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Ergosterol elicits oxidative burst in tobacco cells via phospholipase A2 and protein kinase C signal pathway
Plant Physiology and Biochemistry 42 (2004) 429–435 www.elsevier.com/locate/plaphy
Original article
Ergosterol elicits oxidative burst in tobacco cells via phospholipase A2 and protein kinase C signal pathway Tomas Kasparovsky a, Jean-Pierre Blein b, Vladimir Mikes a,* a
Department of Biochemistry, Masaryk University, Kotlarska 2, 61137 Brno, Czech Republic b INRA/Université de Bourgogne, (UMR 692), BP 86510, 21065 Dijon, cedex France Received 13 January 2004; accepted 5 April 2004 Available online 11 May 2004
Abstract Ergosterol, a typical fungal sterol, induced in tobacco (Nicotiana tabacum L. cv. Xanthi) suspension cells the synthesis of reactive oxygen species and alkalization of the external medium that are dependent on the mobilization of calcium from internal stores. We used specific inhibitors to elucidate the signal pathway triggered by ergosterol compared with cryptogein, a proteinaceous elicitor of Phytophthora cryptogea. Herbimycin A and genistein, inhibitors of tyrosine protein kinases, had no effect on the oxidative burst and pH changes induced by both elicitors. Similarly, H-89, an inhibitor of protein kinase A, had no effect on the induction of these defense reactions. However, the response to both elicitors was completely blocked by NPC-15437, a specific inhibitor of animal protein kinase C (PKC). The responses induced by cryptogein but not those induced by ergosterol were inhibited by U73122 and neomycin, inhibitors of phospholipase C (PLC). On the other hand, the activity of phospholipase A2 (PLA2) measured using a fluorogenic substrate was stimulated by ergosterol and not by cholesterol and cryptogein. A specific inhibitor of PLA2, arachidonic acid trifluoromethyl ketone (AACOCF3), inhibited the pathway stimulated by ergosterol but not that induced by cryptogein. These results suggest that the cryptogein-induced signal pathway leading to the oxidative burst and DpH changes includes PLC and PKC, whereas this response induced by ergosterol includes PLA2 and PKC. © 2004 Elsevier SAS. All rights reserved. Keywords: Cryptogein; Elicitor; Ergosterol; Oxidative burst; PLA2; Tobacco
1. Introduction Plant defense reactions associated with hypersensitive response (HR) are initiated by pathogen-produced signal molecules, called elicitors. The interaction of elicitors with plant cells is accompanied by some specific physiological perturbations such as ion fluxes across the plasma membrane
Abbreviations: AACOCF3, arachidonic acid trifluoromethyl ketone; AOS, active oxygen species; BODIPY FL, 4,4-difluoro-5,7-dimethyl-4bora-3a,4a- diaza-s-indacene-3-pentanoic acid; DAG, diacylglycerol; FW, fresh weight; HR, hypersensitive response; IP3, inositol 1,4,5-trisphosphate; PCD, programmed cell death; PED6, -((6-(2,4-dinitrophenyl)amino) hexanoyl)-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphoethanolamine; PKA, protein kinase A; PKC, protein kinase C; PLA2, phospholipase A2; PLC, phospholipase C; TMB-8, 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride. * Corresponding author. E-mail address: [email protected] (V. Mikes). © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.plaphy.2004.04.003
(namely Ca2+ influx and K+, Cl– efflux), pH changes, plasma membrane depolarization, oxidative burst and induction of rapid cell death. HR is accompanied by defense genes activation, leading to synthesis of phytoalexins and accumulation of pathogen-related proteins. Two types of elicitors are recognized: general or nonspecific elicitors that do not significantly differ in their effect on different cultivars within a plant species and specific elicitors that occur only in a pathogenic race or strain and function only in plant cultivars carrying the matching disease resistance gene. General elicitors include substances typically associated with basic microbial metabolism, such as cell wall glucans, chitin oligomers, lipids and glycopeptides [3]. For many years, natural plant defense reactions have been attracting high attention. Such studies were focused on plant pathogen recognition, signal transduction and induction of resistance as systemic resistance. Ergosterol is a principal component of the fungal plasma membrane. It triggers a defense reaction in tobacco and tomato cells manifested by active oxygen species (AOS) synthesis, changes of ion fluxes and production of the phy-
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toalexin capsidiol [13,16]. The signal pathway induced after ergosterol recognition is not elucidated. In a previous paper we have studied the effect of calcium channel inhibitors on the induction of defense reactions by ergosterol in tobacco cells [16]. We have found that the inhibition of calcium transport through plasma membrane channels did not influence the oxidative burst and the changes of external pH. On the other hand, the elicitation was blocked when the mobilization of internal calcium stores was suppressed. This paper examines the participation of phospholipases and protein kinases in the elicitation by ergosterol in tobacco suspension cells using several inhibitors of phospholipases and protein kinases. We show that the early events elicited by ergosterol in tobacco cells suspensions are activate by PLA2 and PKC in contrast with the proteinaceous elicitor cryptogein that activates mainly the PLC and PKC signal pathway.
2. Results 2.1. Effect of inhibitors of protein kinases on elicitation of tobacco cells Tyrosine and/or serine/threonine protein kinases participate in signal transduction in all living cells. It has been previously shown that the AOS production and pH changes in tobacco cells induced by ergosterol or cryptogein are strongly reduced by co-treatment with staurosporin [16,20], the compound effectively inhibiting protein Ser/Thr protein kinases in plant cell cultures [18,34]. The addition of herbimycin A (1.0 µM), an inhibitor of tyrosine protein kinase, to tobacco cells 30 min prior to elicitation with cryptogein or ergosterol had no significant effect on the AOS production and pH changes (Table 1). Under the same conditions, herbimycin A alone had no effect. In similar experiments with genistein (40 µM), another tyrosine protein kinase inhibitor, only a slight inhibition of pH changes and AOS synthesis induced by ergosterol was observed, and the elicitation with cryptogein was unaffected (Table 1). Thereafter, we tested the participation of PKA or PKC in the transduction of the signal induced by ergosterol or cryptogein in tobacco cells. The 30 min pre-treatment with H-89 (4 µM), a selective inhibitor of PKA, did not lead to the inhibition of AOS production and pH changes elicited with both elicitors (Table 1). On the other hand, NPC-15437 (10 µM), a selective inhibitor of animal PKC, added 30 min prior to the challenge with ergosterol or cryptogein completely inhibited the extent of AOS production and pH changes induced by both compounds (Table 1). H-89 or NPC-15437 alone induced neither AOS production nor pH changes.
