Aluminium ions inhibit the formation of diacylglycerol generated by phosphatidylcholine-hydrolysing phospholipase C in tobacco cells

Authors

  • Přemysl Pejchar,

    1. Institute of Experimental Botany, Academy of Sciences of the Czech Republic, v. v. i., Rozvojová 263, 165 02 Prague 6, Czech Republic
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  • Martin Potocký,

    1. Institute of Experimental Botany, Academy of Sciences of the Czech Republic, v. v. i., Rozvojová 263, 165 02 Prague 6, Czech Republic
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  • Zuzana Novotná,

    1. Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology, Institute of Chemical Technology Prague, Technická 5, 166 28 Prague 6, Czech Republic
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  • Štěpánka Veselková,

    1. Institute of Experimental Botany, Academy of Sciences of the Czech Republic, v. v. i., Rozvojová 263, 165 02 Prague 6, Czech Republic
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  • Daniela Kocourková,

    1. Institute of Experimental Botany, Academy of Sciences of the Czech Republic, v. v. i., Rozvojová 263, 165 02 Prague 6, Czech Republic
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  • Olga Valentová,

    1. Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology, Institute of Chemical Technology Prague, Technická 5, 166 28 Prague 6, Czech Republic
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  • Kateřina Schwarzerová,

    1. Department of Experimental Plant Biology, Faculty of Science, Charles University in Prague, Viničná 5, 128 44 Prague 2, Czech Republic
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  • Jan Martinec

    1. Institute of Experimental Botany, Academy of Sciences of the Czech Republic, v. v. i., Rozvojová 263, 165 02 Prague 6, Czech Republic
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Author for correspondence:
Jan Martinec
Tel: +420 225106416
Email: martinec@ueb.cas.cz

Summary

  • Aluminium ions (Al) have been recognized as a major toxic factor for crop production in acidic soils. This study aimed to assess the impact of Al on the activity of phosphatidylcholine-hydrolysing phospholipase C (PC-PLC), a new member of the plant phospholipase family.
  • We labelled the tobacco cell line BY-2 and pollen tubes with a fluorescent derivative of phosphatidylcholine and assayed for patterns of fluorescently labelled products. Growth of pollen tubes was analysed.
  • We observed a significant decrease of labelled diacylglycerol (DAG) in cells treated with AlCl3. Investigation of possible metabolic pathways that control DAG generation and consumption during the response to Al showed that DAG originated from the reaction catalysed by PC-PLC. The growth of pollen tubes was retarded in the presence of Al and this effect was accompanied by the decrease of labelled DAG similar to the case of the BY-2 cell line. The growth of pollen tubes arrested by Al was rescued by externally added DAG.
  • Our observation strongly supports the role of DAG generated by PC-PLC in the response of tobacco cells to Al.

Introduction

Aluminium ions (Al) released from soils with low pH are recognized as a major limiting factor to plant growth in regions with acid soils (Kochian et al., 2004). Al rapidly halts root growth, and lasting Al stress changes root morphology and causes death of root cells. Studies of the target of Al action in plant tissues and cells have demonstrated that Al enters and binds to the apoplast (Wissemeier & Horst, 1995) and changes the properties of the plasma membrane. Well documented early consequences of Al toxicity are depolarization of the plasma membrane (Sivaguru et al., 2003; Illéšet al., 2006), disruption of ion fluxes (Matsumoto, 2000), disruption of calcium homeostasis (Rengel & Zhang, 2003), inhibition of nitric oxide synthase (Tian et al., 2007) and effects on the cytoskeleton (Sivaguru et al., 1999, 2003; Schwarzerováet al., 2002).

The phospholipid-signalling pathway is now considered to be one of the important plant-signalling mechanisms. Activation of this pathway leads to the hydrolysis of phospholipids by phospholipases which are grouped into three families designated as phospholipase A (PLA), phospholipase C (PLC) and phospholipase D (PLD) according to the site of substrate molecule cleavage. Stimulation of this signalling pathway is involved in many different reactions of plants to environmental factors such as drought, cold, salinity or pathogen attack (Munnik et al., 1998; Wang, 2001, 2004; Meijer & Munnik, 2003; Wang et al., 2006).

Aluminium has been shown to affect the phospholipid-signalling pathway as well. Changes of phosphatidylinositol-specific phospholipase C (PI-PLC) activity after Al treatment have been demonstrated (Jones & Kochian, 1995; Piña-Chable & Hernández-Sotomayor, 2001; Martínez-Estévez et al., 2003). Furthermore, Al caused almost 30% inhibition of phosphatidic acid (PA) formation in an experiment with Coffea arabica cells (Ramos-Díaz et al., 2007). It was shown that PA was generated via PI-PLC and the diacylglycerol kinase pathway, where Al inhibited PI-PLC activity, the first step of the pathway. Recently, we reported that Al inhibits PLD activity both in situ and in vitro in the tobacco cell line BY-2 (Pejchar et al., 2008).

