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American Society of Plant Biologists Elicitor-Activated Phospholipase A2 Generates Lysophosphatidylcholines That Mobilize the Vacuolar H+ Pool for pH Signaling via the Activation of Na+-Dependent Proton FluxesInstitute of Pharmaceutical Biology, Department of Cell Physiology, Martin-Luther-University, 06120 Halle, Germany 1 To whom correspondence should be addressed. E-mail roos{at}pharmazie.uni-halle.de; fax 49-345-5527006
The elicitation of phytoalexin biosynthesis in cultured cells of California poppy involves a shift of cytoplasmic pH via the transient efflux of vacuolar protons. Intracellular effectors of vacuolar proton transport were identified by a novel in situ approach based on the selective permeabilization of the plasma membrane for molecules of  10 kD. Subsequent fluorescence imaging of the vacuolar pH correctly reported experimental changes of activity of the tonoplast proton transporters. Lysophosphatidylcholine (LPC) caused a transient increase of the vacuolar pH by increasing the Na+ sensitivity of a Na+-dependent proton efflux that was inhibited by amiloride. In intact cells, yeast elicitor activated phospholipase A2, as demonstrated by the formation of LPC from fluorescent substrate analogs, and caused a transient increase of endogenous LPC, as determined by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry. It is suggested that LPC generated by phospholipase A2 at the plasma membrane transduces the elicitor-triggered signal into the activation of a tonoplast H+/Na+ antiporter.
Transient shifts of intracellular and apoplastic pH are essential steps in several signal transduction processes of plant cells. Prominent examples include the regulation of stomatal K+ channels by auxin and abscisic acid (Grabov and Blatt, 1997  ; Grill and Himmelbach, 1998), the gravitropic response (Scott and Allen, 1999), the control of apical growth (Feijo et al., 1999), and the induction of pathogen defense reactions in nonhost resistance (Mathieu et al., 1996; Lapous et al., 1998; Roos et al., 1998). Cytoplasmic acidification induced by biotic or abiotic stress is considered a plant-specific trigger for the synthesis of phytoalexins and other secondary metabolites (Sakano, 2001). Regulatory pH shifts may act independent of or in concert with the better investigated Ca2+ signature (for reviews, see Roos, 2000, 2002).
In cultured plant cells, microbial elicitors often trigger rapid alkalinization of the apoplast and the outer medium (Felix et al., 1993
The increase of external pH originates from an influx of protons into the challenged cells, as shown by the simultaneous decrease of intracellular pH and the similar specificities of both pH shifts with respect to elicitor structures such as the chain length of oligogalacturonides (Mathieu et al., 1996
The huge and highly regulated proton pool of the plant vacuole has not yet been considered as a source of protons for cytoplasmic pH changes, although auxin-dependent pH changes were blocked in experimentally evacuolated cells (Frohnmeyer et al., 1998
Acidification of the cytoplasm that was fed by vacuolar protons was described first in elicitor-treated cells of California poppy (Roos et al., 1998
After depletion of the vacuolar proton pool by preincubation with the membrane-permeant base methylamine (i.e., in the absence of a pH gradient across the tonoplast), neither cytoplasmic acidification nor alkaloid biosynthesis could be elicited. Both reactions were restored by withdrawing methylamine and subsequent recovery of vacuolar acidity to a pH of Here, we propose a mechanism by which the signal originating from elicitor contact is relayed to the efflux of vacuolar protons. A product of elicitor-stimulated phospholipase A2 (PLA2) was found to specifically activate proton efflux from the vacuolar pool; thus, it is a likely candidate for the signal transfer between the plasmalemma and the tonoplast.
An in Situ System for Monitoring Vacuolar Proton Transport The search for low-molecular-mass signal molecules that are formed in elicited cells and that trigger vacuolar proton fluxes is central to the present study. It requires an assay of vacuolar transport that is based on free access of micromolecules to the tonoplast and that maintains a maximum of vacuolar functions of the intact cell. An in situ system was developed that, after selective permeabilization of the plasma membrane for low-molecular-mass compounds, kept the vacuole intact within its macromolecular intracellular environment.
In situ measurements appeared superior to experiments with the isolated organelle, because the mechanisms of cellular control over vacuolar proton transport likely include interactions with cytoplasmic proteins and other organelles. Such processes might influence vacuolar (V)-ATPase activity via dissociation/reassociation of its subunits (Yoshida, 1991 By combining osmotic shock with a temperature shift, the plasma membranes of cultured cells could be permeabilized without impairing the known features of vacuolar proton transport (see Methods). The main properties of the resulting in situ system of vacuolar transport are described below.
