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diacylglycerol kinase, requires nitric oxide
Journal of Plant Physiology 168 (2011) 534–539
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Phosphatidic acid production in chitosan-elicited tomato cells, via both phospholipase D and phospholipase C/diacylglycerol kinase, requires nitric oxide Nicolás Raho 1,2 , Leonor Ramirez 1 , M. Luciana Lanteri, Gabriela Gonorazky, Lorenzo Lamattina, Arjen ten Have, Ana M. Laxalt ∗ Instituto de Investigaciones Biológicas-CONICET, Universidad Nacional de Mar del Plata, CC 1245, 7600 Mar del Plata, Argentina
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Article history: Received 2 June 2010 Received in revised form 10 September 2010 Accepted 10 September 2010 Keywords: Lipid signaling Oxidative burst Pathogen associated molecular pattern Plant defense
a b s t r a c t Nitric oxide (NO) and the lipid second messenger phosphatidic acid (PA) are involved in plant defense responses during plant–pathogen interactions. NO has been shown to be involved in the induction of PA production in response to the pathogen associated molecular pattern (PAMP) xylanase in tomato cells. It was shown that NO is critical for PA production induced via phospholipase C (PLC) in concerted action with diacylglycerol kinase (DGK) but not for the xylanase-induced PA via phospholipase D (PLD). In order to study whether this is a general phenomenon during PAMP perception or if it is particular for xylanase, we studied the effect of the PAMP chitosan in tomato cell suspensions. We observed a rapid NO production in tomato cells treated with chitosan. Chitosan induced the formation of PA by activating both PLD and PLC/DGK. The activation of either phospholipase-mediated signaling pathway was inhibited in cells treated with the NO scavenger cPTIO. This indicates that NO is required for PA generation via both the PLD and PLC/DGK pathway during plant defense response in chitosan elicited cells. Responses downstream PA were studied. PLC inhibitors neomycin and U73122 inhibited chitosaninduced ROS production. Differences between xylanase and chitosan-induced phospholipid signaling pathways are discussed. © 2010 Elsevier GmbH. All rights reserved.
Introduction Plants are constantly challenged by different pathogens. In order to resist these pathogens they can activate a battery of responses or defense mechanisms generally referred to as the plant defense response. The first step in the induction of the plant defense response is the recognition of certain pathogen-derived molecules that are referred to as elicitors. Downstream signal transduction cascades become activated upon recognition of these elicitors. One of the second messengers reported to participate in plant defense signaling responses is the lipid phosphatidic acid (PA)
Abbreviations: CFM, cell free medium; cPTIO, 2-(4carboxyphenylalanine)4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium; DAG, diacylglycerol; DGK, diacylglycerol kinase; DGPP, diacylglycerol pyrophosphate; NO, nitric oxide; PA, phosphatidic acid; PAMP, pathogen associated molecular pattern; PBut, phosphatidylbutanol; PIP2 , phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; PLD, phospholipase D; ROS, reactive oxygen species; TLC, thin layer chromatography. ∗ Corresponding author. Tel.: +54 223 4753030; fax: +54 223 4724143. E-mail address: [email protected] (A.M. Laxalt). 1 These authors contribute equally to the article. 2 Present address: Centro de Biología Molecular Severo Ochoa, Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid, Spain. 0176-1617/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2010.09.004
(Laxalt and Munnik, 2002). PA has been shown to accumulate upon treatment with the Pathogen-Associated Molecular Patterns (PAMPs, elicitors that are produced by a broad range of pathogens) xylanase, N-acetylchitotetraose and flagellin in tomato cells (van der Luit et al., 2000) as well as N-acetylchitooligosaccharide in rice cells (Yamaguchi et al., 2003, 2005). PA has also been shown to accumulate upon treatment with race specific elicitor Avr4 in Cf-4 expressing tobacco cells (de Jong et al., 2004), and during AvrRpm1 and AvrRpt2-induced disease resistance responses in Arabidopsis (Andersson et al., 2006). Nod factors have also been reported to induce PA in alfalfa cells (den Hartog et al., 2003). PA can be generated via two pathways. Phospholipase C (PLC) hydrolyses phosphatidylinositol 4,5-bisphosphate (PIP2 ) into two second messengers: inositol 1,4,5-trisphosphate described as a signal for Ca2+ release from internal stores, and diacylglycerol (DAG). In plants, DAG is rapidly phosphorylated by DAG kinase (DGK) generating PA (Testerink and Munnik, 2005). Another PA enzymatic source in plants is the phospholipase D (PLD). PLD hydrolyses structural phospholipids such as phosphatidylcholine (PC), generating PA (Wang, 2000). PA has been shown to be able to trigger an oxidative burst in plants (Testerink and Munnik, 2005). In addition, Ca2+ and PA activate NADPH oxidase in Arabidopsis (Ogasawara et al., 2008; Takeda et al., 2008; Zhang et al., 2009). NADPH oxidase is responsible for reactive oxygen species (ROS) production in plant defense (Torres and Dangl, 2005). The various roles of PA in plant
N. Raho et al. / Journal of Plant Physiology 168 (2011) 534–539
signaling have been extensively reviewed (Testerink and Munnik, 2005). Another second messenger recently established in plants is nitric oxide (NO) (Lamattina et al., 2003; Neill et al., 2003; Delledonne, 2005). NO is involved in the plant defense response of a growing list of plant–pathogen interactions (Distéfano et al., 2010). The interaction between NO and PA has been shown in different physiological plant systems (for review see Distéfano et al., 2010). We showed that in xylanase-treated tomato cells, NO is required for PLC/DGK activation (Laxalt et al., 2007). It has also been demonstrated that chitosan induces NO production in Pisum sativum guard cells (Srivastava et al., 2009). Chitosan is a deacylated derivative of chitin, a major component of the fungal cell wall. It is a linear polysaccharide composed of randomly distributed -(1-4)-linked d-glucosamine (deacetylated unit) and N-acetyl-d-glucosamine (acetylated unit). Chitosan serves as a molecular pattern for the recognition of potential pathogens in the innate immune systems of plants (Nurnberger et al., 2004). Chitosan perception activates plant defense responses, which include ROS production, MAPK activation and extracellular medium alkalization (Iriti and Faoro, 2009). Although a plasma membrane receptor for chitin fragments has been characterized in plants (Iriti and Faoro, 2009), the signal transduction pathway activated by chitosan is not well defined. Here we study the role of NO on phospholipase activation in chitosan treated tomato cells in order to demonstrate whether the interaction between NO and PA is a general PAMP response or whether it is particular for xylanase.