Besides PI-PLC, phosphatidylcholine-hydrolysing phospholipase C (PC-PLC), also known as nonspecific phospholipase C (NPC), is described. This enzyme that generates diacylglycerol (DAG) through glycerophospholipid hydrolysis (mainly phosphatidylcholine (PC)) has been characterized in animals (Exton, 1994) and is well known and characterized in bacteria (Titball, 1993) as well.

Based on the amino acid sequence similarity with the bacterial Gram-negative (nonhaemolytic) PC-PLC family, Nakamura et al. (2005) identified six putative PC-PLC genes in Arabidopsis, designated NPC1NPC6. PC-PLC activity was reported in connection with phospholipid-to-galactosyl DAG exchange in plants (Andersson et al., 2005; Gaude et al., 2008; Tjellström et al., 2008). Down-regulation of PC-PLC was described in relation to elicitor signalling. There was a rapid decrease of DAG concentration in tobacco VBI-0 cells after treatment with the elicitor cryptogein from Phytophthora cryptogea (Scherer et al., 2002). The role of NPC3 and NPC4 in root development and brassinolide signalling was recently shown in Arabidopsis (Wimalasekera et al., 2010).

The main goal of this work was to study the effect of Al on PC-PLC activity. We provide evidence that PC-PLC activity is affected by Al, which results in reduced DAG formation. We suggest that PC-PLC is a target of Al in plants.

Materials and Methods

Chemicals

Bodipy-phosphatidylcholine (2-decanoyl-1-(O-(11-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-proprionyl)amino)undecyl) sn-glycero-3-phosphocholine) (bodipy-PC) was obtained from Invitrogen. Phospholipase A2 (PLA2) from bee venom, PC-PLC from Bacillus cereus, PLD from cabbage, 1,2-dioctanoyl-sn-glycerol (DAG), 1,2-dioctanoyl-sn-glycerol 3-phosphate (PA), D609, anhydrous AlCl3 and Al(NO3)3·9H2O were from Sigma-Aldrich Co. High-performance liquid chromatography-grade chloroform, ethanol, methanol and high-performance thin layer chromatography (HP-TLC) plates were from Merck KGaA (Darmstadt, Germany). All other chemicals were of analytical grade.

Tobacco cell line BY-2

The tobacco cell line BY-2 (Nicotiana tabacum L. cv Bright Yellow 2) (Nagata et al., 1992) was cultured in medium containing 4.3 g l−1 of Murashige–Skoog (MS) salts (Sigma), 1 mg l−1 thiamine, 200 mg l−1 KH2PO4, 100 mg l−1myo-inositol, 30 g l−1 sucrose, and 0.9 μM 2,4-dichlorophenoxyacetic acid at pH 5.8. In a 1-wk interval, 3 ml of cells were transferred to 100 ml of fresh medium and cultured in darkness at 26°C on an orbital shaker (150 rpm; orbital diameter, 30 mm). Based on our previous results (Schwarzerováet al., 2002) showing that Al sensitivity was more pronounced during the exponential phase than during the stationary phase of the cultures, 3-d-old cell cultures were used in all experiments.

Pollen culture

The tobacco (N. tabacum L cv Samsun) pollen was collected from surface-sterilized anthers (before anthesis) dehydrated overnight and stored at −20°C. Before experiments, pollen was kept at room temperature for 15–20 min before resuspending in a simple germination medium containing 10% (w/v) sucrose and 1.6 mM boric acid. The culture was then placed on a rotary shaker at 200 rpm and incubated at room temperature.

Quantification of cell viability

Cell viability was assessed with fluorescein diacetate (FDA) according to Widholm (1972); 40 μl of 0.2% (w/v) FDA stock solution in acetone was diluted with 7 ml of culture medium and an aliquot mixed 1 : 1 (v/v) with cell suspension on a microscopic slide. Viability was determined from at least 10 optical fields on each of three separate slides as a percentage of fluorescing cells (c. 400 cells were counted in each sample).

Measurement of free Al in the medium

Cells were vacuum-filtrated and free Al concentration was measured using pyrocatechol violet (Kerven et al., 1989). Medium was incubated with pyrocatechol violet of final concentration 0.0375% (w/v) for 60 s and the absorbance was immediately measured at 585 nm. The concentration was calculated from a standard calibration curve of 0–25 μM AlCl3.