(1) The formation of "pores" in the plasma membrane was confirmed by the rapid uptake and subsequent incorporation into DNA of propidium iodide, a dye that is impermeant to intact cell membranes (Figure 1)
. After permeabilization,
(2) Nearly all vacuoles, but not the cytoplasmic areas of permeabilized cells, accumulated the pH probes 5-(and-6)-carboxy-2',7'-dimethyl-3'-hydroxy-6'-N-ethylaminospiro[isobenzofuran-1(3H), 9'-(9H)xanthen]-3-one (DM-NERF) and carboxyfluorescein liberated from its permeant acetoxymethyl diacetate ester (Figure 1A). These indicators were used to monitor vacuolar pH via confocal or classic fluorescence ratio imaging during continuous perfusion in a flow-through cell chamber. Under these conditions, the vacuolar proton pool was maintained and controlled by the joint activities of V-ATPase and V-pyrophosphatase (V-PPase), as shown by the response of vacuolar pH to energy supply, specific inhibitors, and changes of the ionic environment.
In suspensions of intact cells, the pH measured in individual vacuoles ranged from 4.3 to 6.4 and was not influenced significantly by adding 1 to 3 mM of either MgATP or Mg pyrophosphate (MgPP). After the permeabilization procedure, vacuoles displayed pH values between 6.2 and 7.2 that did not change by >0.1 unit during 30 min in the absence of energy sources. Upon the addition of MgATP, the vacuoles acidified rapidly, with an initial rate that saturated at
(3) Specific inhibitors of the tonoplast proton pumps triggered a transient loss of vacuolar protons. Among them are bafilomycin A1, a well-characterized inhibitor of V-ATPase (Crider et al., 1994 ; Brauer et al., 1997), and destruxin B, a mycotoxin known for its specific inhibition of V-type ATPases in yeast (Muroi et al., 1994) and mung bean (Bandani et al., 2001). As seen in Figures 5A and 5B
, the effect of destruxin B but not of bafilomycin A1 was fully reversible, which reflects properties of the yeast V-ATPase (Muroi et al., 1994). A transient loss of vacuolar protons also could be evoked by exchanging the Cl- of the perfusion medium for NO3-, confirming the known inhibition of V-ATPase by nitrate. Finally, the diphosphonate etidronate, a specific inhibitor of V-PPase (Zhen et al., 1994), triggered an increase of vacuolar pH if Mg pyrophosphate was present as an energy source (Figure 5C).
In summary, it appears that the in situ vacuoles retained the main characteristics of active and passive proton transport at the tonoplast and thus constitute a sensitive test system. The vacuolar pH reflects a dynamic equilibrium of active accumulation and passive efflux of H+ that is shifted toward a transient net efflux upon the inhibition of proton pumps. Regulatory mechanisms remain intact that achieve the reaccumulation of lost protons even in the presence of pump inhibitors (Figures 5B and 5C). By changing the concentration of the energy source during a perfusion experiment, a slow, nearly constant rate of acidification could be adjusted, facilitating the detection of proton efflux that appeared as an upward deflection of a slightly decreasing pH trace (cf. Figure 5C).
Lysophosphatidylcholine Triggers the Net Efflux of Vacuolar Protons Table 1 shows the results of an intense search for phospholipid-derived compounds that increased the pH of in situ vacuoles when added to the perfusion medium. Among several types of molecules tested, only lysophosphatidylcholine (LPC) triggered a significant loss of vacuolar protons. The ability to evoke a pH shift required a lysophospholipid structure with a choline head group, but there was no discrimination between different fatty acid chain lengths (which varied between 12 and 18 carbon atoms) or their degree of unsaturation. Accordingly, PLA2 digests of plasma membranes prepared from the cell strain used also were active.
The rapid alkaline shift of the vacuolar pH triggered by LPC (between 0.1 and 0.3 units) was transient in nature; that is, the lost protons reappeared in the vacuole within  10 min even in the presence of LPC (Figures 6
, inset, and 7A). Proton efflux also could be initiated in the absence of MgATP or MgPP, provided that a sufficient pH difference toward the extravacuolar medium existed at the time of LPC addition (Figure 7A
, compare top and bottom traces). The dependence of the rate of proton efflux on the LPC concentration could be established by determining the ratio of the decline of vacuolar H+ concentration to the initial pH gradient between the vacuole and the outer medium.
This (remarkably varying) parameter is assumed to be part of the driving force of efflux. As shown in Figure 7B, a minimum concentration of 200 nM LPC proved to be active, and 600 nM was required for the half-maximum effect. At a typical vacuolar pH of 5.2, the decrease of proton concentration caused by 1 µM LPC in vacuoles of different cells ranged from 1 to 2.5 µM. These shifts are in the range found in vacuoles of intact cells after elicitor contact (Roos et al., 1998).