Materials and methods Chemicals Chitosan was prepared from crab shells (Sigma, St. Louis, MO, USA) according to Hadwiger and Beckman (1980) and Shepherd et al. (1997). All control experiments were performed in presence of the corresponding concentration of the vehicle NaAc. Reagents for lipid extractions and subsequent analysis, as well as silica 60-thin layer chromatography (TLC) plates were purchased from Merck (Darmstadt, Germany). The nitric oxide (NO) scavenger 2-(4-carboxyphenyl)-4,4,5,5tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO) and the fluorescent probes 4,5-diaminofluorescein diacetate (DAF-FMDA), DAF-2DA and 2 ,7 -dichlorofluorescein diacetate (H2 DCF-DA) were purchased from Molecular Probes (Eugene, OR, USA). The phospholipase C (PLC) inhibitors U73122 and neomycin were purchased from Sigma. Cell suspensions Suspension cultured tomato cells (Solanum lycopersicum cv. Money Maker; line Msk8) were grown at 25 ◦ C in the dark at 125 rpm in MS medium (Duchefa, Haarlem, The Netherlands) supplemented with 5.4 M NAA, 1 M 6-benzyladenine and vitamins (Duchefa) as described earlier (Laxalt et al., 2007). Treatments Suspension cultured cells of 4–5 days old were exposed to different treatments in 2 mL reaction vials (for microscopy and lipid assays) for different time periods or were incubated in Greiner 96-well plates (for NO and ROS production assays). Inhibitor treatments were performed incubating the cells 30 min prior to the treatment.
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Visualization of NO Endogenous NO was monitored by incubating 100 L of cells with 1 M DAF-2DA (or negative control with fluorophore 4AF DA) for 30 min at 25 ◦ C in the dark. The cells were then subjected to different treatments (for time periods indicated) and mounted on microscope slides. Cells were visualized by fluorescence microscopy with excitation and emission filters of 495 nm and 535 nm respectively, in an Eclipse E 200 microscope (Nikon, Tokyo, Japan). The production of green fluorescence under these conditions was due to NO. Pictures show general phenomena, representative for at least 3 individual experiments.
Quantification of NO and ROS production by fluorometry Fluorometric measurements were performed in a Fluoroskan Ascent microwell fluorometer (Thermo Electron Company, Vantaa, Finland). Eighty microliter batches of cultured cells were pipetted into wells of a Greiner 96-well plate and pre-incubated for 30 min with DAF-FM-DA or H2 DCF-DA (final concentrations of 0.5 M or 4 M respectively) and subsequently incubated with 20 L of cell free medium (CFM) or CFM containing chitosan (final concentration of 50 g mL−1 ) in absence or presence of inhibitors as indicated in figures. The microwell plate was immediately transferred to the fluorometer for measurement using Chroma (Chroma Technology Corp., Rockingham, VT, USA) filters D480-40 and D525-30 for excitation and emission respectively. Fluorescence of each individual well was measured every 1 min over 20 ms at 25 ◦ C. All experiments were performed with three technical replicates.
32 P i
phospholipid labeling and analyses
Eighty-five microliters of Msk8 cells was labeled for 3 h with 5 Ci carrier-free orthophosphate (32 Pi ) (Amersham, Buckinghamshire, UK) prior to treatment with chitosan for time periods and with concentrations as indicated. Control treatments were performed by adding CFM together with NaAc (chitosan vehicle). Incubations were stopped by adding 20 L of 50% perchloric acid. For short-labeling experiments, 85 L of cell suspension was equilibrated in a 2 mL reaction vial for 2 h. Two minutes prior to the addition of 50 g mL−1 chitosan, 20 L of CFM containing 5 Ci 32 P was added to the cells. Incubations were stopped as mentioned i above. Lipids were extracted by adding 3.75 volumes of CHCl3 :MeOH:HCl (50:100:1, v/v) and processed as described before (Laxalt et al., 2007). Lipids were separated on silica-60 TLC plates (Merck) employing EtAc (EtAc/iso-octane/formic acid/H2 O (13:2:3:10, v/v)) as a mobile phase. When indicated, alkaline solvent (CHCl3 :MeOH:[25%, w/v] NH4 OH:H2 O [90:70:4:16, v/v]) and heat-activated impregnated TLC plates (1.2% [w/v] potassium oxalate, 2 mM EDTA in MeOH:H2 O (2:3, v/v)) were used. Radioactivity was visualized by autoradiography. Autoradiographs represent general phenomena, representative for at least 3 individual experiments. Quantification of 32 PA levels was performed by Plot analysis using ImageJ (v1.32j) of non-overexposed autoradiographs. For Fig. 3 32 PA levels were quantified against the standard structural phospholipid levels (32 PA/32 SL) and expressed as fold accumulation taking 32 PA levels of control cells as 1. For Fig. 4 32 PA levels were quantified and the 32 PA levels of chitosan treated cells taken as 100%.
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Fig. 1. Chitosan-treated tomato cells accumulate NO. (A) NO detection by the fluorescent probe DAF-2DA. Cells were treated with 50 g mL−1 chitosan or NaAc (control). Photos were taken 30 min after treatment. A bright-field image is shown for each treatment below the fluorescent image. A representative picture of three independent experiments is shown. Bar = 5 m. (B) Dose– and time–response curves of NO production. Cells were treated with 0, 10, 50 or 100 g mL−1 chitosan in the presence of the NO specific fluorescent probe DAF-FM-DA. Fluorescence was determined using a microwell fluorometer over a 60 min period and expressed as arbitrary fluorescence unit (AU). Error bars represent standard error of means. A representative graph of three independent experiments is shown.
