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Keywords:

  • calcium;
  • diacylglycerol kinase;
  • extracellular ATP;
  • nitric oxide;
  • phosphatidic acid;
  • phospholipase C;
  • phospholipase D;
  • tomato cells

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
  • In animals and plants, extracellular ATP exerts its effects by regulating the second messengers Ca2+, nitric oxide (NO) and reactive oxygen species (ROS). In animals, phospholipid-derived molecules, such as diacylglycerol, phosphatidic acid (PA) and inositol phosphates, have been associated with the extracellular ATP signaling pathway. The involvement of phospholipids in extracellular ATP signaling in plants, as it is established in animals, is unknown.
  • In vivo phospholipid signaling upon extracellular ATP treatment was studied in 32Pi-labeled suspension-cultured tomato (Solanum lycopersicum) cells.
  • Here, we report that, in suspension-cultured tomato cells, extracellular ATP induces the formation of the signaling lipid phosphatidic acid. Exogenous ATP at doses of 0.1 and 1 mm induce the formation of phosphatidic acid within minutes. Studies on the enzymatic sources of phosphatidic acid revealed the participation of both phospholipase D and C in concerted action with diacylglycerol kinase.
  • Our results suggest that extracellular ATP-mediated nitric oxide production is downstream of phospholipase C/diacylglycerol kinase activation.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

In animal cells, ATP is well established as an extracellular signal in a number of cellular responses (Fredholm et al., 1994; Burnstock & Williams, 2000). Extracellular ATP (eATP) is perceived through the purine P2 receptors P2X (ion channels) and P2Y (G protein-coupled receptors) (Ralevic & Burnstock, 1998). Both P2X and P2Y receptors affect directly or indirectly intracellular Ca2+ signaling, resulting in a variety of downstream cellular responses. P2Y receptors act via G protein coupling to activate phospholipase C (PLC), which hydrolyzes phosphatidylinositol 4,5-bisphosphate, leading to the formation of diacylglycerol (DAG) in the membrane and inositol 1,4,5-trisphosphate (IP3) in the cytosol. IP3 triggers Ca2+ release from intracellular stores, stimulating a variety of signaling pathways, including phospholipase A2, Ca2+-dependent K+ channels and nitric oxide synthase (NOS). Downstream DAG, phosphatidylcholine-specific PLC, phospholipase D (PLD), the mitogen-activated protein kinase (MAPK) pathway and Ca2+ influx via voltage-operated Ca2+ channels are being activated. Although the presence of plant P2-like receptors has been suggested (Song et al., 2006), the molecular components involved in the eATP signaling pathway are poorly known.

Phospholipids are emerging as novel second messengers in plant cells. They are formed rapidly in response to a variety of stimuli via the activation of lipid kinases or phospholipases. In particular, phosphatidic acid (PA) has emerged as a second messenger in plants. It accumulates rapidly in response to drought stress, abscisic acid treatments, salt stress, wounding and during pathogenic and mutualistic interactions (Testerink & Munnik, 2005). PA triggers an oxidative burst and the PA activation of NADPH oxidase has been suggested (Sang et al., 2001; de Jong et al., 2004; Park et al., 2004) and recently confirmed (Zhang et al., 2009). PA can be generated directly via PLD activation or via PLC in concerted action with diacylglycerol kinase (DGK). Considering that phospholipases are downstream components of eATP perception in animals (Ralevic & Burnstock, 1998) and that, in plants, phospholipases and lipid-derived molecules regulate diverse responses, we hypothesized a similar scenario in eATP-induced plant responses. In this work, we investigated the effect of eATP on PA production in suspension-cultured tomato cells.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Materials

ATP, ADP, AMP and ATP-γ-S, the PLC inhibitors U73122, its less active analogue U73343 and neomycin, the DGK-specific inhibitor R59022 and LaCl3 were purchased from Sigma (St Louis, MO, USA). Reagents for lipid extractions and subsequent analysis, as well as Silica-60 thin layer chromatography (TLC) plates, were purchased from Merck (Darmstadt, Germany). The fluorescent probe 4,5-diaminofluorescein diacetate was purchased from Molecular Probes (Eugene, OR, USA).