The saturation of H+ efflux at
These data suggest a Na+/H+ exchange process at the vacuolar membrane that is stimulated by LPC. Therefore, we tested the effect of amiloride on the Na+-dependent pH shift. This compound is a well-known inhibitor of Na+/H+ antiporters in animal cells (Counillon and Pouysségur, 2000 ) and was used successfully to inhibit similar transport systems of yeast and plant vacuoles (Darley et al., 2000). As shown in Figure 9A
, the efflux of vacuolar H+ caused by 1 µM LPC plus 10 mM Na+ was abolished completely by 40 µM 5-(N-ethyl-N-isopropyl)-amiloride. The same concentration prevented the proton efflux initiated by 30 mM Na+ in the absence of LPC (Figure 9B).
Other amiloride derivatives, such as 5-(N,N-dimethyl)-amiloride and 5-(N-methyl-N-isobutyl)-amiloride, showed similar inhibition. Although no efflux of vacuolar protons occurred in the presence of amiloride, the rate of acidification increased considerably, giving rise to a more acidic vacuolar pH (Figures 9A and 9B). This finding suggests that an amiloride-sensitive Na+/H+ antiport constitutes a permanent drain of the ATPase-generated H+ gradient across the tonoplast; hence, inhibition of this exchange increases the steady state concentration of vacuolar protons.
In summary, it appears that LPC increases the Na+ sensitivity of a H+/Na+ antiport that is located at the tonoplast and energized by the vacuolar proton gradient. Thus, an efflux of protons is initiated at physiological Na+ concentrations of the cytosol. The effect of LPC did not change upon the removal of Ca2+ from the perfusion medium, as indicated by the absence of any influence of 10 µM of the Ca2+ chelators 1,2-bis(o-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (Kd of
LPC Is Increased Transiently after Elicitor Contact
In the present study, using a gel-based cell chamber that probably minimized mechanical and osmotic stress, elicitor-triggered stimulation of PLA activity was distinguished clearly from the activity of nonelicited cells (Figure 10)
. Elicitor-triggered fluorescence development was prevented by the inhibitors of PLA2 aristolochic acid and 5,8,11,14-eicosatetraynoic acid (ETYA) (Table 2). To identify the substrate specificity of PLA, the conversion of an ether phospholipid was tested that bears one BODIPY fluorophore at the sn-1 alkyl chain: BODIPY-1-O-alkyl-2-acyl-sn-glycerophosphocholine (BEPC; for structure, see Methods). The hydrolysis product indicative of PLA2 activity, BODIPY-1-(O-alkyl)-sn-glycero-3-phosphocholine (BELPC), was formed at a rate that increased after elicitor contact (Figure 11A)
. Thus, the elicitor-stimulated enzyme can be attributed to the A2 group because the ether substrate cannot be converted by PLA1 (Hendrickson et al., 1990
Cellular Steady State Content of LPC Although the hydrolysis of artificial phospholipids indicated the ability of elicited cells to overproduce molecules of the LPC type, the question remained whether elicitor-stimulated cells generated enhanced levels of LPC from genuine phospholipids. When fed to intact cells, BODIPY-labeled LPC (but not its ether homolog) was metabolized rapidly, which indicates a fast turnover of lysophospholipids (Figure 11B). This was not unexpected, because LPC-metabolizing enzymes (e.g., acylases and lysophospholipases) are known from several plant species (Testet et al., 1996 ; Wang, 2001). To gain insight into the steady state content of endogenous LPC, we attempted to use matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS). This method proved a suitable analytic tool, because it allows fast and direct assays of defined phospholipids and related molecules without disturbing signaling processes or metabolism (which might occur during preincubation with radiolabeled lipid precursors). Table 3 lists some of the molecular species of phosphatidylcholine (PC), LPC, and diacylglycerol (DAG) identified in cell extracts and plasma membranes of California poppy. LPCs were represented mainly by the species 18:2 (2 to 3 pmol/mg dry weight). Elicitor contact of cell suspensions caused a transient, significant increase of this molecule that could be differentiated clearly from the slower, continuous increase of the control level (Figure 12A) .
Although less pronounced, an elicitor-dependent increase also was established for the LPC species 16:0. The elicitor-triggered increase of LPC content generated from endogenous sources occurred in the same period as the previously mentioned accumulation of the fluorescent conversion products of the phospholipids BEPC (Figure 11) and BPC (Figure 13 , Table 2). These findings suggest a transient activation of PLA2 by elicitor contact rather than a decrease of LPC metabolism, which in itself would not cause increased fluorescence.