Table 1 Effect of inhibitors of protein kinases, PLC and PLA2 on the AOS production and DpH changes induced by ergosterol or cryptogein. Before adding the elicitor, cells were incubated with the inhibitors for 30 min. AOS concentration and pH changes were monitored for 40 min at 5 and 10 min intervals, respectively. The results are the mean activities of three independent cultures represented in percent relative to the activities of non-inhibited cells, with standard error in parentheses. The values were obtained after 20 and 30 min following elicitation with ergosterol and cryptogein, respectively
Inhibitor 10 µM NPC-15437 2 µM H-89 4 µM H-89 1 µM Herbimycin A 40 µM Genistein 10 µM U-73122 (active) 10 µM U-73343 (inactive) 100 µM Neomycin 28 µM AACOCF3 56 µM AACOCF3
Percentage of activity 50 nM cryptogein AOS (%) DpH (%) 4 (4) 39 (16) 95 (2) 108 (1) 120 (2) 101 (6) 94 (8) 106 (4) 105 (8) 90 (7) 34 (4) 61 (4) 95 (5) 85 (4) 53 (2) 61 (2) 108 (8) 105 (4) 104 (8) 95 (1)
500 nM ergosterol AOS (%) DpH (%) 0 (0) 14 (1) 87 (4) 95 (3) 91 (3) 82 (1) 99 (15) 93 (5) 78 (16) 75 (8) 117 (12) 106 (7) 0 (0) 0 (0) 104 (8) 96 (13) 50 (9) 67 (1) 1 (1) 13 (3)
2.2. Effect of the inhibition of PLC on elicitation of tobacco cells To examine whether the defense response to ergosterol or cryptogein is due to the activation of PLC by elicitor we analyzed the effect of the inhibitors neomycin or U73122. The compound U73343, an inactive analogue, was used as a control [38]. The addition of U73122 (10 µM) 30 min before elicitation with cryptogein suppressed the AOS production by 66% and pH changes by 39% (Table 1), whereas the inactive analogue U73343 had no affect. On the other hand, the pH changes and the production of AOS stimulated by ergosterol were not inhibited by U73122. The effect of the inactive analogue U73343 was contradictory because it totally blocked the elicitation by ergosterol. U73122 or U73343 alone had no effect on AOS production and pH changes. Similar results were obtained with neomycin (100 µM) added 30 min before the elicitation. The AOS production elicited by cryptogein was suppressed by 47% and pH changes by 39% whereas no effect on the elicitation by ergosterol was observed (Table 1). 2.3. Effect of the inhibition of PLA2 on elicitation of tobacco cells To assess the involvement of PLA2 the effect of arachidonic acid trifluoromethyl ketone (AACOCF3) on signal transduction was tested. AACOCF3 (28 µM) caused a 50% decrease in AOS production and a 33% decrease in pH changes when added 30 min prior to the elicitation with ergosterol. These processes were completely blocked by 56 µM AACOCF3. Both concentrations of AACOCF3 alone were inefficient to elicit tobacco cells. In contrast, AACOCF3 at 28 or 56 µM did not inhibit the elicitation induced by cryptogein (Table 1).
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2.4. Measurement of the activity of PLA2 induced by elicitors We examined the effects of ergosterol, cholesterol or cryptogein on the induction of endogenous PLA2 activity in tobacco cells. PED6 is a fluorogenic substrate containing a BODIPY FL dye-labeled sn-2 acyl chain and a dinitrophenyl quencher group. Cleavage of the dye-labeled acyl chain by PLA2 eliminates the intramolecular quenching effect of the dinitrophenyl group, resulting in a corresponding fluorescence increase. The addition of ergosterol induced an obvious increase in PLA2 activity (Fig. 1). Treatment with cryptogein, cholesterol or methanol yielded BODIPY FL fluorescence tracings indistinguishable from controls. In the next experiment, the cell suspension was preincubated for 30 min with AACOCF3 before the addition of ergosterol. The increase in fluorescence of BODIPY FL observed upon the ergosterol treatment was blocked by AACOCF3 confirming that ergosterol-induced PLA2 activity was sensitive to a PLA2 inhibitor (Fig. 2). On the other hand, pretreatment with PKC inhibitor NPC-15437 for 30 min had no effect on ergosterol-induced PLA2 activity (Fig. 2). 2.5. Effect of ergosterol on viability of tobacco cell In many systems, sub-lethal oxidative stress was found to be involved either directly or indirectly in programmed cell death processes. AOS play a key role in the initiation of programmed cell death and analogies were observed between cell death programs in animals and plants [8]. The viability of tobacco cells was measured by use of fluorescein diacetate and propidium iodide. Fluorescein diacetate penetrates into cells where it is hydrolyzed by esterases and the product of hydrolysis, fluorescein, becomes entrapped in living cells with an intact plasma membrane and emits green fluorescence. In contrast, propidium iodide can penetrate the
Fig. 1. Time course of PLA2 activity in suspension cultured tobacco cells measured as the fluorescence of a liberated fatty acid from PED6. The cells were treated with 1.5 µM ergosterol, 1 µM cryptogein, 1.5 µM cholesterol or methanol as a control after 10 min (arrow) following the addition of PED6.
Fig. 2. Effect of PLA2 or PKC inhibitors on the PLA2 activity induced by ergosterol. AACOCF3 (66 µM) or NPC-15437 (10 µM) were added 30 min before time 0 of the experiment. Ergosterol was added after 10 min (arrow) following the addition of PED6. Similar effects were observed in three independent experiments.
damaged plasma membrane of necrotic cells giving rise to an intense red fluorescence [42]. To compare the effect of ergosterol and cryptogein, tobacco cells were incubated with 250– 1250 nM ergosterol or 50–100 nM cryptogein for 24 h. Ergosterol in methanol was added to the bottom of the flask and methanol had to be evaporated (as described in the Section 4) because 0.01% methanol demonstrably diminished cell viability. We verified that the elicitation effect of ergosterol solution after evaporation remained unchanged (data not shown). The exposure of suspension tobacco cells to cryptogein resulted in an increase in death rate (62%) compared with control cells (23%). The addition of methanol alone, although thoroughly evaporated, slightly stimulated the cell death rate, probably due to the traces of impurities in the solvent. The treatment of suspension tobacco cells with ergosterol did not increase the death rate (15–38%) as compared with methanol control (20–35%) (Fig. 3).