Treatment of cells with Al

Three-day-old BY-2 cells were washed with 3% (w/v) sucrose solution for 5 min, and 56 mg (fresh weight) of cells were resuspended in 1 ml of 3% (w/v) sucrose pH 4. Stock solution of fluorescent substrate was prepared by dilution of 1 mg bodipy-PC in 3 ml ethanol. Two microlitres of stock solution were added to the cell suspension (final concentration of bodipy-PC was 0.66 μg ml−1). After 10 min incubation, AlCl3 or Al(NO3)3 from a 50 mM stock solution (pH 4) was added to obtain final concentrations 0–100 μM. After incubation on an orbital shaker at 26°C in the darkness for various time intervals, lipids were extracted and analysed.

Tobacco pollen (4 mg ml−1) was cultivated in a germination medium for 90 min. After this, fresh medium of pH 5 was added 1 : 1 (v/v) to germinating pollen. Fluorescent substrate (0.66 μg ml−1 bodipy-PC) and a different concentration (0–100 μM) of AlCl3 were added to medium. After incubation on an orbital shaker at 26°C in the darkness for 10 min, lipids were extracted and analysed.

Extraction of lipids and HP-TLC analysis of reaction products

Lipids were extracted by the addition of 4 ml of cold methanol : chloroform 2 : 1 (v/v) to harvested cells. After 30 min extraction at room temperature, 2 ml of 0.1 M KCl were added and kept at 4°C for at least 30 min. Samples were then centrifuged for 15 min at 420 g. The lower phase was evaporated to dryness by a vacuum evaporator and redissolved in ethanol.

Samples were applied on the HP-TLC silica gel-60 plates by ATS4 sampler (Camag, Muttenz, Switzerland). After 10 min saturation, plates were developed in the horizontal developing chamber (Camag) in a mobile-phase chloroform : methanol : water 65 : 25 : 4 (v/v/v) (Scherer et al., 2002). Plates were dried and scanned by a video camera (Kodak DC 120) under UV light for computer-assisted quantification (Kodak ds 1D). Identification of individual spots was based on the comparison with fluorescently labelled lipid standards.

Preparation of cellular fractions

Cytosolic, microsomal and plasma membrane-enriched fractions were prepared as described by Novotnáet al. (2003). Briefly, tobacco cells BY-2 were homogenized using sonication, and cytosolic and microsomal fractions were prepared. The plasma membrane-enriched fraction was then prepared by partitioning microsomes in an aqueous dextran–polyethylene glycol two-phase system. Protein concentrations were determined according to Bradford (1976).

In vitro PC-PLC activity assay

PC--PLC activity was measured in vitro with the fluorescent substrate bodipy-PC based on the method of Ella et al. (1994) where bodipy-PC was used in PLA2, PLD and PC-PLC assay. The reaction mixture (50 μl) contained 0.1 mM PC, 0.66 μg bodipy-PC, 0.5% Triton X-100, 20 mM Bis-Tris propane/HCl (pH 6.5), 1 mM CaCl2 and 15 μg of sample proteins. In the case of Al treatment, instead of 20 mM Bis-Tris propane/HCl (pH 6.5), 20 mM homopipes (pH 5.0) was used. The reaction was stopped by the addition of 400 μl of cold methanol : chloroform 2 : 1 (v/v). After 30 min of extraction at room temperature, 200 μl of 0.1 M KCl was added and kept at 4°C for at least 30 min. Samples were then centrifuged for 15 min at 420 g. The lower phase was evaporated to dryness by a vacuum evaporator, redissolved in ethanol and used for HP-TLC analysis.

Preparation of fluorescently labelled lipids

Fluorescently labelled lipids were prepared by digestion of bodipy-PC according to Paul (1999) with slight modifications.

Bodipy-lysophosphatidylcholine (bodipy-LPC) was prepared in 1 ml of reaction mixture containing 42.7 mM Mes-NaOH (pH 5.6), 1% (v/v) Triton X-100, 10 mM CaCl2, 10 μg ml−1 bodipy-PC, and 2.45 U ml−1 bee venom PLA2 (Sigma, P 9279). The reaction was initiated by the addition of PLA2. After incubation for 30 min at 37°C, the reaction was terminated by the addition of 4 ml of cold methanol : chloroform 2 : 1 (v/v).

Bodipy-DAG was prepared in 1 ml of reaction mixture containing 47.7 mM Mes-NaOH (pH 6), 0.05% (v/v) Triton X-100, 5 mM CaCl2, 10 μg ml−1 bodipy-PC, and 0.6 U ml−1 of bacterial PC-PLC (Sigma, P 6135). The reaction was initiated by the addition of PC-PLC. The reaction mixture was incubated for 30 min at 37°C and terminated by the addition of 4 ml of cold methanol : chloroform 2 : 1 (v/v).

Bodipy-PA was prepared in 1 ml of reaction mixture containing 33.5 mM Mes-NaOH (pH 5.6), 1% (v/v) Triton X-100, 50 mM CaCl2, 8 mM SDS, 20 μg ml−1 bodipy-PC, and 100 U ml−1 cabbage PLD (Sigma, P 8398). The reaction was initiated by the addition of PLD. After incubation for 30 min at 30°C, the reaction was terminated by the addition of 4 ml of cold methanol : chloroform 2 : 1 (v/v).