Elicitor Activation of PLA2 in Plasma Membrane Vesicles A calcium-independent PLA2 is known to be active in plasma membrane vesicles prepared from California poppy cell suspensions (Roos et al., 1999 ). Because this membrane preparation did not metabolize fluorescently labeled LPC, it appeared suitable to examine the formation of genuine LPCs from membrane phospholipids. As seen in Figure 12B, the molecular species LPC 16:0, LPC 18:1, and (most abundant) LPC 18:2 could be quantified in MALDI-TOF mass spectra. After elicitor contact, all of them accumulated significantly over the control level. The increase of LPC species started more slowly than in cell suspensions but was not transient as in intact cells, reflecting the absence of LPC metabolism. The compound showing the greatest increase after elicitor contact (LPC 18:2) is identical to the abundant LPC species of intact cells that also peaked in response to elicitor (Figure 12A). Likewise, hydrolysis of the artificial PLA2 substrate BEPC was enhanced in elicitor-treated compared with control vesicles, and this activity was reduced by the PLA2 inhibitor ETYA (Figure 12B, inset). Thus, despite the significant formation of LPC in untreated membrane preparations, whether attributable to a basal level of PLA2 activity or nonspecific hydrolases, the elicitor-triggered production of LPC most probably represents PLA2 activity. This enzyme converts phosphatidylcholines of the plasma membrane (or contaminating membranes) but also the artificial substrates used in this study.
LPC Molecules Are Highly Mobile in Intact Cells
As shown in Figure 13, upon incubation with BELPC, fluorescence was seen over all cytoplasmic areas within 2 min. The fluorescence rapidly reached the tonoplast but did not enter the vacuolar interior. Although these data do not address the mode of transport, they indicate that LPC generated at the plasmalemma can reach the tonoplast easily. This would be consistent with its putative functioning upstream of the vacuolar pH shift: the latter has been imaged in intact cells 2 to 5 min after elicitor contact (Roos et al., 1998
The data presented here suggest a link between two signaling modules of an elicited plant cell: the generation of pH changes and the activation of PLA2. Our earlier suggestions regarding the mobilization of the vacuolar proton pool for the signal transfer (Roos et al., 1998 ) now are more substantiated: LPC that is overproduced transiently by PLA2 at the plasma membrane might convert the elicitor-derived signal into the activation of a vacuolar H+/Na+ exchanger. The evidence supporting this concept is summarized below. (1) Elicitor stimulation of PLA2 activity is indicated by consistent data from two different approaches: monitoring of the hydrolysis of artificial phospholipids (BPC and BEPC) in cell suspensions (Figure 11A) or individual cells (Figure 10) and mass spectrometric detection of LPC species generated from endogenous phospholipids (Figure 12). The finding that intact cells rapidly metabolize fluorescently labeled LPC (Figure 11B) implies tight control over the steady state content of LPC and may explain why the basal level of PLA2 activity detected in control cells did not trigger alkaloid biosynthesis.
(2) The dynamics of LPC, its estimated cytoplasmic concentration, and the extent of vacuolar pH shifts triggered by this compound all fit into the range spanned by the other landmark of the elicitation process, the proton fluxes of intact cells. The level of LPC peaked 2 to 3 min after elicitor contact (Figure 12A); in the same period, maximum shifts of vacuolar and cytoplasmic pH have been observed (see Figure 5 in Roos et al., 1998
The latter was defined as the difference between the total water content of a given cell pellet and the extracellular water volume that is accessible to the nonpermeant protein HSA. It amounts to
The amplitudes of vacuolar pH shifts caused by LPC contact in situ coincide largely with those measured in elicitor-treated intact cells. In the whole cell study (Roos et al., 1998
(3) In purified plasma membranes, a significant portion of the Ca2+-independent, LPC-producing capacity responded to elicitor and was sensitive to ETYA. These findings suggest that the elicitor-stimulated PLA2 activity of intact cells is localized at least in part in this membrane, even though the activation of different lipid-degrading activities during membrane isolation and purification must be taken into account. A Ca2+-insensitive, ETYA-inhibited PLA2 that was isolated as a 48-kD protein from the microsomal membranes of broad bean leaves (Jung and Kim, 2000
The involvement of PLA2 activities in the cellular signaling of plants has been demonstrated repeatedly (for reviews, see Chapman, 1998
With regard to signal functions of the hydrolysis products, fatty acids released by PLA2 are likely second messengers in the transmission of wound- or systemin-triggered signals (Lee et al., 1997
(4) The effect of LPC on vacuolar pH probably reflects the activation of a Na+/H+ antiport system at the vacuolar membrane. This is indicated by the strict dependence on Na+ at concentrations in a range required for the activity of Na+/H+ antiporters in tonoplast vesicles (Barkla et al., 1995
Tonoplast Na+/H+ exchangers were first detected and cloned in plants only a few years ago (Nass et al., 1997
Our experiments suggest a new role of these transporters in the generation of cytoplasmic pH shifts, with implications for pH-controlled signal transfer and pH homeostasis. LPC increases the sensitivity of the Na+/H+ exchange toward cytoplasmic Na+ by a factor of
LPC is known to activate the H+-transporting ATPase of the plasma membrane (Palmgren and Sommarin, 1989
In summary, LPC is a good candidate for an intracytoplasmic messenger that bridges the gap between the activation of a stress-responding enzyme of the plasma membrane and the generation of vacuolar proton fluxes for the expression of the phytoalexin response after contact with low elicitor concentrations (Roos et al., 1998
(5) Finally, it needs to be mentioned that, among the lipid metabolites tested, DAGs also caused an efflux of vacuolar protons (Table 1). However, their cellular content (
This value is 2 orders of magnitude higher than the resting value of cytoplasmic Ca2+ in plant cells (100 to 200 nM) (Trewavas and Malho, 1998
Plant Materials Suspension-cultured cells of California poppy (Eschscholtzia californica) were grown in a medium according to Linsmaier and Skoog (1965) with the hormones 2,4-D and  -naphthalene acetic acid (1 µM each). Cultivation was performed on a gyratory shaker (100 rpm) at 24°C in continuous light (7 µmol·m-2·s-1) in a 9-day growth cycle.