Fig. 3. Effect of cryptogein or ergosterol on viability of tobacco suspension cells. The viability of at least 400 cells was examined using propidium iodide and fluorescein diacetate. The cells with green fluorescence were taken as live and those with red fluorescence as dead. Data represent the mean values from three replicate experiments ± S.D., the control corresponds to water-treated cells.
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3. Discussion Pharmacological studies on defense response signal transduction in plant cell cultures use protein or oligosaccharides elicitors as the inducers [10]. Concerning lipids, Granado et al. [13] previously reported about an elicitation effect of ergosterol from Cladosporium fulvum on tomato cells. Alkanols, alkan-1,3-diols and some hydroxy fatty acids were also potent H2O2 elicitors and enhanced the effect of fungal cell wall fragments, ergosterol and chitosan in cucumber cells [11]. In a previous paper, we have observed that the addition of ergosterol to tobacco cells triggered the synthesis of AOS and accumulation of the phytoalexin capsidiol and activated Ca2+ mainly from internal stores [16]. The production of AOS is lower than that obtained upon elicitation with cryptogein, a very efficient proteinaceous elicitor from Phytophthora cryptogea [35]. Moreover, cryptogein is a sterol carrier protein binding many sterols and fatty acids and the complex sterol-cryptogein could be the active form which triggers defense reactions [28]. In this study, we attempt to elucidate signal pathways induced by ergosterol, including phospholipases and protein kinases. Pharmacological experiments should always be interpreted with caution taking into account the permeability of the cell wall and membrane. In particular, nonspecific side effects are always possible, namely if reagents are used at relatively high concentrations. Tyrosine phosphorylations act upstream the serine/ threonine protein kinases and several studies have documented the involvement of tyrosine-phosphorylated proteins in plant defense reactions (for review see [9]). Herbimycin A is a potent inhibitor of tyrosine phosphorylation and has been used in order to demonstrate the tyrosine kinase involvement in signaling in soybean [31]. Herbimycin A and genistein had no effect on elicitation of AOS production and pH changes by ergosterol or cryptogein (Table 1) so that neither the ergosterol nor the cryptogein signal pathway is linked with tyrosine protein kinases. Serine/threonine protein kinases are necessary for the elicitation of a defense reaction induced by ergosterol and cryptogein because staurosporin, a serine/threonine kinase inhibitor [18,34], annihilated AOS production and strongly reduced pH changes elicited by ergosterol or cryptogein [16]. Protein kinases C (PKC) in animals constitute a family of 11 different types classified into three groups. They include “conventional” or “classic” PKCs activated by Ca2+ and diacylglycerol (DAG) as downstream mediators of PLC signaling, “new” PKCs still activated by the DAG/phorbol ester but Ca2+- insensitive, and “atypical” PKCs which are insensitive to both DAG and Ca2+. However, all PKCs require phosphatidylserine for their activity and some use unsaturated fatty acids and/or lysophospholipids as co-factors [26]. With the use of specific pharmacological agents, the potential biological role of PKCs in plants is beginning to emerge, although no PKC homologous to animal enzymes has been
characterized up to now. The effects of various modulators of PKC activity on the elicitor-induced resistance against a compatible race of Phytophthora infestans implicate this kinase in the overall defense response in potato [36]. Moreover, it has been reported that chitosan induced anthraquinone production in Rubia tinctorum L. cell cultures via Ca2+-dependent PKC [41]. NPC-15437 is a selective inhibitor of PKC interacting at the regulatory domain of the enzyme in animal cells and prevents the binding of DAG/phorbol ester [37]. In this paper, we show that NPC15437 abolished the effect of both ergosterol and cryptogein on the elicitation of AOS production and pH changes (Table 1). This suggests that the PKC playing a crucial role in the elicitation of the oxidative burst and DpH changes by ergosterol or cryptogein in tobacco could be similar to a PKC reported in animal cells (Ca2+ independent, DAG/phorbol ester binding). PKA in contrast to PKC is activated by cAMP. Protein kinase activity regulated by cAMP has been reported in higher plants [30]. H-89 inhibited the root elongation stimulated in A. thaliana [2] and transport of ricin stimulated by protein kinase A in MDCK cells [24]. We found that H-89 had no effect on elicitation of AOS production and pH changes by ergosterol or cryptogein (Table 1) indicating that PKA plays no role in elicitation of the early events by these elicitors. PLA2 represents a class of enzymes that catalyze the hydrolysis of the sn-2 acylester bond of glycerophospholipids resulting in the formation of lysophospholipids and free fatty acids. Eukaryotic PLA2 are divided into several groups. Secreted enzymes (sPLA2), cytosolic Ca2+-dependent PLA2 (cPLA2) responding to different hormonal stimuli, cytosolic Ca2+-independent PLA2 (iPLA2) and platelet activating factor related to the low density lipoprotein PLA2. Several genes of PLA2 of the patatin-like family homologous to animal iPLA2 were identified in Arabidopsis [14]. Their cytosolic or membrane localization was suggested, but the presence of typical cPLA2 in plants was not confirmed. Other known plant PLA2 belong to the family of sPLA2 i.e. secretory proteins. Although it may be hard to envisage them as a transduction signal proteins, their regulatory role was also suggested [1]. Plant PLA2 appears to be associated with plant biological mechanisms, such as auxin signaling, woundinduced signal transduction and pathogenesis. The activity of PLA2 is important for auxin-stimulated growth [29]. Production of alkaloids in poppy (Eschscholtzia californica L.) cells was triggered via PLA2 by yeast elicitor [33]. PLA2 activity increased and the oxidative burst was triggered when cultured soybean cells were challenged with elicitors from Verticillium dahliae or Erwina amylovora [7]. PLA2 activation has been implicated in the response of potato tubers to hyphal wall components of Phytophthora infestans [17]. Wounding promoted PLA2 activity in several plant species [21]. Systemin and oligosaccharide elicitors inducing jasmonic acid synthesis also increase a PLA2 activity without wounding [27]. Although AACOCF3 was described as a specific inhibi-
