene involved in the wound or defense response of plants and expression of this gene is induced by ROS, which is accumulated downstream of physical injury or pathogen infection (Levine et al., 1994
The report of Jeter et al. (2004)
Previous studies have reported that O2– can induce programmed cell death (Levine et al., 1994
We propose a speculative model for the induction of O2– production by eATP and its tight control by NADPH oxidase homologs in Arabidopsis (Fig. 7
). The model depicts the ATP receptor and the linked calcium channel as conceptually two separate entities, but the receptor could also be a P2X type, which is itself a ligand-gated calcium channel. The model predicts that following a wound or other stimulus resulting in the disruption of the plasma membrane (Mehdy et al., 1996
Plant Material
For O2– assays, Arabidopsis (Arabidopsis thaliana) ecotype Wassilewskija or atrbohD/F double mutant and ecotype Columbia-0 were grown on Metro Mix 350 (Hummert) under continuous light for 4 to 5 weeks. The seeds for the atrbohD/F double mutant were obtained from J. Kwak (Kwak et al., 2003
O2– accumulation was detected according to Jabs et al. (1996)
Rosette leaves were pressure infiltrated using a syringe with no needle with 1, 5, 10, 50, or 100 µM ATP, ADP, or AMP. PB alone was infiltrated as the negative control and OGA (10 µg/mL) as the positive control. All leaves were incubated for 1 h at room temperature. We chose 1 h to ensure uniformity of treatment conditions. The leaves were cut off immediately and immersed in 10 mM potassium buffer and 10 mM NaN3, then O2– was detected as described above in "O2– Detection." OGA was obtained from M. Mehdy. For RNA analysis, whole seedlings were submerged gently in 10 mL of MES pH 5.7 (0.5 g/L) alone, and 1, 5, 50, or 100 µM ATP. ATP solutions were dissolved in MES pH 5.7. After submerging the seedlings, they were vacuum infiltrated for 30 s and the vacuum was broken quickly. The seedlings were treated for 30 min and the solutions were poured out. The seedlings were collected at 30, 60, 90, and 180 min after the initial treatment and immediately frozen in liquid nitrogen and stored at –80°C for RNA isolation.
Arabidopsis rosette leaves were infiltrated with 50 µM ATP dissolved in PB pH 7.5 (0.16 mM KH2PO4, 1.1 mM K2HPO4), PB alone, or xanthine (2 mM; Sigma)/xanthine oxidase (5 units; Sigma) as the positive control. Leaves were cut from the plant after 3, 30, 60, 120, 240, and 480 min and immediately immersed in 10 mM potassium buffer and 10 mM NaN3. O2– was detected as described in "O2– Detection."
Full-grown atrbohD/F double knockout rosette leaves or wild-type Columbia-0 rosette leaves were infiltrated with either 50 µM ATP in PB or PB alone. Leaves were incubated at room temperature for 1 h, cut off the plants, and immersed in 10 mM potassium buffer and 10 mM NaN3. O2– was detected as described above in "O2– Detection."
Rosette leaves of mature Arabidopsis plants were detached, placed on a microscope slide, and wounded with a micropipette. Wounds were typically 3 to 4 mm long at the edge of the leaf and cut completely through the leaf. Fluid from the wound site was collected with a micropipette positioned with a manual micromanipulator. The volume of fluid collected was calculated from the height of column of fluid in the micropipette and the measured dimensions of the tip of the micropipette. Fluid volumes typically ranged between 0.1 and 7.0 nL. Immediately after collection, the tip of the micropipette was snapped off in a 1.5 mL microcentrifuge tube and plunged in liquid N2. Typically, less than 3 min elapsed between wounding and freezing of the collected sample. A new wound site was created for each fluid collection, although the same leaf was used for more than one wound. Two to four collections were pooled together in the same microcentrifuge tube and stored at –80°C for [ATP] determination. The concentration of ATP present in each of the samples was determined using a bioluminescent detection reagent (ENLITEN rLuciferase/Luciferin; Promega). Frozen pooled collections were resuspended in 10 mL of buffer (10 mM HEPES, pH 7.7), vortexed, spun down quickly, and transferred to a 12 x 50 mm test tube for measurement. Fifty microliters of rLuciferase/Luciferin reagent was added to the resuspended sample and luminescence was measured in a luminometer (Turner Designs 20/20; Turner BioSystems) using a 2 s delay and a 10 s integration. The amount of ATP present in the sample was calculated from the measured relative light units using a standard curve spanning the relative light unit range obtained from the samples. The accuracy of this approach was validated by sampling from source pipettes containing known ATP standards. The methods for collection and concentration determination were the same as described above for the wound samples and the calculated concentrations were compared against the known concentrations of the sample.