Yeast Elicitor Preparation
Permeabilization and Preparation of Cells for Microscopy After adding 2 µM propidium iodide from a 1 mM aqueous stock solution, a 10-µL cell suspension was mixed with 50 µL of medium 2 that contained 3% of low-melting-point agarose (type VII; Sigma) and was kept at 40°C. The mixture was spread rapidly on a cover glass kept at 20°C. The resulting agarose film with the embedded cells was placed in a flow-through cell chamber (volume of 140 µL; Bachofer, Reutlingen, Germany), mounted at the microscope stage, and perfused with perfusion medium at a flow rate of 300 µL/min. Medium 1 consisted of 300 mM KCl, 20 mM Hepes, and 5 mM glutathione. Medium 2 consisted of 100 mM KCl, 20 mM Hepes, and 5 mM glutathione. Perfusion medium consisted of 100 mM KCl, 10 mM NaCl, 50 mM 3-(N-morpholino)-propanesulfonic acid Bis-tris propane, 5 mM glutathione, 0.5 mM Na citrate, 0.5 mM Na2HPO4, 2 mM ascorbic acid, 100 µM KCN, and 0.1% BSA, Na2ATP, or Na4 pyrophosphate plus equimolar amounts of MgCl2 indicated in the relevant figures. For Na+-free perfusion medium, Na+ was replaced by K+ and KCl + NaCl was replaced by 70 mM KCl + 80 mM sorbitol. All solutions were adjusted to pH 7.4 with KOH. Effectors and inhibitors were added from stock solutions in DMSO (destruxin and bafilomycin), chloroform (phospholipids, lysophospholipids, fatty acids, and diacylglycerol), or water (Na etidronate). The final concentration of each solvent was kept at <0.05% and did not affect the vacuolar pH in a standard perfusion experiment.
Microscopy and pH Mapping Confocal imaging was performed with a Leica TCS-SP confocal microscope (Wetzlar, Germany). Fluorescence images of vacuoles accumulating DM-NERF (a rhodol-based pH indicator from Molecular Probes, Eugene, OR) were obtained by excitation with the 488-nm argon laser line and simultaneous scanning at emission of 493 to 525 nm (channel 1) and emission of 527 to 560 nm (channel 2). Ratio images (channel 1:channel 2) were calculated using Corel Photopaint software (Toronto, Canada).
Calibration of ratio images to pH was done with calibration graphs established for DM-NERF (pK of 5.4) and carboxyfluorescein (pK of 6.4). For this purpose, cells preincubated with the respective pH probe were permeabilized and resuspended in 40 mM Mes buffers of varied pH. These buffers contained either 80 mM methylamine (pH 7 to 5.5) or 5 mM Na pivalate, 100 mM KCl, and 10 µM nigericine (pH 5.2 to 4.0) to facilitate the equilibration of external and vacuolar pH, according to Brauer et al. (1997)
Detection of Changes in Vacuolar pH during Perfusion Experiments
Assay of Phospholipase A Activity
In Cell Suspensions The chloroform phase was removed with a syringe, concentrated by evaporation under nitrogen at 20°C and resuspension in a smaller volume of chloroform, and used for TLC analysis. Samples for matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were prepared similarly but in the absence of external substrates, and 200 µL methanol:chloroform (4:1, v/v) and 160 µL of additional chloroform were used.