Cell suspensions

Suspension-cultured cells (Solanum lycopersicum L. cv Money Maker; line Msk8; Felix et al., 1991) were grown at 24°C in the dark at 125 rpm in Murashige and Skoog medium (Duchefa, Haarlem, the Netherlands) supplemented with 5.4 μm 1-naphthalene acetic acid, 1 μm 6-benzyladenine and vitamins (Duchefa), as described previously (Felix et al., 1991).

32P phospholipid labeling and analyses

Eighty-five microliters of Msk8 cells were labeled for 3 h with 5 μCi carrier-free 32PO43− (Perkin Elmer, Boston, USA) in 2 ml reaction vials before treatment with 85 μl of ATP or ATP-γ-S for the time periods and at the concentrations indicated. Controls were performed by the addition of cell free medium to the cells. The inhibitors neomycin, ethylene glycol tetraacetic acid (EGTA) and LaCl3 were pre-incubated for 10 min before ATP treatments. When indicated, treatments were performed in the presence of 0.5% (v/v) n-butanol. Incubations were stopped and lipids were extracted and processed as described previously (Munnik et al., 1996). Lipids were separated on Silica-60 TLC plates (Merck) employing ethyl acetate (EtAc)/iso-octane/formic acid/H2O (13 : 2 : 3 : 10, v/v) (Munnik et al., 1998) as mobile phase. Radioactivity was visualized by autoradiograph. Autoradiographs represent general phenomena, and are representative of at least three individual experiments. Radiolabeled phospholipids were quantified by phosphoimaging (Molecular Dynamics, Sunnyvale, CA, USA). PA levels were quantified against total radiolabeled lipids; the PA level of each sample was subsequently expressed as the fold change, taking the PA level of the control as one.

Quantification of nitric oxide (NO) production by fluorometry

Fluorometric measurements were performed in a Fluoroskan Ascent microwell fluorometer (Thermo Electron Company, Vantaa, Finland) according to Laxalt et al. (2007). Cultured cells (100 μl) were pre-incubated with 0.5 μm 4,5-diaminofluorescein diacetate for 30 min in the absence or presence of inhibitors. Thereafter, the cells were subjected to ATP treatments, as indicated in the figures. The gradient of NO production throughout all of the experiments of each treatment was expressed as the fold increase, taking the NO level of the control as unity. All experiments were performed in triplicate. At least four independent experiments were performed.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

ATP induces PA formation

To investigate whether exogenously added ATP induces PA formation, cultured cells were labeled with 32Pi for 3 h and then stimulated with different concentrations of ATP for 30 min. In vivo levels of phospholipids were analyzed by TLC (Fig. 1). ATP-treated cells showed no variation in the structural phospholipids, such as phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI) and phosphatidylcholine (PC), compared with nontreated cells (Fig. 1a). However, the levels of PA varied according to the ATP doses applied (Fig. 1a). The quantification of these variations showed biphasic PA accumulation with the highest levels detected at 0.1 and 1 mm ATP (Fig. 1b). Tomato cells were treated with the nonhydrolyzable ATP (ATP-γ-S) as an agonist of the P2-like receptor. Fig. 1(a,b) shows that 0.1 mm ATP-γ-S clearly stimulates PA production. A 10-times lower dose of 0.1 mm ATP-γ-S induces PA production (data not shown), suggesting a receptor mediation of eATP-induced PA production in tomato cells. ADP and AMP treatments did not induce a significant increase in PA levels (Fig. 1b). The ATP doses used did not modify cell viability measured at 120 min or overnight (data not shown). The formation of PA was also time dependent (Fig. 1c). Using 0.1 and 1 mm ATP, increases in PA levels were detected from 30 min, reaching 1.5- and 2.5-fold at 60 min, respectively (Fig. 1d). Time course experiments with 0.1 mm ATP-γ-S showed an earlier PA accumulation (data not shown). Our results indicate that exogenous applications of ATP unequivocally induce PA production.