Lipids were then extracted as described earlier. To remove noncleaved bodipy-PC, extracted lipids were separated by HP-TLC and the corresponding spot was scrapped from the plate and extracted overnight at 4°C in 3 ml of methanol. After extraction, 3 ml of chloroform and 3 ml of water were added and samples were centrifuged for 15 min at 420 g. The lower phase was evaporated to dryness by a vacuum evaporator and redissolved in ethanol.

To prepare bodipy-monoacylglycerol (bodipy-MAG), the tobacco BY-2 cells (c. 0.56 g for assay) were incubated with 6.6 μg ml−1 bodipy-PC. The reaction was stopped and bodipy-MAG was purified as described earlier. Bodipy-MAG was identified by mass spectrometry (data not shown).

Measurement of pollen tube elongation

To study the effects of Al, DAG (dimethyl sulfoxide-soluble 1,2-dioctanoyl-sn-glycerol) and PA (water-soluble 1,2-dioctanoyl-sn-glycerol 3-phosphate) on pollen tube elongation, various concentrations of tested substances were added to the suspension of growing pollen tubes 1.5 h after imbibition and the culture was grown for an additional 1.5 h. The cells were fixed in 0.37% (v/v) formaldehyde and the mean tube growth rate was evaluated using a light microscope with the image analysis software Analysis (Olympus, Tokyo, Japan (http://www.olympus-global.com)).

Results

The viability of BY-2 cells was not affected by Al within experimental time

Aluminium is known to affect a wide range of cellular processes (Panda et al., 2009) and the long exposure time finally leads to cell death. In our experiments we were interested in early events associated with Al toxicity. Therefore, the viability of BY-2 cells during the first hours of Al treatment and their ability to recover after Al treatment were analysed.

There were no significant changes in the cell viability up to 2 h and a slight decrease by 10% was observed after 6 h of treatment with 100 μM AlCl3 (Fig. 1). Cells treated for 1 h with 100 μM AlCl3 were normally able to grow further when removed from Al-containing medium; after 5 h of growth in the control medium the viability of treated cells was comparable to untreated controls. A similar situation was observed in the case of 2 h treatment with Al (data not shown). These results indicate that within the frame of our experiments there were no irreversible changes of cellular metabolism.

Figure 1.

 Viability of 3-d-old tobacco BY-2 cells during 24 h of exposure to 100 μM AlCl3 in 3% (w/v) sucrose (triangles). Controls were grown in Murashige–Skoog (MS) medium (closed circles) or in 3% (w/v) sucrose (open circles). Each value represents the mean of the viability determined from at least 10 optical fields on each of three separate slides as a percentage of fluorescing cells (c. 400 cells were counted in each sample in total) ± SD. This experiment was repeated twice with similar results.

To prevent Al complexation and the formation of various Al forms in full MS medium, all Al treatments were carried out in 3% (w/v) sucrose at pH 4. Since the simple sucrose solution was not buffered, cell suspension pH was measured during the experiment. The pH rose within the first 10 min of incubation by c. 1.3 units. After 120 min of treatment, the pH was c. 5.5 in both control and treated suspensions. The concentration of free inorganic Al was measured in the medium of treated cell suspension. The concentrations of free inorganic Al at 10, 20 and 120 min were 6.0 ± 0.4, 5.8 ± 0.6 and 6.2 ± 1.1 μM, respectively.

Bodipy-PC incorporation into BY-2 cells was not affected by Al

To study the changes in the phospholipid pattern under Al stress, we used a fluorescent derivative of PC (bodipy-PC) as a phospholipase substrate. Bodipy-PC was quickly incorporated into cellular membranes of control nontreated cells. Fluorescence was detected within 5 min of incorporation (Fig. 2a) and the maximum of the fluorescence was reached after 15 min (Fig. 2b). The process of incorporation was not affected by Al treatment and the incorporation dynamics was comparable to the control cells (Fig. 2d,e). Bodipy-PC was not entrapped into the cell wall, as was demonstrated by plasmolysis of the cells with 1 M NaCl (Fig. 2c,f). All further analyses of bodipy-PC metabolism in BY-2 cells were performed after 10 min of substrate incorporation.

Figure 2.