In Plasma Membrane Vesicles For MALDI-TOF-MS, 50-µL samples were mixed with 100 µL of methanol:chloroform (4:1, v/v) and vortexed for 30 min. After adding 50 µL of 150 mM NaCl and 80 µL of chloroform, samples were treated as described for cell suspensions.
Preparation of Plasma Membrane Vesicles
TLC of Phospholipid Samples
MALDI-TOF Assays of Phospholipids, Lysophospholipids, and Diacylglycerols
Chloroform extract (1.8 µL) was mixed with the same volume of matrix solution (0.5 M 2,5-dihydrobenzoic acid and 0.1% [v/v] trifluoroacetic acid in methanol) and spotted onto the sample plate, and the solvent was removed by drying under a warm air stream. MALDI-TOF mass spectra were obtained on a Voyager Biospectrometry work station (PerSeptive Biosystems, Framingham, MA) using the protocol published by Schiller et al. (1999) In brief, the ions liberated by a pulsed nitrogen laser at 337 nm were accelerated by a 20-kV voltage within the ion source, and the total field-free time-of-flight distance was 2 m. Internal calibration was performed by setting the mass peak of the protonated 2,5-dihydrobenzoic acid matrix to 155.034 D. Each mass spectrum was calculated by averaging the results of 128 single laser shots and was repeated two times. Positive ion mode spectra were used throughout.
Quantification of the compounds detected was based on signal-to-noise (S/N) ratios as described by Müller et al. (2001) In our experiments, the S/N ratios of LPC and diacylglycerol (DAG) peaks were calibrated to the concentration of these compounds by adding authentic LPC 16:0 or DAG 18:0/18:0 as internal standards to the chloroform extracts in amounts that yielded S/N ratios of a similar magnitude as those of the molecule to be analyzed. The increments in the S/N ratios caused by the authentic compounds were averaged and used to convert the measured S/N ratios of LPC and DAG to their respective concentrations. Peaks with S/N ratios of <3 were excluded from analysis.
Structures of the Artificial Phospholipase Substrates
In this substrate, the vicinity of the fluorophores located at either fatty acid results in intramolecular quenching. Separation of one or two fatty acid(s) upon hydrolysis causes an increase of fluorescence that can be visualized by fluorescence microscopy, as shown in zebrafish embryos (Farber et al., 1999
We are grateful to M. Petkovi  , J. Schiller, and K. Arnold (Institute of Medical Physics and Biophysics, University of Leipzig) for their expert help with the MALDI-TOF MS and to A. Nitzsche and W. Schwartze from our department for corroborating the fluorescence assays of phospholipase A. Destruxin B was a kind gift of A. Takatsuki (RIKEN Institute, Saitama, Japan). The work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 363) and by the Fonds der Chemischen Industrie.
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.002329. Received February 14, 2002; accepted April 1, 2002.
Apse, M.P., Aharon, G.S., Snedden, W.A., and Blumwald, E. (1999). Salt tolerance conferred by overexpression of a vacuolar Na+/H+ antiport in Arabidopsis. Science 285, 1256–1258. Arz, M.C., and Grambow, H.J. (1995). Elicitor and suppressor effects on phospholipase C in isolated plasma membranes correlate with alterations in phenylalanine ammonia-lyase activity of wheat leaves. J. Plant Physiol. 146, 64–70. Bandani, A.R., Amiri, B., Butt, T.M., and Gordon-Weeks, R. (2001). Effects of efrapeptin and destruxin, metabolites of entomogenous fungi, on the hydrolytic activity of a vacuolar type ATPase identified on the brush border membrane vesicles of Galleria mellonella midgut and on plant membrane bound hydrolytic enzymes. Biochim. Biophys. Acta 1510, 367–377.[Medline] Barkla, B.J., Zingarelli, L., Blumwald, E., and Smith, J.A.C. (1995). Tonoplast Na+/H+ antiport activity and its energization by the vacuolar H+ ATPase in the halophytic plant Mesembryanthemum crystallinum. Plant Physiol. 109, 549–556.[Abstract]
Blechert, S., Brodschelm, W., Hölder, S., Kammerer, L., Kutchan, T.M., Müller, M.J., Xia, Z.-Q., and Zenk, M.H. (1995). The octadecanoic pathway: Signal molecules for the regulation of secondary pathways. Proc. Natl. Acad. Sci. USA 92, 4099–4105. Blumwald, E. (2000). Sodium transport and salt tolerance in plants. Curr. Opin. Cell Biol. 12, 431–434.[CrossRef][Web of Science][Medline] Brauer, D., Uknalis, J., Triana, R., and Tu, S.I. (1997). Effects of external pH and ammonium on vacuolar pH in maize roothair cells. Plant Physiol. Biochem. 35, 31–39. Chandra, S., Heinstein, P.F., and Low, P.S. (1996). Activation of phospholipase A by plant defense elicitors. Plant Physiol. 110, 979–986.[Abstract] Chapman, K.D. (1998). Phospholipase activation during plant growth and development and in response to environmental stress. Trends Plant Sci. 11, 419–426.[CrossRef]
Counillon, L., and Pouysségur, J. (2000). The expanding family of eucaryotic Na+/H+ exchangers. J. Biol. Chem. 276, 1–4.