Results Chitosan triggers NO production in tomato cell suspensions To investigate whether the elicitor chitosan is able to trigger NO formation in tomato cell suspensions, we used the NO-specific fluorophore DAF-2DA. Fig. 1A shows that 50 g mL−1 chitosan induced NO production, as compared to control cells. A quantitative dose–response experiment was performed using a fluorometer and the NO specific probe DAF-FM-DA (Fig. 1B). Fig. 1B shows that NO production increased steadily from 10 g mL−1 until 100 g mL−1 chitosan. Chitosan induces the formation of PA via PLD and PLC/DGK activation We then characterized the PA formation in chitosan-treated tomato cells by performing dose–response and time-course experiments. Phospholipids were labeled by incubating the cells with 32 P for 3 h, and subsequently treated with chitosan. The lipids were i extracted and separated by alkaline TLC. Fig. 2A (representative for three independent experiments) shows the phospholipid profile obtained after 30 min treatment with different doses of chitosan. Chitosan-treated cells showed no variation of the structural phospholipids phosphatidylglycerol (PG), phosphatidylethanolamine (PE), PC or phosphatidylinositol (PI) when compared with non-
Fig. 2. Chitosan stimulates PA formation via PLD and PLC/DGK activation. (A and B) Suspension-cultured tomato cells were labeled with 32 Pi for 3 h and then treated with or without chitosan. Lipids were extracted and separated by TLC. (A) Cells were treated with different doses of chitosan for 30 min. Lipids were separated in an alkaline TLC system. PG, phosphatidylglycerol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PI, phosphatidylinositol; DGPP, diacylglycerol pyrophosphate; PIP, phosphatidylinositol monophosphate; PIP2 , phosphatidylinositol 4,5-bisphosphate. (B) Cells were treated with 50 g mL−1 of chitosan for different times (min) in the presence or absence of 0.5% 1-butanol. Lipids were separated in EtAc TLC system. SL, structural phospholipids, PBut, phosphatidylbutanol. (C) Suspension-cultured tomato cells were labeled with 32 Pi for 2 min and then treated with or without 50 g mL−1 chitosan for the indicated time (min). Lipids were extracted and separated by alkaline TLC system. A representative autoradiograph is shown (n = 3).
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Fig. 4. Chitosan-induced PLC/DGK pathway requires NO. Suspension-cultured tomato cells were labeled with 32 Pi for 2 min and then treated with or without 50 g mL−1 chitosan for 10 min in presence or absence of 1 mM cPTIO. Lipids were extracted and separated by EtAc TLC. PA levels were quantified and expressed as percentage, 100% corresponding to the relative PA levels in chitosan-treated cells in the absence of cPTIO. Error bars represent standard error of means (n = 4).
Fig. 3. NO is required for chitosan-induced PA production and PLD activation. Suspension-cultured tomato cells were labeled with 32 Pi for 3 h and then treated with 50 g mL−1 chitosan for 30 min in presence of different doses of the NO scavenger cPTIO. All experiments were performed in 0.5% 1-butanol. Lipids were extracted and separated by EtAc TLC. (A) A representative autoradiograph is shown. SL, structural phospholipids. PA (B) and PBut (C) levels were quantified and expressed as fold increase in relation to control samples. Error bars represent standard error of means (n = 4).
treated cells (Fig. 2A). However, the levels of PA increased according to the chitosan doses applied, with the highest levels of PA in 50 g mL−1 chitosan treated cells (Fig. 2A). In addition, the levels of diacylglycerol pyrophosphate (DGPP), a product of PA phosphorylation, increased in a dose dependent manner (Fig. 2A). Phosphatidylinositol monophosphate (PIP) and PIP2 levels decreased with 50 and 100 g mL−1 chitosan (Fig. 2A), suggesting that its metabolism could be linked to the increase in PA and DGPP. PA can be generated via two pathways, PLD and/or PLC/DGK activation. We first tested whether PLD contributes to the chitosaninduced PA formation shown in Fig. 2A. PLD activity is measured in vivo, which consists in the quantification of the transfer of the phosphatidyl group from its substrate to a primary alcohol, such as 1-butanol. The level of the product, phosphatidylbutanol (PBut), is a relative measure of PLD activity (Munnik, 2001). Therefore, cells were pre-labeled with 32 Pi for 3 h and subsequently treated with
50 g mL−1 chitosan for different periods of time in the presence of 0.5% (v/v) 1-butanol. Fig. 2B (right panel) shows higher PBut levels in 30 min chitosan treated cells compared to the levels in control cells. Thus, chitosan induces PLD activation in tomato cells. As a control of 1-butanol treatments, cells were treated with chitosan in absence of 1-butanol. PA levels increased gradually in chitosan treated cells throughout all the experiment regardless the presence of 1-butanol (Fig. 2B). This suggests that 0.5% 1-butanol does not affect chitosan-induced PA formation. We performed a short labeling strategy (Munnik, 2001) in order to elucidate whether chitosan induces PA formation via PLC/DGK. The strategy is based on the fact that 32 Pi is slowly incorporated into structural phospholipids, but rapidly into the ATP pool, which is subsequently used by DGK to phosphorylate the PLC-derived DAG to 32 PA. In contrast, the 32 PA derived from PLD requires long 32 Pi labeling times. Accordingly, tomato cells were labeled for 2 min and treated with 50 g mL−1 chitosan. Fig. 2C shows the pattern of 32 P -short labeled phospholipids. In contrast to long labeling experi iments (Fig. 2A) the radioactivity in the structural phospholipids gradually increases throughout the experiment (Fig. 2C, control, see also van der Luit et al., 2000). When cells were treated with chitosan, the level of 32 PA rapidly accumulated compared to the level in the control (Fig. 2C). This response coincided with a decrease in the levels of PIP and PIP2 , putative substrates for PLC (Fig. 2C). Moreover, the PLC inhibitor neomycin (de Jong et al., 2004) reduced the chitosan-induced PA accumulation (data not shown). These results show that chitosan induces PLC/DGK activation in tomato cells. NO is required for chitosan-induced PA production via PLD and PLC/DGK We further studied whether NO was required for PA production. For that, PA production was measured in presence of the NO scavenger, cPTIO. Chitosan-induced PA levels decreased when cells were co-incubated with cPTIO in a dose dependent manner (Fig. 3A and B). Fig. 3A and C shows that PBut levels are reduced in the presence of cPTIO in a dose dependent manner. This demonstrates that NO is required for PLD activation during chitosan treatments. We also studied whether NO was required for the chitosaninduced PLC/DGK activation. PLC/DGK activities were assayed in the same conditions as indicated in Fig. 2C. Chitosan-induced PA levels were quantified at 10 min after chitosan treatments in presence of 1 mM cPTIO (Fig. 4). The NO scavenger cPTIO blocked chitosantriggered PA production. These studies indicate that NO is required for the activation of PLD and PLC/DGK.