 Incorporation of bodipy-phosphatidylcholine (bodipy-PC) into 3-d-old tobacco BY-2 cells. Bodipy-PC with or without 100 μM AlCl3 was added to the cell suspension: (a) 5 min of incorporation, nontreated cells; (b) 15 min of incorporation, nontreated cells; (c) 15 min of incorporation, nontreated cells after plasmolysis with 1 M NaCl; (d) 5 min of incorporation, Al-treated cells; (e) 15 min of incorporation, Al-treated cells; (f) 15 min of incorporation, Al-treated cells after plasmolysis with 1 M NaCl. The cells were observed using an epifluorescence microscope (Nikon Eclipse E600, Tokyo, Japan). For observation of bodipy fluorescence, a filter for excitation at 450–490 nm and a 520 nm barrier filter were used. Pictures were collected and processed using the image analysis Lucia G/F system (Laboratory Imaging, Prague, Czech Republic). This experiment was repeated twice with similar results. Bar, 30 μm.

Al inhibited production of bodipy-DAG

To study lipid turnover during Al treatment, tobacco BY-2 cells were prelabelled with bodipy-PC for 10 min and then treated with 100 μM AlCl3 for 2 h. HP-TLC analysis of the labelled products showed significantly lower production of bodipy-DAG in cells treated with AlCl3, while bodipy-PA, bodipy-MAG and bodipy-LPC were only slightly affected (Fig. 3). Comparable results were observed with Al(NO3)3 (data not shown). The effect of Al on DAG was concentration-dependent (Fig. 4a). The decrease of DAG was very rapid and was already detected after 10 min of Al treatment; DAG dropped to 40% within 20 min when compared with the nontreated cells (Fig. 4b). When cells were treated for longer periods (up to 4 h), there was no further decrease of DAG (data not shown).

Figure 3.

 Profile of bodipy-labelled lipid products after Al treatment of tobacco BY-2 cells. (a) Bodipy-phosphatidylcholine (bodipy-PC) was added to the 3-d-old cell suspension and after 10 min of incubation, 100 μM AlCl3 was added. Lipids were extracted after 2 h of incubation and separated by high-performance thin layer chromatography (HP-TLC). (b) Standards of bodipy-labelled products. Monoacylglycerol (MAG) was identified by mass spectrometry. This experiment was repeated five times with similar results. DAG, diacylglycerol; LPC, lysophosphatidylcholine; PA, phosphatidic acid; PC, phosphatidylcholine.

Figure 4.

 Concentration dependence (a) and time course (b) of bodipy-diacylglycerol (bodipy-DAG) production in tobacco BY-2 cells treated with Al. (a) Three-day-old BY-2 cells were prelabelled for 10 min with bodipy-phosphatidylcholine (bodipy-PC) and treated for 2 h with different concentrations in the range 0–100 μM AlCl3. Lipids were extracted, separated by high-performance thin layer chromatography (HP-TLC) and quantified. Each value is related to the control nontreated cells (100%). The plotted values are the means + SE from independently analysed parallel samples. This experiment was repeated three times with similar results. (b) Three-day-old BY-2 cells were prelabelled for 10 min with bodipy-PC and treated with 100 μM AlCl3. Lipids were extracted at the time intervals indicated, separated by HP-TLC and quantified. Each value is related to the control nontreated cells (100%). The plotted values are the means + SE from independently analysed samples run in triplicate. This experiment was repeated three times with similar results.

Bodipy-DAG was generated by PC-PLC

In respect of the fact that DAG can be generated via several routes, a question arose as to which of these pathways is responsible for the decrease of DAG in Al-treated cells. The observed decrease of DAG in response to Al treatment can occur in response to either down-regulation of enzymes producing DAG or up-regulation of enzymes metabolizing DAG (Supporting Information, Fig. S1).

Thus, at first, we considered PC-PLC, PLD/PA-phosphatase pathway or MAG-acyltransferase.

Bodipy-DAG was not generated via a two-step PLD/PA-phosphatase pathway  We tested whether the observed inhibition of bodipy-DAG concentration is the result of inhibition of a two-step PLD/PA-phosphatase pathway. In this pathway, PLD cleaves PC to produce PA, which is then dephosphorylated by PA-phosphatase to DAG. In this experiment we used a PLD-specific transphosphatidylation assay (Munnik et al., 1995; Wang, 1999) based on the ability of PLD to preferentially use primary alcohols instead of water as an acceptor of phosphatidyl group. Therefore, in the presence of n-butanol, phosphatidylbutanol is formed instead of PA and less DAG is formed. In our experiments, however, no significant effect on bodipy-DAG formation was detected when bodipy-PC-labelled BY-2 cells were incubated with n-butanol (0.4% v/v) for 2 h (Fig. 5a).

Figure 5.

 Profile of bodipy-labelled lipid products after n-butanol and Al treatment of tobacco BY-2 cells. (a) Three-day-old BY-2 cells were prelabelled for 10 min with bodipy-phosphatidylcholine (bodipy-PC) and treated with 0.4%n-butanol; or (b) pre-labelled for 10 min with bodipy-PA and treated with 100 μM AlCl3. Lipids were extracted after 2 h of incubation and separated by high-performance thin layer chromatography (HP-TLC). (c) Standards of bodipy-labelled products. Monoacylglycerol (MAG) was identified by mass spectrometry. This experiment was repeated twice with similar results. ButOH, butanol; DAG, diacylglycerol; LPC, lysophosphatidylcholine; PA, phosphatidic acid; PBut, phosphatidylbutanol; PC, phosphatidylcholine.