Crider, B.P., Xie, X.-S., and Stones, D.K. (1994). Bafilomycin inhibits proton flow through the H+ channel of vacuolar proton pumps. J. Biol. Chem. 269, 17379–17381. Darley, C.P., van Wuytswinkel, O.C., van der Woude, K., Mager, W.H., and de Boer, A.H. (2000). Arabidopsis thaliana and Saccharomyces cerevisiae NHX1 genes encode amiloride sensitive electroneutral Na+/H+ exchangers. Biochem. J. 351, 241–249.[CrossRef][Web of Science][Medline]
Farber, S.A., Olson, E.S., Clark, J.D., and Halpern, M.E. (1999). Characterization of Ca2+-dependent phospholipase A2 activity during zebrafish embryogenesis. J. Biol. Chem. 274, 19338–19346.
Feijo, J.A., Sainhas, J., Hackett, G.R., Kunkel, J.G., and Hepler, P.K. (1999). Growing pollen tubes possess a constitutive alkaline band in the clear zone and a growth-dependent acidic tip. J. Cell Biol. 144, 483–496. Felix, G., Regenass, M., and Boller, T. (1993). Specific perception of subnanomolar concentrations of chitin fragments by tomato cells: Induction of extracellular alkalinization, changes in protein phosphorylation, and establishment of a refractory state. Plant J. 4, 307–316.[CrossRef][Web of Science]
Feng, Y., and Forgac, M. (1994). Inhibition of vacuolar H+-ATPase by disulfide bond formation between cysteine 254 and cysteine 532 in subunit A. J. Biol. Chem. 269, 13224–13230. Frohnmeyer, H., Grabov, A., and Blatt, M.R. (1998). A role for the vacuole in auxin-mediated control of cytosolic pH by Vicia mesophyll and guard cells. Plant J. 13, 109–116. Fukuda, A., Nakamura, A., and Tanaka, Y. (1999). Molecular cloning and expression of the Na+/H+ exchanger gene in Oryza sativa. Biochim. Biophys. Acta 1446, 149–155.[Medline]
Gaxiola, R.A., Rao, R., Sherman, A., Grisafi, P., Alper, S.L., and Fink, G.R. (1999). The Arabidopsis thaliana proton transporters A6tNhx1 and Avp1 can function in cation detoxification in yeast. Proc. Natl. Acad. Sci. USA 96, 1480–1485. Grabov, A., and Blatt, M.R. (1997). Parallel control of the inward-rectifier K+ channel by cytosolic free Ca2+ and pH in Vicia guard cells. Planta 201, 84–95.[CrossRef][Web of Science] Grill, E., and Himmelbach, A. (1998). ABA signal transduction. Curr. Opin. Plant Biol. 1, 412–418.[CrossRef][Web of Science][Medline] Hanju, Y., Doonam, P., and Youngsook, L. (1996). In vivo evidence for the involvement of phospholipase A and protein kinase in the signal transduction pathway for auxin induced corn coleoptile elongation. Physiol. Plant. 96, 359–368.[CrossRef] He, D.Y., Yazaki, Y., Nishizawa, Y., Takai, R., Yamada, K., Sakano, K., Shibuya, N., and Minami, E. (1998). Gene activation by cytoplasmic acidification in suspension-cultured rice cells in response to the potent elicitor, N-acetylchitoheptaose. Mol. Plant-Microbe Interact. 11, 1167–1174.[CrossRef] Hendrickson, H.S., Kotz, K.J., and Hendrickson, E.K. (1990). Evaluation of fluorescent and colored phosphatidylcholine analogs as substrates for the assay of phospholipase A2. Anal. Biochem. 185, 80–83.[CrossRef][Medline]
Jung, K.M., and Kim, D.K. (2000). Purification and characterization of a membrane-associated 48-kilodalton phospholipase A2 in leaves of broad bean. Plant Physiol. 123, 1057–1068.
Kane, P.M. (1995). Disassembly and reassembly of the yeast vacuolar H+-ATPase in vivo. J. Biol. Chem. 270, 17025–17032.