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Fig. 5. PLC inhibitors block chitosan-induced ROS production. Cells were treated for 30 min with 50 g mL−1 chitosan in the absence or presence of different doses of U73122 or neomycin (M). ROS levels were determined in a microwell fluorometer, using H2 DCF-DA. The gradient of ROS production throughout 30 min was calculated and expressed as % of maximal production taking the levels of chitosan treated cells as 100%. Error bars represent standard error of means (n = 4). Means denoted with the same letter do not significantly differ at P < 0.05 according to one-way ANOVA.
ROS in chitosan treated cells ROS production is one of the earliest responses to pathogen attack and has been shown to occur upon chitosan elicitation (Iriti and Faoro, 2009). We studied whether PA was required for ROS production in chitosan treated cells. ROS generation was quantified in a fluorometer using the ROS fluorescent probe H2 DCF-DA (Laxalt et al., 2007). We analyzed the effect of inhibiting PLC/DGK derived PA on chitosan-induced ROS production. For this, we employed the PLC inhibitors neomycin and U73122 (de Jong et al., 2004). Fig. 5 shows that both PLC-inhibitors reduced the chitosan-induced ROS production. Inhibition of PA formation via PLD is achieved using a primary alcohol. Fig. 2B shows that 0.5% of 1-butanol does not significantly changed chitosan-induced PA levels compared to the PA levels in absence of the alcohol. Accordingly, 0.5% of 1-butanol does not affect ROS production triggered by chitosan (data not shown). Higher doses of 1-butanol induced ROS production in tomato cells (data not shown). Therefore, we could not measure whether PLD activity is required for chitosan-induced ROS production. Discussion NO and PA have emerged as second messengers in signaling in plants and in plant defense signaling in particular. We have shown that NO and PA interact in plant signaling (for recent review see Distéfano et al., 2010). In this report we show the induction of NO and PA in tomato cells by chitosan, a widely used elicitor of plant defense responses. Chitosan induces NO in tomato cells (Fig. 1). Furthermore chitosan induces PA formation via two pathways, PLD and PLC/DGK (Fig. 2) and both require NO for their activation (Figs. 3 and 4). Finally, chitosan induces ROS for which PLC activation appears to be required. Previously, chitosan was shown to induce NO production in guard cells of Pisum sativum and to be involved in chitosan-induced systemic resistance in pearl millet against downy mildew disease (Manjunatha et al., 2009; Srivastava et al., 2009). Chitotetraose, a tetramer of N-acetyl-d-glucosamine, induces PA production via PLC/DGK activation and not via PLD in tomato and alfalfa cells (van der Luit et al., 2000; den Hartog et al., 2003). In rice cell suspensions, N-acetylchito-oligosaccharide treatment activates rapidly and transiently PLD and PLC (Yamaguchi et al., 2003, 2005). It is not known whether NO is required for these PA inductions. Irrespective of the exact role that NO plays in response to chitin oligosaccharides, there are differences on PA generating
pathways that are being activated, depending on the type of the chitin-derived molecules, even though recognition seems to be widely conserved among plant species (Kaku et al., 2006). Also other PAMPs have been shown to induce NO and PA. Xylanase treatment induces PLC/DGK and PLD activation in tomato cells (van der Luit et al., 2000). However, only the PLC/DGK and not the PLD activation requires NO (Laxalt et al., 2007). Besides PAMPs, also certain race specific elicitors induce NO and PA formation. Recognition of the race-specific elicitor AVR4 from Cladosporium fulvum, activates PLC/DGK and not PLD (de Jong et al., 2004) and the formation of PA also appears to be NO-dependent (Laxalt et al., unpublished). Thus, the interaction between NO and PA is a general PAMP and race specific response. However, depending on the elicitor, it seems that there is specificity on the activation of the phospholipase pathway and the requirement of NO. Chitosan not only induces PLC/DGK activation via NO, like in xylanase- or AVR4-treated cells, but also PLD. This corresponds to the idea that during plant perception of the pathogen, many signaling components appear to be shared between race-specific and non-race specific defense responses (Nurnberger et al., 2004). A number of questions remain to be answered. Firstly, how NO activates phospholipases is still unknown. NO could act directly on PLDs, PLCs and/or DGKs either by nitrosylation of cystein residues or nitration of tyrosine residues. Indirect activation can be envisaged by means of regulation of either Ca2+ homeostasis or protein kinases (extensively discussed by Distéfano et al., 2010). Secondly, it remains unclear how PA is able to induce such variety of responses. One of the downstream PA targets is the activation of the NADPH oxidase, with a subsequent ROS production. In xylanase treated cells, scavenging of NO or inhibition of PLC/DGK pathway diminished xylanase-induced ROS production (Laxalt et al., 2007). Previously, Yamaguchi and collaborators showed that PLD and PLC/DGK activation triggered by N-acetyl-chitooligosaccharide treatment was required for the first phase of ROS production (Yamaguchi et al., 2005). In addition, recognition of the specific elicitors AVR4, AvrRpm1 or AvrRpt2 induced PA accumulation and downstream, ROS production (de Jong et al., 2004; Andersson et al., 2006). Our results show that chitosan induces ROS production within minutes and PLC activity is required for this response (Fig. 5). We suggest that NO-dependent, PLC/DGK-generated PA is involved in the induction of ROS production during race and non-race specific plant defense. It is however not clear whether NO-induced PLD derived PA is also capable of inducing ROS. In tomato cell suspensions, extracellular ATP induces both PLD and PLC/DGK pathways (Sueldo et al., 2010) and NO production (Foresi et al., 2007). Converse to the signaling during plant-defense response, in extracellular ATP signaling PA lies upstream of NO production (Sueldo et al., 2010). Moreover, extracellular ATP triggers ROS production via NADPH oxidase, and NO has been shown to be downstream the NADPH oxidase AtRBOH in abscisic acid-treated guard cells (Zhang et al., 2009). Genetic approaches would be required to unequivocally demonstrate the proposed order of events following elicitor’s perception in plants. Acknowledgments This work was financially supported by Universidad Nacional de Mar del Plata (UNMdP) (LL, AtH, AML), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) (MLL, LL, AtH, AML) and Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) (NR, LR, MLL, GG, LL, AtH, AML). References Andersson MX, Kourtchenko O, Dangl JL, Mackey D, Ellerstrom M. Phospholipasedependent signalling during the AvrRpm1- and AvrRpt2-induced disease resistance responses in Arabidopsis thaliana. Plant J 2006;47:947–59.