We also looked for PA-phosphatase dephosphorylation of PA to DAG. Bodipy-PA was incorporated into BY-2 cells for 10 min and the incorporation was not influenced by Al (Fig. S2). Cells were then treated with 100 μM AlCl3 for 2 h. An expected product of bodipy-PA metabolism, bodipy-DAG was not detected in either control or Al-treated cells (Fig. 5b).

Results of both experiments with n-butanol and with bodipy-PA clearly showed that the PLD/PA-phosphatase pathway was not involved in the formation of bodipy-DAG.

Al treatment did not influence MAG-acyltransferase activity  Diacylglycerol is the product of MAG-acyltransferase activity. Therefore, down-regulation of this enzyme could be involved in the decrease of bodipy-DAG concentration during Al treatment. Relatively high amounts of bodipy-MAG were formed from bodipy-PC in both control and Al-treated cells (Fig. 3). However, the amount of bodipy-DAG in Al-treated cells (100 μM AlCl3, 2 h) labelled with externally applied bodipy-MAG (Fig. S2) remained unchanged when compared with the control cells (Fig. S3a), suggesting that this pathway was not involved in the response to Al treatment.

The decrease of DAG in Al-treated cells was not caused by up-regulation of enzymes which use DAG as a substrate  The decrease of DAG can also be caused by up-regulation of DAG-lipase, DAG-kinase or choline phosphotransferase, enzymes that use DAG as their substrate (Fig. S1). To sort out whether these enzymes are up-regulated after Al treatment, cells were labelled with bodipy-DAG and treated with 100 μM AlCl3 for 2 h. Bodipy-MAG as the product of DAG-lipase was detected; however, its concentration was the same in control and Al-treated cells. Other expected products (bodipy-PA and bodipy-PC) were not detected in either the control or Al-treated cells (Fig. S3b). These results clearly indicated that the up-regulation of DAG-consuming enzymes was not involved in the observed decrease of bodipy-DAG in Al-treated cells.

Activity of PC-PLC in vitro slightly sensitive to Al was detected in the BY-2 cellular membranes

In the next step we wanted to show directly the presence of PC-PLC activity in vitro in the cell homogenate. Soluble, microsomal and plasma membrane-enriched fractions from tobacco BY-2 cells were prepared and the activity of PC-PLC in these fractions was tested using the fluorescent substrate bodipy-PC (see the Materials and Methods section). No detectable PC-PLC activity was found in the soluble fraction. As expected, bodipy-DAG was generated in the assay in both microsomal and plasma membrane fractions, and the highest specific PC-PLC activity was found in the plasma membrane fraction (Fig. 6a). We measured in vitro PC-PLC activity of the plasma membrane fraction treated by 100 μM AlCl3. The activity was slightly sensitive to Al at high AlCl3 concentrations (Fig. 6b).

Figure 6.

 Phosphatidylcholine-hydrolysing phospholipase C (PC-PLC) activity in vitro. Cytosolic, microsomal and enriched plasma membrane fractions were prepared from 3-d-old tobacco BY-2 cells. PC-PLC activity was determined in vitro using fluorescent substrate bodipy-phosphatidylcholine (bodipy-PC). (a) Cellular distribution of PC-PLC activity. (b) PC-PLC activity of plasma membrane fraction treated with AlCl3. The plotted values are the means + SE from independently analysed parallel samples. This experiment was repeated twice with similar results. CF, cytosolic fraction; DAG, diacylglycerol; MF, microsomal fraction; PM, enriched plasma membrane fraction.

Al treatment inhibited growth and caused a decrease of bodipy-DAG in tobacco pollen tubes

An immediate consequence of exposure of roots to Al is the inhibition of root growth which subsequently affects nutrients and water uptake. Along the root apex, the meristematic, transition, and apical elongation zones are the most sensitive to Al toxicity (Sivaguru et al., 1999). Besides these root cell types, there are other highly Al-sensitive cell types, including root hairs (Care, 1995; Jones et al., 1998), pollen tubes (Konishi & Miyamoto, 1983; Zhang et al., 1999) and filamentous algae (Alessa & Oliveira, 2001).

We chose pollen tubes to study the effect of Al on DAG changes and we intended to confirm results gathered using tobacco cell culture on another Al-sensitive cell type. An advantage of this experimental material is that it permits the same experimental approach to be used as in the case of BY-2 cell culture.