Rosette leaves were infiltrated with PB alone, 50 µM ATP, 50 µM ATP plus inhibitor, or inhibitor alone. The leaves were incubated at room temperature for 1 h, cut, and immersed in 10 mM potassium buffer and 10 mM NaN3. O2– was detected as described in "O2– Detection." The inhibitors used were 250 µM DPI, 250 µM PPADS, 30 µM RB2, 10 µM adenosine, 1 mM lanthanum chloride (LaCl3), 1 mM 1,2-Bis(2-amino-5-bromophenoxy)ethane-N,N,N',N'-tetraacetic acid, 50 µM W7, and 50 µM W5. All inhibitors were dissolved in distilled deionized water except DPI, which was dissolved in dimethyl sulfoxide, and for this treatment a dimethyl sulfoxide-only control was added. All inhibitors were obtained from Sigma except for W5 (Calbiochem). Seedlings were pretreated with inhibitor or MES. Ten milliliter solutions of MES or inhibitor (PPADS, RB, or adenosine) were poured gently into the petri dishes containing seedlings. They were briefly vacuum infiltrated (30 s) and the vacuum was broken quickly. The seedlings incubated in the solutions for 10 min, and the solutions were poured out. Treatments of either 50 µM ATP or MES for the negative control were added to the seedlings. The seedlings were again vacuum infiltrated for a brief time (30 s) and incubated in the solutions for 30 min. The solutions were poured out, and the seedlings were collected at 30, 60, 90, and 180 min. The seedlings were immediately frozen in liquid nitrogen and stored at –80°C for RNA isolation.
RNA was isolated using the RNeasy kit (Qiagen) according to the manufacturer's instructions. Ten micrograms of RNA was denatured by incubation with a glyoxyl loading dye (Ambion) for 30 min at 50°C. All of the RNA was separated by electrophoresis and transferred onto a Bright star nylon membrane (Ambion). The RNA was cross-linked to the membrane using short UV light for 2 min. The northern analysis was done using the northern Max-Gly kit (Ambion) according to the manufacturer's instructions. Radioactivity was detected using a Phosphorimager (model 445SI; Molecular Dynamics). Specific cDNA probes for AtrbohD, PAL1, LOX2, or ACS6 were hybridized to the membranes. Probes were randomly labeled with dCTP-
The AtAPY2 cDNA (GenBank accession no. AF141671) existed as an insert in the TA cloning site of the vector pCR2.1 (Invitrogen; Steinebrunner et al., 2000
All leaves were analyzed with ImageJ (http://rsb.info.nih.gov/ij/index.html). Stained areas were measured by measuring pixels of staining and divided by total leaf area to normalize from leaf to leaf. This ratio was multiplied by 100 to obtain the percentage of stained leaf area. The northern analysis with inhibitor treatments were also analyzed using Image J. The areas of the bands from the northern image were measured, as well as the bands from the equal loading gel. The expression levels were adjusted according to the equal loading bands. The MES buffer treatment was designated as 100 and the other treatments are relative to the MES buffer. The resulting graphs show the relative expression levels. Statistical analyses were done using the Student's t-test in Microsoft Excel. Error bars are all SD. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AF141671 (AtAPY1), AF055357 (AtrbohD), L23968 (LOX2), NM_129260 (PAL1), and NM_117199 (ACS6).
We thank Dr. M. Mehdy for the OGA and for thoughtful discussions about the manuscript and research, T. Butterfield for independent confirmation of [ATP] at wound sites, and Dr. W. Tang for sharing primers and labeled probe for ACS6. Received October 19, 2005; returned for revision January 2, 2006; accepted January 2, 2006.
1 This work was supported by grants from the National Science Foundation (IBN–0080363 and IBN–0344221 to S.J.R.) and from the National Aeronautics and Space Administration (NGT5–50371 to S.C.S.). C.J.S. was supported in part by a grant to June M. Kwak from the National Research Initiative of the U.S. Department of Agriculture (2004–35100–14909). 
2 Present address: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland 20742–5815.
3 Present address: Section of Molecular Biotechnology, Technical University of Dresden, 01069 Dresden, Germany. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Stanley J. Roux (sroux{at}uts.cc.utexas.edu). Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.073072. * Corresponding author; e-mail sroux{at}uts.cc.utexas.edu; fax 512–232–3402.
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ABSTRACT
RESULTS
8; two independent experiments). All ATP and ADP treatments are statistically significant compared to the buffer or AMP treatments (P 
[Ca2+]cyt in mediating the effects of ATP on gene expression. Similarly, our finding that PPADS and two other antagonists of P2-like receptors block the accumulation of transcripts for AtrbohD and PAL lends support to the idea that these ATP effects could be mediated by a detection system with pharmacological properties similar to the animal P2 receptors. 
32P (NEN-Perkin Elmer) using the DECAprime II kit (Ambion) according to the manufacturer's instructions. The primers used for AtrbohD (