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tor of animal cPLA2, it was shown to inhibit PLA2 activity induced by systemin in tomato leaves [27]. In Arabidopsis cells, iPLA2 was sensitive to inhibitors of animal iPLA2 and cPLA2 [15]. In our study, AACOCF3 annihilated elicitation of AOS production and pH changes induced by ergosterol. On the other hand, AACOCF3 had no effect on elicitation of AOS production and pH changes induced by cryptogein (Table 1). This suggests that PLA2 plays a key role in the ergosterol-triggered signal pathway that leads to the oxidative burst whereas this signal pathway of cryptogein does not include PLA2. This was confirmed by an increase in activity of PLA2 stimulated by ergosterol and not by cryptogein and cholesterol using a fluorescent substrate of PLA2 (Fig. 1). NPC-15437, an inhibitor of PKC, had no effect on PLA2 activation by ergosterol (Fig. 2) so that PKC should act downstream PLA2. The best known function of PLC is the hydrolysis of phosphatidylinositol 4,5-bisphosphate to produce cellular messengers, inositol 1,4,5-trisphosphate (IP3) and DAG. IP3 binds to a receptor and mediates Ca2+ release to the cytoplasm, whereas DAG activates PKC. DAG can be rapidly converted to phosphatidic acid. Many components in the animal PLC cascade have been identified in plant cells (for review see [26]). Polygalacturonate and mastoparan induced an increase in IP3 levels in cell suspensions of soybean [22], and it has been reported that chitooligosaccharides triggered defense reactions and increased phosphatidic acid levels probably via PLC in suspension-cultured tomato cells [40]. U73122 and neomycin inhibited PI-PLC in Arabidopsis cells in comparison with U73343 as its inactive analogue [38]. These two inhibitors reduced the elicitation of AOS production and the pH changes induced by cryptogein, whereas U73343 had no effect (Table 1). This suggests that cryptogein elicits the oxidative burst and DpH changes involving PLC, and confirms the observation that neomycin, an inhibitor of PLC, partially inhibited an increase in cytosolic calcium concentration induced by cryptogein in tobacco cells [19]. The AOS production and pH changes induced by cryptogein were not sensitive to TMB-8, an inhibitor of calcium release from vacuoles [16]. However, cryptogein induces a massive transport of extra-cellular calcium through the plasma membrane [39] so that the secondary release of calcium from vacuoles may not participate in cryptogeintriggered responses. On the other hand, U73122 was inefficient in inhibiting the AOS production and pH changes elicited by ergosterol (Table 1), indicating that PLC plays only a minor role in elicitation by ergosterol. Interestingly, the inefficient derivative U73343 abolished completely the effects of ergosterol (Table 1). U73343, contrary to U73122, possesses a strong estrogenic activity mediated by the estrogen receptors [5]. So, we hypothesize that U73343 could interfere with ergosterol binding to a putative receptor in tobacco cells like an ergosterol antagonist. We have shown that ergosterol initiates only insignificant cell death in suspension tobacco cells compared to the methanol control (Fig. 3). AOS production appears to be a
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part of active defense against pathogens, and AOS may act themselves as secondary messengers for the initiation of host cell death [23] The death of infected and stressed cells in order to prevent systemic spread of a pathogen appears to be a conserved strategy in both plants and animals. It has been shown previously that AOS induces PCD in soybean and Arabidopsis thaliana cell cultures [8,23]. H2O2 signal involved in HR and systemic acquired resistance can induce PCD in tobacco BY-2 by a process similar to apoptosis in a dose-dependent manner [15]. However, some recent evidence suggests that although cell death and the induction of defense genes are activated by the same signal, they are regulated by separate mechanisms (for review [32]). To date, we have no evidence about the activation of defense genes by ergosterol. It was demonstrated that ergosterol induced a rapid accumulation of a nonspecific lipid transfer protein in grape [12]. In order to understand the importance of ergosterol signaling in plant defense the expression of defense genes in whole plants should be studied.
4. Methods 4.1. Chemicals Sterols, inhibitors and other chemicals were obtained from Sigma-Aldrich (Prag, Czechia). PED6 was purchased from Molecular Probes, Oregon, USA. Cryptogein was isolated as previously described [4]. Ergosterol, U73122, U73343, AACOCF3, herbimycin, H-89, and genistein were dissolved in methanol, PED6 in ethanol. Cryptogein and NPC-15437 were dissolved in water. All stock solutions were stored at –20 °C. 4.2. Cultures Tobacco suspension cells (Nicotiana tabacum L. cv. Xanthi) were grown at a constant temperature (25 °C), under a photoperiod of 12 h with a light intensity of 80 µE m–2 s–1 on a rotary shaker (125 rpm). They were subcultured weekly in Chandler’s medium [6]. 4.3. Elicitation of tobacco suspension cells Plant cells harvested during exponential phase of growth were filtered, washed and resuspended in 2 mM MES buffer (pH 5.75) containing 175 mM mannitol, 0.5 mM K2SO4 and 0.5 mM CaCl2 (elicitation buffer). The concentration of cells was 0.1 g FW ml–1. Samples of 20 ml suspensions were aliquoted in 50 ml Erlenmeyer flasks. After a 3 h equilibration period the cells were treated with the elicitors and the inhibitors. Control cells (blank) were treated with methanol at concentrations that did not exceed 0.2% (v/v). The pH changes were registered every 10 min after the addition of
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elicitor with a pH electrode. The AOS concentrations were monitored every 5 min in 250 µl aliquots as described before [16].
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Ergosterol dissolved in methanol or methanol alone was added to 25 ml Erlenmeyer flasks and the solvent was evaporated. Plant cells harvested during the exponential phase of growth were filtered, washed and resuspended in fresh Chandler’s medium (0.1 g FW ml–1). Samples of 10 ml suspensions were aliquoted in 25 ml Erlenmeyer flasks with the evaporated ergosterol solution. In the case of cryptogein, the elicitor was added directly to 10 ml of the suspension. After 24 h incubation, the cells were harvested, washed in phosphate buffer solution and incubated for 10 min at room temperature in phosphate buffer solution containing fluorescein diacetate (2 µg ml–1). Propidium iodide (20 µg ml–1) was then added and incubation proceeded another 5 min. The percentage of living and necrotic cells was evaluated using fluorescence microscopy. The number of living cells was calculated on the basis of green fluorescence of fluorescein and that of necrotic cells on the basis of red fluorescence of propidium iodide as described earlier [25]. Four hundred cells were observed in each preparation and numbers were expressed as a percentage of total cells.
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4.4. Determination of PLA2 activity PLA2 activity was measured in vivo by a method described by Chandra et al. [7] using PED6 as a fluorogenic substrate. Plant cells harvested in exponential phase of growth were filtered, washed, resuspended in fresh culture medium and equilibrated for 6 h as described above. To 2 ml of cell suspension in fluorescence cuvette, 10 µl of PED6 (1 mg ml–1 in methanol) were added and equilibrated for 10 min. Suspension was maintained under mild stirring at 25 °C. Fluorescence was measured on a Shimadzu RF 5301 PC spectrofluorimeter (excitation and emission wavelengths were 485 and 517 nm, respectively). Elicitors were added after 10 min following the addition of PED6.