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Phosphatidic acid production in chitosan-elicited tomato cells, via both phospholipase D and phospholipase C/diacylglycerol kinase, requires nitric oxide Nicolás Raho 1,2 , Leonor Ramirez 1 , M. Luciana Lanteri, Gabriela Gonorazky, Lorenzo Lamattina, Arjen ten Have, Ana M. Laxalt ∗ Instituto de Investigaciones Biológicas-CONICET, Universidad Nacional de Mar del Plata, CC 1245, 7600 Mar del Plata, Argentina
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Article history: Received 2 June 2010 Received in revised form 10 September 2010 Accepted 10 September 2010 Keywords: Lipid signaling Oxidative burst Pathogen associated molecular pattern Plant defense
a b s t r a c t Nitric oxide (NO) and the lipid second messenger phosphatidic acid (PA) are involved in plant defense responses during plant–pathogen interactions. NO has been shown to be involved in the induction of PA production in response to the pathogen associated molecular pattern (PAMP) xylanase in tomato cells. It was shown that NO is critical for PA production induced via phospholipase C (PLC) in concerted action with diacylglycerol kinase (DGK) but not for the xylanase-induced PA via phospholipase D (PLD). In order to study whether this is a general phenomenon during PAMP perception or if it is particular for xylanase, we studied the effect of the PAMP chitosan in tomato cell suspensions. We observed a rapid NO production in tomato cells treated with chitosan. Chitosan induced the formation of PA by activating both PLD and PLC/DGK. The activation of either phospholipase-mediated signaling pathway was inhibited in cells treated with the NO scavenger cPTIO. This indicates that NO is required for PA generation via both the PLD and PLC/DGK pathway during plant defense response in chitosan elicited cells. Responses downstream PA were studied. PLC inhibitors neomycin and U73122 inhibited chitosaninduced ROS production. Differences between xylanase and chitosan-induced phospholipid signaling pathways are discussed. © 2010 Elsevier GmbH. All rights reserved.
Introduction Plants are constantly challenged by different pathogens. In order to resist these pathogens they can activate a battery of responses or defense mechanisms generally referred to as the plant defense response. The first step in the induction of the plant defense response is the recognition of certain pathogen-derived molecules that are referred to as elicitors. Downstream signal transduction cascades become activated upon recognition of these elicitors. One of the second messengers reported to participate in plant defense signaling responses is the lipid phosphatidic acid (PA)
Abbreviations: CFM, cell free medium; cPTIO, 2-(4carboxyphenylalanine)4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium; DAG, diacylglycerol; DGK, diacylglycerol kinase; DGPP, diacylglycerol pyrophosphate; NO, nitric oxide; PA, phosphatidic acid; PAMP, pathogen associated molecular pattern; PBut, phosphatidylbutanol; PIP2 , phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; PLD, phospholipase D; ROS, reactive oxygen species; TLC, thin layer chromatography. ∗ Corresponding author. Tel.: +54 223 4753030; fax: +54 223 4724143. E-mail address: [email protected] (A.M. Laxalt). 1 These authors contribute equally to the article. 2 Present address: Centro de Biología Molecular Severo Ochoa, Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid, Spain. 0176-1617/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2010.09.004
(Laxalt and Munnik, 2002). PA has been shown to accumulate upon treatment with the Pathogen-Associated Molecular Patterns (PAMPs, elicitors that are produced by a broad range of pathogens) xylanase, N-acetylchitotetraose and flagellin in tomato cells (van der Luit et al., 2000) as well as N-acetylchitooligosaccharide in rice cells (Yamaguchi et al., 2003, 2005). PA has also been shown to accumulate upon treatment with race specific elicitor Avr4 in Cf-4 expressing tobacco cells (de Jong et al., 2004), and during AvrRpm1 and AvrRpt2-induced disease resistance responses in Arabidopsis (Andersson et al., 2006). Nod factors have also been reported to induce PA in alfalfa cells (den Hartog et al., 2003). PA can be generated via two pathways. Phospholipase C (PLC) hydrolyses phosphatidylinositol 4,5-bisphosphate (PIP2 ) into two second messengers: inositol 1,4,5-trisphosphate described as a signal for Ca2+ release from internal stores, and diacylglycerol (DAG). In plants, DAG is rapidly phosphorylated by DAG kinase (DGK) generating PA (Testerink and Munnik, 2005). Another PA enzymatic source in plants is the phospholipase D (PLD). PLD hydrolyses structural phospholipids such as phosphatidylcholine (PC), generating PA (Wang, 2000). PA has been shown to be able to trigger an oxidative burst in plants (Testerink and Munnik, 2005). In addition, Ca2+ and PA activate NADPH oxidase in Arabidopsis (Ogasawara et al., 2008; Takeda et al., 2008; Zhang et al., 2009). NADPH oxidase is responsible for reactive oxygen species (ROS) production in plant defense (Torres and Dangl, 2005). The various roles of PA in plant
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signaling have been extensively reviewed (Testerink and Munnik, 2005). Another second messenger recently established in plants is nitric oxide (NO) (Lamattina et al., 2003; Neill et al., 2003; Delledonne, 2005). NO is involved in the plant defense response of a growing list of plant–pathogen interactions (Distéfano et al., 2010). The interaction between NO and PA has been shown in different physiological plant systems (for review see Distéfano et al., 2010). We showed that in xylanase-treated tomato cells, NO is required for PLC/DGK activation (Laxalt et al., 2007). It has also been demonstrated that chitosan induces NO production in Pisum sativum guard cells (Srivastava et al., 2009). Chitosan is a deacylated derivative of chitin, a major component of the fungal cell wall. It is a linear polysaccharide composed of randomly distributed -(1-4)-linked d-glucosamine (deacetylated unit) and N-acetyl-d-glucosamine (acetylated unit). Chitosan serves as a molecular pattern for the recognition of potential pathogens in the innate immune systems of plants (Nurnberger et al., 2004). Chitosan perception activates plant defense responses, which include ROS production, MAPK activation and extracellular medium alkalization (Iriti and Faoro, 2009). Although a plasma membrane receptor for chitin fragments has been characterized in plants (Iriti and Faoro, 2009), the signal transduction pathway activated by chitosan is not well defined. Here we study the role of NO on phospholipase activation in chitosan treated tomato cells in order to demonstrate whether the interaction between NO and PA is a general PAMP response or whether it is particular for xylanase.