image

Figure 1.  Exogenous ATP induces phosphatidic acid (PA) production in tomato cells. Suspension-cultured tomato cells were labeled with 32Pi for 3 h and then treated: (a, b) with different doses of ATP, 0.1 mm ATP-γ-S, 1 mm AMP or 1 mm ADP for 30 min; (c, d) for different times with 0 (circles), 0.1 (squares) or 1 mm (triangles) ATP. Lipids were extracted and separated by ethyl acetate thin-layer chromatography (EtAc TLC). (a, c) Representative autoradiographs are shown. The structural phospholipids phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI) and phosphatidylcholine (PC) are indicated. (b, d) Quantification of six independent experiments is plotted. PA levels are expressed as the fold increase in relation to control samples (b) or in relation to t0 (d). Error bars indicate the standard error (SE) of the means (= 6). (b) Asterisks indicate treatments that were significantly different with respect to the control treatment (Student’s t-test, < 0.05).

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PA production can occur via PLC/DGK and/or PLD. First, we tested whether PLD contributes to ATP-induced PA formation. The assay involved the measurement of the in vivo transfer of the phosphatidyl group from PLD’s substrate to the primary alcohol 1-butanol. The subsequent formation of phosphatidylbutanol (PBut) is a relative measure of PLD activity (Munnik et al., 1995). PLD activity was therefore measured in cells prelabeled with 32Pi for 3 h, and subsequently treated with 0.1 and 1 mm ATP in the presence of 0.5% (v/v) 1-butanol for different periods of time. Fig. 2(a) shows the accumulation of PBut upon 0.1 or 1 mm ATP treatments. In 1 mM ATP-treated cells PBut was detected within 5 min, implying rapid PLD activation. In 0.1 mm ATP-treated cells, PBut production was low at the beginning of the experiment, reaching maximum levels from 30 min onwards. Thus, exogenous ATP induces the activation of PLD in tomato cells.

image

Figure 2.  Extracellular ATP (eATP) activates phospholipase D (PLD) and phospholipase C (PLC)/diacylglycerol kinase (DGK) pathways. Suspension-cultured tomato cells were labeled with 32Pi for 3 h and then treated with 0 (open circles), 0.1 (closed circles) or 1 mm (squares) ATP. (a) Treatments were performed in the presence of 0.5% (v/v) 1-butanol, and PLD activity was assayed by the accumulation of phosphatidylbutanol (PBut) at the times indicated. Lipids were separated by ethyl acetate thin layer chromatography (EtAc TLC) and the radioactivity in PBut was quantified by phosphoimaging. PBut levels are expressed relative to the control cells. Error bars indicate the standard error (SE) of the means (n = 6). (b) Treatments were performed in the presence of different concentrations of the PLC inhibitor neomycin (0 μm, black bars; 10 μm, grey bars; 100 μm, white bars). Lipids were extracted and separated by EtAc TLC and the radioactivity in phosphatidic acid (PA) was quantified by phosphoimaging. PA levels are expressed as the fold increase in relation to control samples. Error bars indicate SE of the means (= 3). Means denoted with the same letter do not differ significantly at < 0.05 according to one-way ANOVA.

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To test whether ATP induces PA formation via the PLC/DGK pathway, the PLC inhibitor neomycin was assayed. Neomycin inhibits the formation of PLC-generated PA by chelating the PLC substrates PIP and PIP2 (de Jong et al., 2004). Cells labeled with 32Pi for 3 h were pre-incubated for 10 min with different concentrations of neomycin, and subsequently treated with 0.1 or 1 mm ATP for 30 min. Fig. 2(b) shows that neomycin inhibits ATP-induced PA accumulation. In conclusion, the addition of exogenous ATP activated the PLC/DGK and PLD pathways, generating the second messenger PA.