Acknowledgements We thank Prof. L. Havel (Mendel University of Agriculture and Forestry, Brno, CZ) for his help in fluorescence microscopy. This work was supported by the grant Barrande 2002-042-2 (Czech Republic, France) and 522/02/0925 (Grant Agency of Czech Republic).
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Original article
Ergosterol elicits oxidative burst in tobacco cells via phospholipase A2 and protein kinase C signal pathway Tomas Kasparovsky a, Jean-Pierre Blein b, Vladimir Mikes a,* a
Department of Biochemistry, Masaryk University, Kotlarska 2, 61137 Brno, Czech Republic b INRA/Université de Bourgogne, (UMR 692), BP 86510, 21065 Dijon, cedex France Received 13 January 2004; accepted 5 April 2004 Available online 11 May 2004
Abstract Ergosterol, a typical fungal sterol, induced in tobacco (Nicotiana tabacum L. cv. Xanthi) suspension cells the synthesis of reactive oxygen species and alkalization of the external medium that are dependent on the mobilization of calcium from internal stores. We used specific inhibitors to elucidate the signal pathway triggered by ergosterol compared with cryptogein, a proteinaceous elicitor of Phytophthora cryptogea. Herbimycin A and genistein, inhibitors of tyrosine protein kinases, had no effect on the oxidative burst and pH changes induced by both elicitors. Similarly, H-89, an inhibitor of protein kinase A, had no effect on the induction of these defense reactions. However, the response to both elicitors was completely blocked by NPC-15437, a specific inhibitor of animal protein kinase C (PKC). The responses induced by cryptogein but not those induced by ergosterol were inhibited by U73122 and neomycin, inhibitors of phospholipase C (PLC). On the other hand, the activity of phospholipase A2 (PLA2) measured using a fluorogenic substrate was stimulated by ergosterol and not by cholesterol and cryptogein. A specific inhibitor of PLA2, arachidonic acid trifluoromethyl ketone (AACOCF3), inhibited the pathway stimulated by ergosterol but not that induced by cryptogein. These results suggest that the cryptogein-induced signal pathway leading to the oxidative burst and DpH changes includes PLC and PKC, whereas this response induced by ergosterol includes PLA2 and PKC. © 2004 Elsevier SAS. All rights reserved. Keywords: Cryptogein; Elicitor; Ergosterol; Oxidative burst; PLA2; Tobacco
1. Introduction Plant defense reactions associated with hypersensitive response (HR) are initiated by pathogen-produced signal molecules, called elicitors. The interaction of elicitors with plant cells is accompanied by some specific physiological perturbations such as ion fluxes across the plasma membrane
Abbreviations: AACOCF3, arachidonic acid trifluoromethyl ketone; AOS, active oxygen species; BODIPY FL, 4,4-difluoro-5,7-dimethyl-4bora-3a,4a- diaza-s-indacene-3-pentanoic acid; DAG, diacylglycerol; FW, fresh weight; HR, hypersensitive response; IP3, inositol 1,4,5-trisphosphate; PCD, programmed cell death; PED6, -((6-(2,4-dinitrophenyl)amino) hexanoyl)-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphoethanolamine; PKA, protein kinase A; PKC, protein kinase C; PLA2, phospholipase A2; PLC, phospholipase C; TMB-8, 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride. * Corresponding author. E-mail address: [email protected] (V. Mikes). © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.plaphy.2004.04.003
(namely Ca2+ influx and K+, Cl– efflux), pH changes, plasma membrane depolarization, oxidative burst and induction of rapid cell death. HR is accompanied by defense genes activation, leading to synthesis of phytoalexins and accumulation of pathogen-related proteins. Two types of elicitors are recognized: general or nonspecific elicitors that do not significantly differ in their effect on different cultivars within a plant species and specific elicitors that occur only in a pathogenic race or strain and function only in plant cultivars carrying the matching disease resistance gene. General elicitors include substances typically associated with basic microbial metabolism, such as cell wall glucans, chitin oligomers, lipids and glycopeptides [3]. For many years, natural plant defense reactions have been attracting high attention. Such studies were focused on plant pathogen recognition, signal transduction and induction of resistance as systemic resistance. Ergosterol is a principal component of the fungal plasma membrane. It triggers a defense reaction in tobacco and tomato cells manifested by active oxygen species (AOS) synthesis, changes of ion fluxes and production of the phy-
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toalexin capsidiol [13,16]. The signal pathway induced after ergosterol recognition is not elucidated. In a previous paper we have studied the effect of calcium channel inhibitors on the induction of defense reactions by ergosterol in tobacco cells [16]. We have found that the inhibition of calcium transport through plasma membrane channels did not influence the oxidative burst and the changes of external pH. On the other hand, the elicitation was blocked when the mobilization of internal calcium stores was suppressed. This paper examines the participation of phospholipases and protein kinases in the elicitation by ergosterol in tobacco suspension cells using several inhibitors of phospholipases and protein kinases. We show that the early events elicited by ergosterol in tobacco cells suspensions are activate by PLA2 and PKC in contrast with the proteinaceous elicitor cryptogein that activates mainly the PLC and PKC signal pathway.
2. Results 2.1. Effect of inhibitors of protein kinases on elicitation of tobacco cells Tyrosine and/or serine/threonine protein kinases participate in signal transduction in all living cells. It has been previously shown that the AOS production and pH changes in tobacco cells induced by ergosterol or cryptogein are strongly reduced by co-treatment with staurosporin [16,20], the compound effectively inhibiting protein Ser/Thr protein kinases in plant cell cultures [18,34]. The addition of herbimycin A (1.0 µM), an inhibitor of tyrosine protein kinase, to tobacco cells 30 min prior to elicitation with cryptogein or ergosterol had no significant effect on the AOS production and pH changes (Table 1). Under the same conditions, herbimycin A alone had no effect. In similar experiments with genistein (40 µM), another tyrosine protein kinase inhibitor, only a slight inhibition of pH changes and AOS synthesis induced by ergosterol was observed, and the elicitation with cryptogein was unaffected (Table 1). Thereafter, we tested the participation of PKA or PKC in the transduction of the signal induced by ergosterol or cryptogein in tobacco cells. The 30 min pre-treatment with H-89 (4 µM), a selective inhibitor of PKA, did not lead to the inhibition of AOS production and pH changes elicited with both elicitors (Table 1). On the other hand, NPC-15437 (10 µM), a selective inhibitor of animal PKC, added 30 min prior to the challenge with ergosterol or cryptogein completely inhibited the extent of AOS production and pH changes induced by both compounds (Table 1). H-89 or NPC-15437 alone induced neither AOS production nor pH changes.