Materials and methods Chemicals Chitosan was prepared from crab shells (Sigma, St. Louis, MO, USA) according to Hadwiger and Beckman (1980) and Shepherd et al. (1997). All control experiments were performed in presence of the corresponding concentration of the vehicle NaAc. Reagents for lipid extractions and subsequent analysis, as well as silica 60-thin layer chromatography (TLC) plates were purchased from Merck (Darmstadt, Germany). The nitric oxide (NO) scavenger 2-(4-carboxyphenyl)-4,4,5,5tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO) and the fluorescent probes 4,5-diaminofluorescein diacetate (DAF-FMDA), DAF-2DA and 2 ,7 -dichlorofluorescein diacetate (H2 DCF-DA) were purchased from Molecular Probes (Eugene, OR, USA). The phospholipase C (PLC) inhibitors U73122 and neomycin were purchased from Sigma. Cell suspensions Suspension cultured tomato cells (Solanum lycopersicum cv. Money Maker; line Msk8) were grown at 25 ◦ C in the dark at 125 rpm in MS medium (Duchefa, Haarlem, The Netherlands) supplemented with 5.4 M NAA, 1 M 6-benzyladenine and vitamins (Duchefa) as described earlier (Laxalt et al., 2007). Treatments Suspension cultured cells of 4–5 days old were exposed to different treatments in 2 mL reaction vials (for microscopy and lipid assays) for different time periods or were incubated in Greiner 96-well plates (for NO and ROS production assays). Inhibitor treatments were performed incubating the cells 30 min prior to the treatment.
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Visualization of NO Endogenous NO was monitored by incubating 100 L of cells with 1 M DAF-2DA (or negative control with fluorophore 4AF DA) for 30 min at 25 ◦ C in the dark. The cells were then subjected to different treatments (for time periods indicated) and mounted on microscope slides. Cells were visualized by fluorescence microscopy with excitation and emission filters of 495 nm and 535 nm respectively, in an Eclipse E 200 microscope (Nikon, Tokyo, Japan). The production of green fluorescence under these conditions was due to NO. Pictures show general phenomena, representative for at least 3 individual experiments.
Quantification of NO and ROS production by fluorometry Fluorometric measurements were performed in a Fluoroskan Ascent microwell fluorometer (Thermo Electron Company, Vantaa, Finland). Eighty microliter batches of cultured cells were pipetted into wells of a Greiner 96-well plate and pre-incubated for 30 min with DAF-FM-DA or H2 DCF-DA (final concentrations of 0.5 M or 4 M respectively) and subsequently incubated with 20 L of cell free medium (CFM) or CFM containing chitosan (final concentration of 50 g mL−1 ) in absence or presence of inhibitors as indicated in figures. The microwell plate was immediately transferred to the fluorometer for measurement using Chroma (Chroma Technology Corp., Rockingham, VT, USA) filters D480-40 and D525-30 for excitation and emission respectively. Fluorescence of each individual well was measured every 1 min over 20 ms at 25 ◦ C. All experiments were performed with three technical replicates.
32 P i
phospholipid labeling and analyses
Eighty-five microliters of Msk8 cells was labeled for 3 h with 5 Ci carrier-free orthophosphate (32 Pi ) (Amersham, Buckinghamshire, UK) prior to treatment with chitosan for time periods and with concentrations as indicated. Control treatments were performed by adding CFM together with NaAc (chitosan vehicle). Incubations were stopped by adding 20 L of 50% perchloric acid. For short-labeling experiments, 85 L of cell suspension was equilibrated in a 2 mL reaction vial for 2 h. Two minutes prior to the addition of 50 g mL−1 chitosan, 20 L of CFM containing 5 Ci 32 P was added to the cells. Incubations were stopped as mentioned i above. Lipids were extracted by adding 3.75 volumes of CHCl3 :MeOH:HCl (50:100:1, v/v) and processed as described before (Laxalt et al., 2007). Lipids were separated on silica-60 TLC plates (Merck) employing EtAc (EtAc/iso-octane/formic acid/H2 O (13:2:3:10, v/v)) as a mobile phase. When indicated, alkaline solvent (CHCl3 :MeOH:[25%, w/v] NH4 OH:H2 O [90:70:4:16, v/v]) and heat-activated impregnated TLC plates (1.2% [w/v] potassium oxalate, 2 mM EDTA in MeOH:H2 O (2:3, v/v)) were used. Radioactivity was visualized by autoradiography. Autoradiographs represent general phenomena, representative for at least 3 individual experiments. Quantification of 32 PA levels was performed by Plot analysis using ImageJ (v1.32j) of non-overexposed autoradiographs. For Fig. 3 32 PA levels were quantified against the standard structural phospholipid levels (32 PA/32 SL) and expressed as fold accumulation taking 32 PA levels of control cells as 1. For Fig. 4 32 PA levels were quantified and the 32 PA levels of chitosan treated cells taken as 100%.
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Fig. 1. Chitosan-treated tomato cells accumulate NO. (A) NO detection by the fluorescent probe DAF-2DA. Cells were treated with 50 g mL−1 chitosan or NaAc (control). Photos were taken 30 min after treatment. A bright-field image is shown for each treatment below the fluorescent image. A representative picture of three independent experiments is shown. Bar = 5 m. (B) Dose– and time–response curves of NO production. Cells were treated with 0, 10, 50 or 100 g mL−1 chitosan in the presence of the NO specific fluorescent probe DAF-FM-DA. Fluorescence was determined using a microwell fluorometer over a 60 min period and expressed as arbitrary fluorescence unit (AU). Error bars represent standard error of means. A representative graph of three independent experiments is shown.