PLC, DGK and extracellular Ca2+ influx are required for ATP-induced NO production

In animals, one of the downstream effectors of eATP-induced PLC activation is the production of NO (Ralevic & Burnstock, 1998). Since in plants, NO production on eATP treatment has been reported in tomato cell suspensions, as well as in Salvia miltiorrhiza hairy roots and in Arabidopsis pollen tubes (Foresi et al., 2007; Wu & Wu, 2008; Reichler et al., 2009), we evaluated whether ATP-induced NO production requires PLC/DGK activation. The activities of PLC and DGK, and the downstream responses, are often probed using the inhibitors U73122 and neomycin to inhibit PLC and R59022 to inhibit DGK (den Hartog et al., 2001; de Jong et al., 2004; Laxalt et al., 2007). U73122 inhibits the hydrolysis of phosphatidylinositol 4,5-bisphosphate by a recombinant plant PI-PLC (Staxen et al., 1999), and R59022 inhibits the activity of recombinant Arabidopsis DGK 2 and 7 (Gomez-Merino et al., 2004, 2005). Cultured cells of 4–5 d old were exposed to ATP treatments in the presence or absence of the PLC inhibitors neomycin or U73122, and NO production was measured. Fig. 3 shows that 0.1 and 1 mm ATP induces NO production, as reported previously (Foresi et al., 2007). The PLC inhibitors diminish ATP-induced NO production (Fig. 3a,b), whereas the inactive analogue U73343 has no effect on ATP-induced NO production (Fig. 3c). Fig. 3(d) shows that the DGK inhibitor blocks eATP-induced NO production (Fig. 3d), indicating that NO production requires PLC and DGK activation in ATP-treated tomato cells.

image

Figure 3.  Phospholipase C (PLC) and diacylglycerol kinase (DGK) inhibitors reduce nitric oxide (NO) levels. Tomato cells were pre-incubated for 30 min with different concentrations of the PLC inhibitors (a) neomycin or (b) U73122, (c) the inactive analogue U73433 or (d) the DGK inhibitor R59022, and then treated with 0.1 or 1 mm ATP in the presence of the NO-specific fluorescent probe (DAF-FM-DA). The gradient of NO production throughout 60 min was calculated and expressed as the fold change with respect to non-treated cells. Error bars indicate the standard error (SE) of the means (= 4). Means denoted with the same letter do not differ significantly at < 0.05 according to one-way ANOVA.

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As NO production has been shown to require Ca2+ influx from the extracellular environment in ATP-treated hairy roots (Wu & Wu, 2008), NO production in tomato cells was measured in the presence of La3+ as a plasma membrane Ca2+-permeable channel inhibitor (White, 2000). La3+ competes for Ca2+-binding sites on the plasma membrane. Previously, Gelli & Blumwald (1997) have reported, in Msk8 tomato-cultured cells, a Ca2+ voltage-dependent channel sensitive to La3+ (Gelli & Blumwald, 1997). Fig. 4(a) shows that La3+ inhibits ATP-induced NO production in a dose-dependent manner (see Supporting Information Fig. S1(a)). As it has been reported that La3+ has intracellular effects (Polisensky & Braam, 1996; Liu & Hasenstein, 2005) we also performed experiments in the presence of EGTA as an extracellular Ca2+ chelator. As expected, EGTA inhibits ATP-induced NO production in a dose-dependent manner (see Supporting Information Fig. S1(a)). Subsequently, we studied whether ATP-induced PA production was dependent on Ca2+ influx through the plasma membrane. Fig. 4(b) shows that La3+ is unable to inhibit ATP-induced PA production. Surprisingly, the incubation of cells with La3+ was sufficient to raise PA levels above the control (Fig. 4b). Moreover, the addition of 5 or 10 mm La3+ to 1 mm ATP-treated cells increased PA levels up to three-fold, clearly showing no inhibition of ATP-induced PA production, whereas NO levels were effectively reduced (Fig. 4a,b). Similar effects of La3+ treatments have been reported previously (Polisensky & Braam, 1996), but still the role of La3+ as an extracellular Ca2+ channel inhibitor has been questioned (Liu & Hasenstein, 2005). The extracellular Ca2+ chelator EGTA did not affect ATP-induced PA levels (Fig. S1b). Thus, pharmacological evidence suggests that ATP-induced NO accumulation, but not PA, requires the activation of plasma membrane Ca2+-permeable channels.