Table 1 Effect of inhibitors of protein kinases, PLC and PLA2 on the AOS production and DpH changes induced by ergosterol or cryptogein. Before adding the elicitor, cells were incubated with the inhibitors for 30 min. AOS concentration and pH changes were monitored for 40 min at 5 and 10 min intervals, respectively. The results are the mean activities of three independent cultures represented in percent relative to the activities of non-inhibited cells, with standard error in parentheses. The values were obtained after 20 and 30 min following elicitation with ergosterol and cryptogein, respectively
Inhibitor 10 µM NPC-15437 2 µM H-89 4 µM H-89 1 µM Herbimycin A 40 µM Genistein 10 µM U-73122 (active) 10 µM U-73343 (inactive) 100 µM Neomycin 28 µM AACOCF3 56 µM AACOCF3
Percentage of activity 50 nM cryptogein AOS (%) DpH (%) 4 (4) 39 (16) 95 (2) 108 (1) 120 (2) 101 (6) 94 (8) 106 (4) 105 (8) 90 (7) 34 (4) 61 (4) 95 (5) 85 (4) 53 (2) 61 (2) 108 (8) 105 (4) 104 (8) 95 (1)
500 nM ergosterol AOS (%) DpH (%) 0 (0) 14 (1) 87 (4) 95 (3) 91 (3) 82 (1) 99 (15) 93 (5) 78 (16) 75 (8) 117 (12) 106 (7) 0 (0) 0 (0) 104 (8) 96 (13) 50 (9) 67 (1) 1 (1) 13 (3)
2.2. Effect of the inhibition of PLC on elicitation of tobacco cells To examine whether the defense response to ergosterol or cryptogein is due to the activation of PLC by elicitor we analyzed the effect of the inhibitors neomycin or U73122. The compound U73343, an inactive analogue, was used as a control [38]. The addition of U73122 (10 µM) 30 min before elicitation with cryptogein suppressed the AOS production by 66% and pH changes by 39% (Table 1), whereas the inactive analogue U73343 had no affect. On the other hand, the pH changes and the production of AOS stimulated by ergosterol were not inhibited by U73122. The effect of the inactive analogue U73343 was contradictory because it totally blocked the elicitation by ergosterol. U73122 or U73343 alone had no effect on AOS production and pH changes. Similar results were obtained with neomycin (100 µM) added 30 min before the elicitation. The AOS production elicited by cryptogein was suppressed by 47% and pH changes by 39% whereas no effect on the elicitation by ergosterol was observed (Table 1). 2.3. Effect of the inhibition of PLA2 on elicitation of tobacco cells To assess the involvement of PLA2 the effect of arachidonic acid trifluoromethyl ketone (AACOCF3) on signal transduction was tested. AACOCF3 (28 µM) caused a 50% decrease in AOS production and a 33% decrease in pH changes when added 30 min prior to the elicitation with ergosterol. These processes were completely blocked by 56 µM AACOCF3. Both concentrations of AACOCF3 alone were inefficient to elicit tobacco cells. In contrast, AACOCF3 at 28 or 56 µM did not inhibit the elicitation induced by cryptogein (Table 1).
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2.4. Measurement of the activity of PLA2 induced by elicitors We examined the effects of ergosterol, cholesterol or cryptogein on the induction of endogenous PLA2 activity in tobacco cells. PED6 is a fluorogenic substrate containing a BODIPY FL dye-labeled sn-2 acyl chain and a dinitrophenyl quencher group. Cleavage of the dye-labeled acyl chain by PLA2 eliminates the intramolecular quenching effect of the dinitrophenyl group, resulting in a corresponding fluorescence increase. The addition of ergosterol induced an obvious increase in PLA2 activity (Fig. 1). Treatment with cryptogein, cholesterol or methanol yielded BODIPY FL fluorescence tracings indistinguishable from controls. In the next experiment, the cell suspension was preincubated for 30 min with AACOCF3 before the addition of ergosterol. The increase in fluorescence of BODIPY FL observed upon the ergosterol treatment was blocked by AACOCF3 confirming that ergosterol-induced PLA2 activity was sensitive to a PLA2 inhibitor (Fig. 2). On the other hand, pretreatment with PKC inhibitor NPC-15437 for 30 min had no effect on ergosterol-induced PLA2 activity (Fig. 2). 2.5. Effect of ergosterol on viability of tobacco cell In many systems, sub-lethal oxidative stress was found to be involved either directly or indirectly in programmed cell death processes. AOS play a key role in the initiation of programmed cell death and analogies were observed between cell death programs in animals and plants [8]. The viability of tobacco cells was measured by use of fluorescein diacetate and propidium iodide. Fluorescein diacetate penetrates into cells where it is hydrolyzed by esterases and the product of hydrolysis, fluorescein, becomes entrapped in living cells with an intact plasma membrane and emits green fluorescence. In contrast, propidium iodide can penetrate the
Fig. 1. Time course of PLA2 activity in suspension cultured tobacco cells measured as the fluorescence of a liberated fatty acid from PED6. The cells were treated with 1.5 µM ergosterol, 1 µM cryptogein, 1.5 µM cholesterol or methanol as a control after 10 min (arrow) following the addition of PED6.
Fig. 2. Effect of PLA2 or PKC inhibitors on the PLA2 activity induced by ergosterol. AACOCF3 (66 µM) or NPC-15437 (10 µM) were added 30 min before time 0 of the experiment. Ergosterol was added after 10 min (arrow) following the addition of PED6. Similar effects were observed in three independent experiments.
damaged plasma membrane of necrotic cells giving rise to an intense red fluorescence [42]. To compare the effect of ergosterol and cryptogein, tobacco cells were incubated with 250– 1250 nM ergosterol or 50–100 nM cryptogein for 24 h. Ergosterol in methanol was added to the bottom of the flask and methanol had to be evaporated (as described in the Section 4) because 0.01% methanol demonstrably diminished cell viability. We verified that the elicitation effect of ergosterol solution after evaporation remained unchanged (data not shown). The exposure of suspension tobacco cells to cryptogein resulted in an increase in death rate (62%) compared with control cells (23%). The addition of methanol alone, although thoroughly evaporated, slightly stimulated the cell death rate, probably due to the traces of impurities in the solvent. The treatment of suspension tobacco cells with ergosterol did not increase the death rate (15–38%) as compared with methanol control (20–35%) (Fig. 3).