Results Chitosan triggers NO production in tomato cell suspensions To investigate whether the elicitor chitosan is able to trigger NO formation in tomato cell suspensions, we used the NO-specific fluorophore DAF-2DA. Fig. 1A shows that 50 g mL−1 chitosan induced NO production, as compared to control cells. A quantitative dose–response experiment was performed using a fluorometer and the NO specific probe DAF-FM-DA (Fig. 1B). Fig. 1B shows that NO production increased steadily from 10 g mL−1 until 100 g mL−1 chitosan. Chitosan induces the formation of PA via PLD and PLC/DGK activation We then characterized the PA formation in chitosan-treated tomato cells by performing dose–response and time-course experiments. Phospholipids were labeled by incubating the cells with 32 P for 3 h, and subsequently treated with chitosan. The lipids were i extracted and separated by alkaline TLC. Fig. 2A (representative for three independent experiments) shows the phospholipid profile obtained after 30 min treatment with different doses of chitosan. Chitosan-treated cells showed no variation of the structural phospholipids phosphatidylglycerol (PG), phosphatidylethanolamine (PE), PC or phosphatidylinositol (PI) when compared with non-
Fig. 2. Chitosan stimulates PA formation via PLD and PLC/DGK activation. (A and B) Suspension-cultured tomato cells were labeled with 32 Pi for 3 h and then treated with or without chitosan. Lipids were extracted and separated by TLC. (A) Cells were treated with different doses of chitosan for 30 min. Lipids were separated in an alkaline TLC system. PG, phosphatidylglycerol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PI, phosphatidylinositol; DGPP, diacylglycerol pyrophosphate; PIP, phosphatidylinositol monophosphate; PIP2 , phosphatidylinositol 4,5-bisphosphate. (B) Cells were treated with 50 g mL−1 of chitosan for different times (min) in the presence or absence of 0.5% 1-butanol. Lipids were separated in EtAc TLC system. SL, structural phospholipids, PBut, phosphatidylbutanol. (C) Suspension-cultured tomato cells were labeled with 32 Pi for 2 min and then treated with or without 50 g mL−1 chitosan for the indicated time (min). Lipids were extracted and separated by alkaline TLC system. A representative autoradiograph is shown (n = 3).
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Fig. 4. Chitosan-induced PLC/DGK pathway requires NO. Suspension-cultured tomato cells were labeled with 32 Pi for 2 min and then treated with or without 50 g mL−1 chitosan for 10 min in presence or absence of 1 mM cPTIO. Lipids were extracted and separated by EtAc TLC. PA levels were quantified and expressed as percentage, 100% corresponding to the relative PA levels in chitosan-treated cells in the absence of cPTIO. Error bars represent standard error of means (n = 4).
Fig. 3. NO is required for chitosan-induced PA production and PLD activation. Suspension-cultured tomato cells were labeled with 32 Pi for 3 h and then treated with 50 g mL−1 chitosan for 30 min in presence of different doses of the NO scavenger cPTIO. All experiments were performed in 0.5% 1-butanol. Lipids were extracted and separated by EtAc TLC. (A) A representative autoradiograph is shown. SL, structural phospholipids. PA (B) and PBut (C) levels were quantified and expressed as fold increase in relation to control samples. Error bars represent standard error of means (n = 4).
treated cells (Fig. 2A). However, the levels of PA increased according to the chitosan doses applied, with the highest levels of PA in 50 g mL−1 chitosan treated cells (Fig. 2A). In addition, the levels of diacylglycerol pyrophosphate (DGPP), a product of PA phosphorylation, increased in a dose dependent manner (Fig. 2A). Phosphatidylinositol monophosphate (PIP) and PIP2 levels decreased with 50 and 100 g mL−1 chitosan (Fig. 2A), suggesting that its metabolism could be linked to the increase in PA and DGPP. PA can be generated via two pathways, PLD and/or PLC/DGK activation. We first tested whether PLD contributes to the chitosaninduced PA formation shown in Fig. 2A. PLD activity is measured in vivo, which consists in the quantification of the transfer of the phosphatidyl group from its substrate to a primary alcohol, such as 1-butanol. The level of the product, phosphatidylbutanol (PBut), is a relative measure of PLD activity (Munnik, 2001). Therefore, cells were pre-labeled with 32 Pi for 3 h and subsequently treated with
50 g mL−1 chitosan for different periods of time in the presence of 0.5% (v/v) 1-butanol. Fig. 2B (right panel) shows higher PBut levels in 30 min chitosan treated cells compared to the levels in control cells. Thus, chitosan induces PLD activation in tomato cells. As a control of 1-butanol treatments, cells were treated with chitosan in absence of 1-butanol. PA levels increased gradually in chitosan treated cells throughout all the experiment regardless the presence of 1-butanol (Fig. 2B). This suggests that 0.5% 1-butanol does not affect chitosan-induced PA formation. We performed a short labeling strategy (Munnik, 2001) in order to elucidate whether chitosan induces PA formation via PLC/DGK. The strategy is based on the fact that 32 Pi is slowly incorporated into structural phospholipids, but rapidly into the ATP pool, which is subsequently used by DGK to phosphorylate the PLC-derived DAG to 32 PA. In contrast, the 32 PA derived from PLD requires long 32 Pi labeling times. Accordingly, tomato cells were labeled for 2 min and treated with 50 g mL−1 chitosan. Fig. 2C shows the pattern of 32 P -short labeled phospholipids. In contrast to long labeling experi iments (Fig. 2A) the radioactivity in the structural phospholipids gradually increases throughout the experiment (Fig. 2C, control, see also van der Luit et al., 2000). When cells were treated with chitosan, the level of 32 PA rapidly accumulated compared to the level in the control (Fig. 2C). This response coincided with a decrease in the levels of PIP and PIP2 , putative substrates for PLC (Fig. 2C). Moreover, the PLC inhibitor neomycin (de Jong et al., 2004) reduced the chitosan-induced PA accumulation (data not shown). These results show that chitosan induces PLC/DGK activation in tomato cells. NO is required for chitosan-induced PA production via PLD and PLC/DGK We further studied whether NO was required for PA production. For that, PA production was measured in presence of the NO scavenger, cPTIO. Chitosan-induced PA levels decreased when cells were co-incubated with cPTIO in a dose dependent manner (Fig. 3A and B). Fig. 3A and C shows that PBut levels are reduced in the presence of cPTIO in a dose dependent manner. This demonstrates that NO is required for PLD activation during chitosan treatments. We also studied whether NO was required for the chitosaninduced PLC/DGK activation. PLC/DGK activities were assayed in the same conditions as indicated in Fig. 2C. Chitosan-induced PA levels were quantified at 10 min after chitosan treatments in presence of 1 mM cPTIO (Fig. 4). The NO scavenger cPTIO blocked chitosantriggered PA production. These studies indicate that NO is required for the activation of PLD and PLC/DGK.