Growing tobacco pollen tubes incubated in cultivation medium containing various concentrations of AlCl3 showed reduced tube length after 1.5 h of treatment (Fig. 7a), confirming that the pollen tube growth was an Al-sensitive process. Further, tobacco pollen tubes were labelled with bodipy-PC and treated with different concentrations of Al (0–100 μM AlCl3) for 10 min. The reaction was terminated by the addition of the extraction solution and the lipids were extracted as described in the Materials and Methods section. HP-TLC analysis of the labelled products showed a significantly lower production of bodipy-DAG in pollen tube cells treated with Al and the effect of Al on bodipy-DAG was concentration-dependent (Fig. 7b).

Figure 7.

 Effect of Al on the growth rate and diacylglycerol (DAG) production in tobacco pollen tubes. (a) Effect of Al treatment on the growth of tobacco pollen tubes. Tobacco pollen was grown in a germination medium. After 1.5 h, fresh medium pH 5 was added 1 : 1 (v/v) together with different concentrations (0–100 μM) of AlCl3 and the culture was grown for an additional 1.5 h. The cells were fixed and mean tube growth rate was evaluated using a light microscope. At least 100 cells were evaluated for each point. Data shown are from one of three independent experiments and represent means + SE. (b) Production of bodipy-DAG in pollen tubes is dependent on the concentration of AlCl3. Tobacco pollen was grown in a germination medium. After 1.5 h, fresh medium at pH 5 was added 1 : 1 (v/v) and bodipy-phosphatidylcholine (bodipy-PC) and different concentrations (0–100 μM) of AlCl3 were added to the medium. Lipids were extracted after 10 min incubation, separated by high-performance thin layer chromatography (HP-TLC) and quantified. Each value is related to the control nontreated cells (100%). The plotted values are the means + SE from independently analysed parallel samples. This experiment was repeated three times with similar results.

DAG restored growth inhibition caused by Al treatment

To test if the pollen tube growth inhibition was associated with DAG decrease during Al treatment, exogenous DAG was added to pollen tubes treated with Al. Pollen tube elongation was rescued when the culture was incubated in a medium containing DAG simultaneously added with AlCl3, while the effect of exogenous PA (which can be produced from DAG by DAG-kinase) in the presence of AlCl3 was less pronounced (Fig. 8). Exogenous DAG and PA alone did not influence the pollen tube growth in our control experimental conditions.

Figure 8.

 Effect of exogenous diacylglycerol (DAG) on the growth rate of tobacco pollen tubes. Tobacco pollen was grown in a germination medium. After 1.5 h, fresh medium at pH 5 was added 1 : 1 (v/v) and 50 μM AlCl3 was added to the medium together with exogenous 5 μM DAG or phosphatidic acid (PA) and the culture was grown for an additional 1.5 h. The cells were fixed and the mean tube growth rate was evaluated using a light microscope. At least 100 cells were evaluated for each point. Data shown are from one of three independent experiments and represent means + SE.

Discussion

Several cellular processes are affected by Al. However, the exact molecular mechanism and time sequence of individual changes followed by Al treatment is still under investigation. Phospholipases, especially PI-PLC and PLD, are some of the components which are affected within minutes after Al treatment (Piña-Chable & Hernández-Sotomayor, 2001; Martínez-Estévez et al., 2003; Ramos-Díaz et al., 2007; Pejchar et al., 2008). Here we show that a new member of the plant phospholipase family, PC-PLC, is involved in the response of tobacco cells to Al exposure as well.

At the beginning we showed that there is a rapid decrease in the concentration of labelled DAG after Al treatment and that this decrease is dependent on the concentration of Al used (Figs 3, 7). Next, we studied in detail several alternative pathways implicated in the generation and consumption of DAG. The slight decrease in the concentration of bodipy-PA (Fig. 3) indicated that PLD could be inhibited during 2 h of Al stress and that the PLD/PA-phosphatase pathway might therefore be involved. In addition, we recently reported that Al also inhibited PIP2-dependent PLD activity in vitro (Pejchar et al., 2008). Nevertheless, the decreased DAG concentration shown here cannot be caused by inhibition of the PLD/PA-phosphatase pathway because we did not observe any active transformation of bodipy-PA to bodipy-DAG (Fig. 5b). In addition, in the presence of n-butanol, the concentration of bodipy-DAG was not changed (Fig. 5a), which means that PLD is not involved in the production of bodipy-DAG. We also excluded other possible pathways that could be involved in DAG production or consumption. Therefore, the DAG decrease during Al treatment was the result of the inhibition of PC-PLC activity acting directly on PC. It is important to emphasize that our results do not imply that the PLD/PA-phosphatase pathway or DAG production via the MAG-acyltransferase are not active in plant cells. These pathways are well documented elsewhere (Pierrugues et al., 2001; Testerink & Munnik, 2005; Wang et al., 2006). However, our findings signify that, by using bodipy-PC as substrate, we can monitor specifically the direct conversion of PC to DAG that is mediated by the enzyme PC-PLC.