Fig. 3. Effect of cryptogein or ergosterol on viability of tobacco suspension cells. The viability of at least 400 cells was examined using propidium iodide and fluorescein diacetate. The cells with green fluorescence were taken as live and those with red fluorescence as dead. Data represent the mean values from three replicate experiments ± S.D., the control corresponds to water-treated cells.
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3. Discussion Pharmacological studies on defense response signal transduction in plant cell cultures use protein or oligosaccharides elicitors as the inducers [10]. Concerning lipids, Granado et al. [13] previously reported about an elicitation effect of ergosterol from Cladosporium fulvum on tomato cells. Alkanols, alkan-1,3-diols and some hydroxy fatty acids were also potent H2O2 elicitors and enhanced the effect of fungal cell wall fragments, ergosterol and chitosan in cucumber cells [11]. In a previous paper, we have observed that the addition of ergosterol to tobacco cells triggered the synthesis of AOS and accumulation of the phytoalexin capsidiol and activated Ca2+ mainly from internal stores [16]. The production of AOS is lower than that obtained upon elicitation with cryptogein, a very efficient proteinaceous elicitor from Phytophthora cryptogea [35]. Moreover, cryptogein is a sterol carrier protein binding many sterols and fatty acids and the complex sterol-cryptogein could be the active form which triggers defense reactions [28]. In this study, we attempt to elucidate signal pathways induced by ergosterol, including phospholipases and protein kinases. Pharmacological experiments should always be interpreted with caution taking into account the permeability of the cell wall and membrane. In particular, nonspecific side effects are always possible, namely if reagents are used at relatively high concentrations. Tyrosine phosphorylations act upstream the serine/ threonine protein kinases and several studies have documented the involvement of tyrosine-phosphorylated proteins in plant defense reactions (for review see [9]). Herbimycin A is a potent inhibitor of tyrosine phosphorylation and has been used in order to demonstrate the tyrosine kinase involvement in signaling in soybean [31]. Herbimycin A and genistein had no effect on elicitation of AOS production and pH changes by ergosterol or cryptogein (Table 1) so that neither the ergosterol nor the cryptogein signal pathway is linked with tyrosine protein kinases. Serine/threonine protein kinases are necessary for the elicitation of a defense reaction induced by ergosterol and cryptogein because staurosporin, a serine/threonine kinase inhibitor [18,34], annihilated AOS production and strongly reduced pH changes elicited by ergosterol or cryptogein [16]. Protein kinases C (PKC) in animals constitute a family of 11 different types classified into three groups. They include “conventional” or “classic” PKCs activated by Ca2+ and diacylglycerol (DAG) as downstream mediators of PLC signaling, “new” PKCs still activated by the DAG/phorbol ester but Ca2+- insensitive, and “atypical” PKCs which are insensitive to both DAG and Ca2+. However, all PKCs require phosphatidylserine for their activity and some use unsaturated fatty acids and/or lysophospholipids as co-factors [26]. With the use of specific pharmacological agents, the potential biological role of PKCs in plants is beginning to emerge, although no PKC homologous to animal enzymes has been
characterized up to now. The effects of various modulators of PKC activity on the elicitor-induced resistance against a compatible race of Phytophthora infestans implicate this kinase in the overall defense response in potato [36]. Moreover, it has been reported that chitosan induced anthraquinone production in Rubia tinctorum L. cell cultures via Ca2+-dependent PKC [41]. NPC-15437 is a selective inhibitor of PKC interacting at the regulatory domain of the enzyme in animal cells and prevents the binding of DAG/phorbol ester [37]. In this paper, we show that NPC15437 abolished the effect of both ergosterol and cryptogein on the elicitation of AOS production and pH changes (Table 1). This suggests that the PKC playing a crucial role in the elicitation of the oxidative burst and DpH changes by ergosterol or cryptogein in tobacco could be similar to a PKC reported in animal cells (Ca2+ independent, DAG/phorbol ester binding). PKA in contrast to PKC is activated by cAMP. Protein kinase activity regulated by cAMP has been reported in higher plants [30]. H-89 inhibited the root elongation stimulated in A. thaliana [2] and transport of ricin stimulated by protein kinase A in MDCK cells [24]. We found that H-89 had no effect on elicitation of AOS production and pH changes by ergosterol or cryptogein (Table 1) indicating that PKA plays no role in elicitation of the early events by these elicitors. PLA2 represents a class of enzymes that catalyze the hydrolysis of the sn-2 acylester bond of glycerophospholipids resulting in the formation of lysophospholipids and free fatty acids. Eukaryotic PLA2 are divided into several groups. Secreted enzymes (sPLA2), cytosolic Ca2+-dependent PLA2 (cPLA2) responding to different hormonal stimuli, cytosolic Ca2+-independent PLA2 (iPLA2) and platelet activating factor related to the low density lipoprotein PLA2. Several genes of PLA2 of the patatin-like family homologous to animal iPLA2 were identified in Arabidopsis [14]. Their cytosolic or membrane localization was suggested, but the presence of typical cPLA2 in plants was not confirmed. Other known plant PLA2 belong to the family of sPLA2 i.e. secretory proteins. Although it may be hard to envisage them as a transduction signal proteins, their regulatory role was also suggested [1]. Plant PLA2 appears to be associated with plant biological mechanisms, such as auxin signaling, woundinduced signal transduction and pathogenesis. The activity of PLA2 is important for auxin-stimulated growth [29]. Production of alkaloids in poppy (Eschscholtzia californica L.) cells was triggered via PLA2 by yeast elicitor [33]. PLA2 activity increased and the oxidative burst was triggered when cultured soybean cells were challenged with elicitors from Verticillium dahliae or Erwina amylovora [7]. PLA2 activation has been implicated in the response of potato tubers to hyphal wall components of Phytophthora infestans [17]. Wounding promoted PLA2 activity in several plant species [21]. Systemin and oligosaccharide elicitors inducing jasmonic acid synthesis also increase a PLA2 activity without wounding [27]. Although AACOCF3 was described as a specific inhibi-