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Fig. 5. PLC inhibitors block chitosan-induced ROS production. Cells were treated for 30 min with 50 g mL−1 chitosan in the absence or presence of different doses of U73122 or neomycin (M). ROS levels were determined in a microwell fluorometer, using H2 DCF-DA. The gradient of ROS production throughout 30 min was calculated and expressed as % of maximal production taking the levels of chitosan treated cells as 100%. Error bars represent standard error of means (n = 4). Means denoted with the same letter do not significantly differ at P < 0.05 according to one-way ANOVA.
ROS in chitosan treated cells ROS production is one of the earliest responses to pathogen attack and has been shown to occur upon chitosan elicitation (Iriti and Faoro, 2009). We studied whether PA was required for ROS production in chitosan treated cells. ROS generation was quantified in a fluorometer using the ROS fluorescent probe H2 DCF-DA (Laxalt et al., 2007). We analyzed the effect of inhibiting PLC/DGK derived PA on chitosan-induced ROS production. For this, we employed the PLC inhibitors neomycin and U73122 (de Jong et al., 2004). Fig. 5 shows that both PLC-inhibitors reduced the chitosan-induced ROS production. Inhibition of PA formation via PLD is achieved using a primary alcohol. Fig. 2B shows that 0.5% of 1-butanol does not significantly changed chitosan-induced PA levels compared to the PA levels in absence of the alcohol. Accordingly, 0.5% of 1-butanol does not affect ROS production triggered by chitosan (data not shown). Higher doses of 1-butanol induced ROS production in tomato cells (data not shown). Therefore, we could not measure whether PLD activity is required for chitosan-induced ROS production. Discussion NO and PA have emerged as second messengers in signaling in plants and in plant defense signaling in particular. We have shown that NO and PA interact in plant signaling (for recent review see Distéfano et al., 2010). In this report we show the induction of NO and PA in tomato cells by chitosan, a widely used elicitor of plant defense responses. Chitosan induces NO in tomato cells (Fig. 1). Furthermore chitosan induces PA formation via two pathways, PLD and PLC/DGK (Fig. 2) and both require NO for their activation (Figs. 3 and 4). Finally, chitosan induces ROS for which PLC activation appears to be required. Previously, chitosan was shown to induce NO production in guard cells of Pisum sativum and to be involved in chitosan-induced systemic resistance in pearl millet against downy mildew disease (Manjunatha et al., 2009; Srivastava et al., 2009). Chitotetraose, a tetramer of N-acetyl-d-glucosamine, induces PA production via PLC/DGK activation and not via PLD in tomato and alfalfa cells (van der Luit et al., 2000; den Hartog et al., 2003). In rice cell suspensions, N-acetylchito-oligosaccharide treatment activates rapidly and transiently PLD and PLC (Yamaguchi et al., 2003, 2005). It is not known whether NO is required for these PA inductions. Irrespective of the exact role that NO plays in response to chitin oligosaccharides, there are differences on PA generating
pathways that are being activated, depending on the type of the chitin-derived molecules, even though recognition seems to be widely conserved among plant species (Kaku et al., 2006). Also other PAMPs have been shown to induce NO and PA. Xylanase treatment induces PLC/DGK and PLD activation in tomato cells (van der Luit et al., 2000). However, only the PLC/DGK and not the PLD activation requires NO (Laxalt et al., 2007). Besides PAMPs, also certain race specific elicitors induce NO and PA formation. Recognition of the race-specific elicitor AVR4 from Cladosporium fulvum, activates PLC/DGK and not PLD (de Jong et al., 2004) and the formation of PA also appears to be NO-dependent (Laxalt et al., unpublished). Thus, the interaction between NO and PA is a general PAMP and race specific response. However, depending on the elicitor, it seems that there is specificity on the activation of the phospholipase pathway and the requirement of NO. Chitosan not only induces PLC/DGK activation via NO, like in xylanase- or AVR4-treated cells, but also PLD. This corresponds to the idea that during plant perception of the pathogen, many signaling components appear to be shared between race-specific and non-race specific defense responses (Nurnberger et al., 2004). A number of questions remain to be answered. Firstly, how NO activates phospholipases is still unknown. NO could act directly on PLDs, PLCs and/or DGKs either by nitrosylation of cystein residues or nitration of tyrosine residues. Indirect activation can be envisaged by means of regulation of either Ca2+ homeostasis or protein kinases (extensively discussed by Distéfano et al., 2010). Secondly, it remains unclear how PA is able to induce such variety of responses. One of the downstream PA targets is the activation of the NADPH oxidase, with a subsequent ROS production. In xylanase treated cells, scavenging of NO or inhibition of PLC/DGK pathway diminished xylanase-induced ROS production (Laxalt et al., 2007). Previously, Yamaguchi and collaborators showed that PLD and PLC/DGK activation triggered by N-acetyl-chitooligosaccharide treatment was required for the first phase of ROS production (Yamaguchi et al., 2005). In addition, recognition of the specific elicitors AVR4, AvrRpm1 or AvrRpt2 induced PA accumulation and downstream, ROS production (de Jong et al., 2004; Andersson et al., 2006). Our results show that chitosan induces ROS production within minutes and PLC activity is required for this response (Fig. 5). We suggest that NO-dependent, PLC/DGK-generated PA is involved in the induction of ROS production during race and non-race specific plant defense. It is however not clear whether NO-induced PLD derived PA is also capable of inducing ROS. In tomato cell suspensions, extracellular ATP induces both PLD and PLC/DGK pathways (Sueldo et al., 2010) and NO production (Foresi et al., 2007). Converse to the signaling during plant-defense response, in extracellular ATP signaling PA lies upstream of NO production (Sueldo et al., 2010). Moreover, extracellular ATP triggers ROS production via NADPH oxidase, and NO has been shown to be downstream the NADPH oxidase AtRBOH in abscisic acid-treated guard cells (Zhang et al., 2009). Genetic approaches would be required to unequivocally demonstrate the proposed order of events following elicitor’s perception in plants. Acknowledgments This work was financially supported by Universidad Nacional de Mar del Plata (UNMdP) (LL, AtH, AML), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) (MLL, LL, AtH, AML) and Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) (NR, LR, MLL, GG, LL, AtH, AML). References Andersson MX, Kourtchenko O, Dangl JL, Mackey D, Ellerstrom M. Phospholipasedependent signalling during the AvrRpm1- and AvrRpt2-induced disease resistance responses in Arabidopsis thaliana. Plant J 2006;47:947–59.
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