image

Figure 4.  Requirements of extracellular Ca2+ on nitric oxide (NO) and phosphatidic acid (PA) production in ATP-treated tomato cells. Cells were pre-incubated for 15 min with different concentrations of La3+ and then treated with 0.1 or 1 mm ATP. (a) The gradient of NO production throughout 60 min was calculated and expressed as the fold change with respect to non-treated cells. Error bars indicate the standard error (SE) of the means (= 4). (b) PA levels after 30 min of treatment are expressed as the fold increase in relation to control samples. Error bars indicate SE of the means (= 3). Means denoted with the same letter do not differ significantly at < 0.05 according to one-way ANOVA.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

In this article, we have provided evidence for PA production upon eATP treatment in tomato cells. The results show that ATP increases PA levels via the activation of PLD and PLC/DGK, demonstrating a novel signaling pathway in eATP-treated tomato cells. In addition, our results show that PLC and DGK are required for ATP-induced NO production.

In animals, eATP is perceived through the purine P2 receptors P2X (ion channels) and P2Y (G protein-coupled receptors) (Ralevic & Burnstock, 1998). ATP, ADP, UTP and UDP are agonists of P2Y receptors, whereas only ATP is the main agonist of P2X (Ralevic & Burnstock, 1998). Efforts have been directed towards the identification of eATP receptors in plants. The genomes of Arabidopsis and rice do not encode proteins with obvious similarity to either P2X or P2Y receptors (Moriyama et al., 2006; Roux & Steinebrunner, 2007; Lu et al., 2009). Thus, higher plants seem to use different or highly divergent receptors to sense extracellular nucleotides. Plants respond to ATP and ADP (Roux & Steinebrunner, 2007) or only to ATP (Kim et al., 2006; Wu & Wu, 2008). In suspension-cultured tomato cells, PA production is detectable upon addition of exogenous ATP or the agonist ATP-γ-S, but is not detected by the addition of ADP or AMP. Similarly, ADP and AMP induce slight NO production in suspension-cultured tomato cells (Foresi et al., 2007).

In suspension-cultured tomato cells, eATP induces biphasic PA accumulation with peaks at 0.1 and 1 mm. Unexpectedly, 0.5 mm ATP does not induce an intermediate PA response. Bimodal effects of ATP have been reported in mesangial cells and interpreted as the activation of different receptors depending on the ATP doses applied (Schulman et al., 1999). Roux & Steinebrunner (2007), Wu & Wu (2008) and Tonón et al. (2010) have suggested that eATP is beneficial at certain concentrations, but becomes inhibitory at excessive concentrations. ATP concentrations of 0.1 and 1 mm induce PA accumulation and NO production in tomato cultured cells. The half-life of ATP in the cell medium of tomato cultured cells, measured according to Weerasinghe et al. (2009), is approx. 30 min (data not shown). This could explain the differences in the ATP doses used and the times of the responses compared with other biological systems. Hence, experiments with ATP-γ-S have shown the accumulation of PA at earlier time points (data not shown). In Arabidopsis roots, sequential touch stimulation revealed a strong refractory period for ATP release in response to mechanostimulation (Weerasinghe et al., 2009). Thus, the ATP doses applied in order to generate a physiological response are dependent on the microenvironment location and the level of hydrolytic activity known to be present in plant cell walls. Accordingly, examples of millimolar doses that trigger a response have been reported in plants (Roux & Steinebrunner, 2007). Physiologically, eATP concentrations may increase depending on the situation (Jeter et al., 2004). During wounding, the cell membrane is broken and millimolar ATP concentrations may be released into the extracellular space (Jeter et al., 2004). In addition, other stress conditions, such as mechanical stimuli and osmotic stress, induce ATP release (Jeter et al., 2004; Weerasinghe et al., 2009). More recently, eATP levels have been closely correlated with regions of active growth and cell expansion (Kim et al., 2006). Remarkably, increases in PA production have been reported in the above-mentioned processes (Munnik & Meijer, 2001; Bargmann et al., 2009a,b; Han & Yuan, 2009; Zhang et al., 2009). Nevertheless, a direct link between eATP and PA has never been shown in plants. In this article, we report PA as a second messenger in eATP signaling in tomato cells. The enzymatic source of eATP-induced PA production was also studied. Our data show that ATP triggers PA production via PLD and PLC in concerted action with DGK.