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tor of animal cPLA2, it was shown to inhibit PLA2 activity induced by systemin in tomato leaves [27]. In Arabidopsis cells, iPLA2 was sensitive to inhibitors of animal iPLA2 and cPLA2 [15]. In our study, AACOCF3 annihilated elicitation of AOS production and pH changes induced by ergosterol. On the other hand, AACOCF3 had no effect on elicitation of AOS production and pH changes induced by cryptogein (Table 1). This suggests that PLA2 plays a key role in the ergosterol-triggered signal pathway that leads to the oxidative burst whereas this signal pathway of cryptogein does not include PLA2. This was confirmed by an increase in activity of PLA2 stimulated by ergosterol and not by cryptogein and cholesterol using a fluorescent substrate of PLA2 (Fig. 1). NPC-15437, an inhibitor of PKC, had no effect on PLA2 activation by ergosterol (Fig. 2) so that PKC should act downstream PLA2. The best known function of PLC is the hydrolysis of phosphatidylinositol 4,5-bisphosphate to produce cellular messengers, inositol 1,4,5-trisphosphate (IP3) and DAG. IP3 binds to a receptor and mediates Ca2+ release to the cytoplasm, whereas DAG activates PKC. DAG can be rapidly converted to phosphatidic acid. Many components in the animal PLC cascade have been identified in plant cells (for review see [26]). Polygalacturonate and mastoparan induced an increase in IP3 levels in cell suspensions of soybean [22], and it has been reported that chitooligosaccharides triggered defense reactions and increased phosphatidic acid levels probably via PLC in suspension-cultured tomato cells [40]. U73122 and neomycin inhibited PI-PLC in Arabidopsis cells in comparison with U73343 as its inactive analogue [38]. These two inhibitors reduced the elicitation of AOS production and the pH changes induced by cryptogein, whereas U73343 had no effect (Table 1). This suggests that cryptogein elicits the oxidative burst and DpH changes involving PLC, and confirms the observation that neomycin, an inhibitor of PLC, partially inhibited an increase in cytosolic calcium concentration induced by cryptogein in tobacco cells [19]. The AOS production and pH changes induced by cryptogein were not sensitive to TMB-8, an inhibitor of calcium release from vacuoles [16]. However, cryptogein induces a massive transport of extra-cellular calcium through the plasma membrane [39] so that the secondary release of calcium from vacuoles may not participate in cryptogeintriggered responses. On the other hand, U73122 was inefficient in inhibiting the AOS production and pH changes elicited by ergosterol (Table 1), indicating that PLC plays only a minor role in elicitation by ergosterol. Interestingly, the inefficient derivative U73343 abolished completely the effects of ergosterol (Table 1). U73343, contrary to U73122, possesses a strong estrogenic activity mediated by the estrogen receptors [5]. So, we hypothesize that U73343 could interfere with ergosterol binding to a putative receptor in tobacco cells like an ergosterol antagonist. We have shown that ergosterol initiates only insignificant cell death in suspension tobacco cells compared to the methanol control (Fig. 3). AOS production appears to be a
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part of active defense against pathogens, and AOS may act themselves as secondary messengers for the initiation of host cell death [23] The death of infected and stressed cells in order to prevent systemic spread of a pathogen appears to be a conserved strategy in both plants and animals. It has been shown previously that AOS induces PCD in soybean and Arabidopsis thaliana cell cultures [8,23]. H2O2 signal involved in HR and systemic acquired resistance can induce PCD in tobacco BY-2 by a process similar to apoptosis in a dose-dependent manner [15]. However, some recent evidence suggests that although cell death and the induction of defense genes are activated by the same signal, they are regulated by separate mechanisms (for review [32]). To date, we have no evidence about the activation of defense genes by ergosterol. It was demonstrated that ergosterol induced a rapid accumulation of a nonspecific lipid transfer protein in grape [12]. In order to understand the importance of ergosterol signaling in plant defense the expression of defense genes in whole plants should be studied.
4. Methods 4.1. Chemicals Sterols, inhibitors and other chemicals were obtained from Sigma-Aldrich (Prag, Czechia). PED6 was purchased from Molecular Probes, Oregon, USA. Cryptogein was isolated as previously described [4]. Ergosterol, U73122, U73343, AACOCF3, herbimycin, H-89, and genistein were dissolved in methanol, PED6 in ethanol. Cryptogein and NPC-15437 were dissolved in water. All stock solutions were stored at –20 °C. 4.2. Cultures Tobacco suspension cells (Nicotiana tabacum L. cv. Xanthi) were grown at a constant temperature (25 °C), under a photoperiod of 12 h with a light intensity of 80 µE m–2 s–1 on a rotary shaker (125 rpm). They were subcultured weekly in Chandler’s medium [6]. 4.3. Elicitation of tobacco suspension cells Plant cells harvested during exponential phase of growth were filtered, washed and resuspended in 2 mM MES buffer (pH 5.75) containing 175 mM mannitol, 0.5 mM K2SO4 and 0.5 mM CaCl2 (elicitation buffer). The concentration of cells was 0.1 g FW ml–1. Samples of 20 ml suspensions were aliquoted in 50 ml Erlenmeyer flasks. After a 3 h equilibration period the cells were treated with the elicitors and the inhibitors. Control cells (blank) were treated with methanol at concentrations that did not exceed 0.2% (v/v). The pH changes were registered every 10 min after the addition of
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elicitor with a pH electrode. The AOS concentrations were monitored every 5 min in 250 µl aliquots as described before [16].
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4.4. Determination of PLA2 activity PLA2 activity was measured in vivo by a method described by Chandra et al. [7] using PED6 as a fluorogenic substrate. Plant cells harvested in exponential phase of growth were filtered, washed, resuspended in fresh culture medium and equilibrated for 6 h as described above. To 2 ml of cell suspension in fluorescence cuvette, 10 µl of PED6 (1 mg ml–1 in methanol) were added and equilibrated for 10 min. Suspension was maintained under mild stirring at 25 °C. Fluorescence was measured on a Shimadzu RF 5301 PC spectrofluorimeter (excitation and emission wavelengths were 485 and 517 nm, respectively). Elicitors were added after 10 min following the addition of PED6.
Acknowledgements We thank Prof. L. Havel (Mendel University of Agriculture and Forestry, Brno, CZ) for his help in fluorescence microscopy. This work was supported by the grant Barrande 2002-042-2 (Czech Republic, France) and 522/02/0925 (Grant Agency of Czech Republic).
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