The use of an inhibitor of PC-PLC activity would be another tool to study involvement of PC-PLC in bodipy-DAG formation. D609 is an inhibitor of PC-PLC activity known from animal cell studies. However, this inhibitor is known to be active towards a zinc-dependent type of PC-PLC. Plant PC-PLC and a zinc-dependent type of PC-PLC differ significantly in sequence and secondary protein structure (Fig. S4a,b); thus it is not very probable that D609 will inhibit plant PC-PLC activity. This assumption was experimentally confirmed, as inhibition of bodipy-DAG formation was not detected in BY-2 cells treated with 20 μM D609 (Fig. S4c).

Using the same substrate, we detected activity of PC-PLC in the membrane fractions prepared from tobacco BY-2 cells. The highest specific PC-PLC activity was found in the plasma membrane fraction, which is in agreement with the results of Nakamura et al. (2005). However, the in vitro PC-PLC activity was sensitive only to high concentrations of AlCl3. This result either suggests that Al causes reduction of PC-PLC indirectly or it may reflect experimental conditions of the in vitro assay, which are certainly different from intracellular conditions. Alteration of the substrate leading to less availability as a result of the structural changes caused by binding with Al must also be considered as a possible indirect inhibitory mechanism.

Rapid inhibition of both growth and DAG formation after Al treatment was found in tobacco pollen tubes as well. When exogenous DAG was added to Al-treated pollen tubes, the inhibition of growth was rescued while PA had a minor effect, suggesting an important role for DAG/PC-PLC in Al toxicity. Neither DAG nor PA had an effect on pollen growth when added without AlCl3.

Although the inhibition of the PC-PLC product, DAG, after Al treatment and the remediation of Al-inhibited pollen tube growth strongly suggest an important role of this lipid in Al toxicity, the exact nature of this role remains to be determined.

Diacylglycerol has a unique function in lipid-mediated signalling, as an intermediate in lipid metabolism and a component of membranes. In recent years, there has been great progress in understanding the DAG metabolism in human cells at the molecular level. Deregulation of the DAG metabolism has been linked to the pathophysiology of several human diseases, such as cancer, diabetes, immune system disorders and Alzheimer’s disease. Impaired DAG generation and/or consumption also have severe effects on organ development and cell growth (Carrasco & Mérida, 2007).

The signalling role of DAG in plant cells is not obvious. Munnik (2001) and Meijer & Munnik (2003) reported that DAG as a product of phosphatidylinositol 4,5-bisphosphate hydrolysis was rapidly phosphorylated by DAG-kinase to PA, which plays active roles in the plant signalling processes. However, DAG is likely to act as a signalling molecule in some systems, such as tobacco pollen tubes (Helling et al., 2006). Protein kinase C (PKC) is the principal target of DAG regulation in animal cells (Griner & Kazanietz, 2007). Although sequence homologues of PKC have not been identified in the Arabidopsis genome, functional homologues were described in potato tuber (Subramaniam et al., 1997) and in Brassica juncea seedlings (Deswal et al., 2004). These data indicate that DAG may act as a signalling molecule in plant cells as well, yet the molecular mechanism remains unknown.

Diacylglycerol is also important in the structure and dynamics of biological membranes. DAG, when concentrated in small membrane areas, can influence membrane curvature and induce unstable, asymmetric regions in membrane bilayers. These are essential for membrane fusion and fission processes (Carrasco & Mérida, 2007; Haucke & Di Paolo, 2007). Calcium-induced fusion of phospholipid vesicles was enhanced by DAG in vitro. The fusion was stimulated specifically by 1,2-sn-DAG, whereas 1,3-isomers were not effective. It has also been described (Goñi & Alonso, 1999) that the rate of fusion increased with PC-PLC activity within a certain range of activities. It is clear that the catalytic action of PLC producing DAG, not the mere presence of the enzyme molecule, was responsible for the effect on fusion (Goñi & Alonso, 1999; Gómez-Fernández & Corbalán-García, 2007). Membrane fusion events occur in many physiological processes, such as exocytosis, endocytosis, membrane biogenesis and cell division. Thus, it is worthwhile assuming that the decrease of DAG content during Al stress might affect the processes described earlier and rapidly inhibit root growth.

In summary, we report a rapid inhibition of DAG formation in tobacco cells during Al treatment. The DAG decrease during Al stress was ascribed to the inhibition of the novel plant phospholipase, PC-PLC. Such findings suggest an important role of this recently revealed plant enzyme in the perception of environmental signals in plant cells. Both downstream and upstream molecular events are currently being investigated.

Acknowledgements

The authors thank Hana Fialová for her technical assistance. The work was supported by the Czech Science Foundation (grant no. 522/07/1614) and the Ministry of Education, Youth and Sports (project no. MSM 6046137305).

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