In animals, eATP-induced NO production is downstream of PLC activation (Burnstock & Williams, 2000). In tomato cell suspensions, eATP induces both PLD and PLC/DGK pathways, and ATP-induced NO production is downstream of PLC/DGK activation. However, our previous results have shown that NO is upstream of PA formation during plant defense responses, stomatal closure and adventitious root formation. During elicitor treatments in tomato cell suspensions, NO regulates PA production and subsequent downstream responses via activation of the PLC/DGK pathway, independent of PLD activation (Laxalt et al., 2007). PLC inhibitors affecting PA levels reduce reactive oxygen species (ROS) production, but not NO, during the plant defense response in tomato cell suspensions (Laxalt et al., 2007). In guard cells, PLC and PLD activities are required for NO-induced PA formation and stomatal closure (Distéfano et al., 2008). In adventitious root formation, auxin-induced NO production triggers PA formation via PLD and not via PLC/DGK (Lanteri et al., 2008). In eATP signalling, PA lies upstream of NO production. As Arabidopsis has 12 PLD, seven PLC and seven DGK genes (Munnik, 2001), it is a subject for future studies to elucidate which phospholipase and kinase isoforms are being activated upon eATP perception.

As more data become available, it is becoming evident that eATP regulates different physiological processes through the second messengers Ca2+, ROS and NO. Demidchik et al. (2009) have shown that eATP causes the production of ROS through the activation of the specific plasma membrane NADPH oxidase. ROS, in turn, activates plasma membrane voltage-dependent Ca2+ channels. The authors have proposed that an eATP-induced release of Ca2+ from internal stores (by an unknown mechanism) lies at the beginning of [Ca2+]cyt elevation, and this activates NADPH oxidase (Demidchik et al., 2009). Yet another molecule involved in NADPH oxidase activation is PA (Zhang et al., 2009).

PA is known to act as a key mediator of signaling pathways, and a number of PA targets have been identified, suggesting that PA is involved in many processes (Testerink & Munnik, 2005). PA has been shown to activate OXI1, a protein kinase implicated in oxidative burst-mediated signaling in Arabidopsis (Deak et al., 1999; Anthony et al., 2004). Stress conditions that cause ATP release, such as touch, low temperature and salinity, involve AtMAPK3, which functions downstream of OXI1 (Rentel et al., 2004). eATP causes NADPH oxidase-dependent ROS production, and AtMAPK3 transcription lies downstream of the ATP activation of the NADPH oxidase RHD2/AtRBOHC (Demidchik et al., 2009). In macrophages, PA and Ca2+ are involved in the activation of NADPH oxidase (McPhail et al., 1999). In plants, PA is able to trigger an oxidative burst, suggesting a similar activation of NADPH oxidase (de Jong et al., 2004; Park et al., 2004), and Ca2+ and PA have been shown to activate NADPH oxidase in Arabidopsis (Ogasawara et al., 2008; Takeda et al., 2008; Zhang et al., 2009). ROS production in Arabidopsis root hairs by RHD2 NADPH oxidase stimulates Ca2+ influx which, in turn, activates RHD2 NADPH oxidase, constituting a positive feedback regulation in the expanding root hair cells (Takeda et al., 2008). Moreover, PLDα1-derived PA interacts and activates the NADPH oxidase AtRBOH and downstream NO production in abscisic acid-treated guard cells (Zhang et al., 2009). In addition, eATP-induced NO accumulation requires the activation of plasma membrane Ca2+ channels in tomato cells (our results) and in hairy roots (Wu & Wu, 2008). The above-presented evidence, together with our results, suggests that, during eATP perception, the second messengers PA, Ca2+, ROS and NO are interconnected. We have shown that ATP-induced NO production requires both Ca2+ and PA. However, whether Ca2+ is downstream or independent of PA remains to be elucidated. More precise information about the nature of the mechanisms that mediate eATP-induced PA production will contribute to an understanding of the role of PA in this signaling pathway.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank Drs L. Lanteri and G. Gonorazky for critical reading of the manuscript. This work was financially supported by Universidad Nacional de Mar del Plata (UNMdP) (AML, LL, CAC), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) (NPF, CAC, LL, AML) and Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) (DJS, CAC, LL, AML).

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  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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