Kuchitsu, K., Yazaki, Y., Sakano, K., and Shibuya, N. (1997). Transient cytoplasmic pH change and ion fluxes through the plasma membrane in suspension-cultured rice cells triggered by N-acetylchitooligosaccharide elicitor. Plant Cell Physiol. 38, 1012–1018. Lapous, D., Mathieu, Y., Guern, J., and Lauriére, C. (1998). Increase of defense gene transcripts by cytoplasmic acidification in tobacco cell suspensions. Planta 205, 452–458.[CrossRef] Larsson, C., Widell, S., and Sommarin, M. (1994). Isolation of highly purified plant plasma membranes and separation of inside-out and right-side-out vesicles. FEBS Lett. 229, 289–292.[CrossRef]
Laxalt, A.M., ter Riet, B., Verdonk, J.C., Parigi, L., Tameling, W.I.L., Vossen, J., Haring, M., Musgrave, A., and Munnik, T. (2001). Characterization of five tomato phospholipase D cDNAs: Rapid and specific expression of LePLD. Lebrun-Garcia, A., Bourque, S., Binet, M.N., Ouaked, F., Wendehenne, D., Chiltz, A., Schaffner, A., and Pugin, A. (1999). Involvement of plasma membrane proteins in plant defense responses: Analysis of the cryptogein signal transduction in tobacco. Biochimie 6, 663–668.[CrossRef] Lee, S., Suh, S., Kim, S., Crain, R.C., and Kwak, J.M. (1997). Systemic elevation of phosphatidic acid and lysophospholipid levels in wounded plants. Plant J. 12, 547–556.[CrossRef][Web of Science] Linsmaier, E.M., and Skoog, F. (1965). Organic growth factor requirements of tobacco tissue cultures. Physiol. Plant. 18, 100–127.[CrossRef] Mathieu, Y., Lapous, D., Thomine, S., Laurière, C., and Guern, J. (1996). Cytoplasmic acidification as an early phosphorylation-dependent response of tobacco cells to elicitors. Planta 199, 416–424.[Web of Science] Meijer, H.J.G., Arisz, S.A., van Himbergen, J.A.J., Musgrave, A., and Munnik, T. (2001). Hyperosmotic stress rapidly generates lyso-phosphatidic acid in Chlamydomonas. Plant J. 25, 541–548.[CrossRef][Web of Science][Medline] Miège, C., and Maréchall, E. (1999). 1,2-sn-Diacylglycerol in plant cells: Product, substrate and regulator. Plant Physiol. Biochem. 37, 795–808.[Medline]
Müller, M., Schiller, J., Petkovi
Müller, M.J., Brodschelm, W., Spannagl, E., and Zenk, M.H. (1993). Signaling in the elicitation process is mediated through the octadecanoid pathway leading to jasmonic acid. Proc. Natl. Acad. Sci. USA 90, 7490–7494. Munnik, T. (2001). Phosphatidic acid: An emerging plant lipid second messenger. Trends Plant Sci. 6, 227–233.[CrossRef][Web of Science][Medline] Munnik, T., Irvine, R.F., and Musgrave, A. (1998). Phospholipid signalling in plants. Biochim. Biophys. Acta 1389, 222–272.[Medline] Muroi, M., Shiragami, N., and Takatsuki, A. (1994). Destruxin B, specific and readily reversible inhibitor of vacuolar-type H+-translocating ATPase. Biochem. Biophys. Res. Commun. 205, 1358–1365.[CrossRef][Medline]
Narvaez-Vasquez, J., Florin-Christensen, J., and Ryan, C.A. (1999). Positional specificity of a phospholipase A activity induced by wounding, systemin, and oligosaccharide elicitors in tomato leaves. Plant Cell 11, 2249–2260.
Nass, R., Cunningham, K.W., and Rao, R. (1997). Intracellular sequestration of sodium by a novel Na+/H+ exchanger in yeast is enhanced by mutations in the plasma membrane H+-ATPase. J. Biol. Chem. 272, 26145–26152.
Nass, R., and Rao, R. (1999). The yeast endosomal Na+/H+ exchanger, NHx1, confers osmotolerance following acute hypertonic shock. Microbiology 145, 3221–3228. Padan, E. (2001). Na+/H+ antiporters. Biochim. Biophys. Acta 1505, 144–157.[Medline]
Palmgren, M.G., and Sommarin, M. (1989). Lysophosphatidylcholine stimulates ATP dependent proton accumulation in isolated oat root plasma-membrane vesicles. Plant Physiol. 90, 1009–1014. Paul, R.U., Holk, A., and Scherer, G.F. (1998). Fatty acids and lysophospholipids as potential second messengers in auxin action: Rapid activation of a phospholipase A2 activity by auxin in suspension-cultured parsley and soybean cells. Plant J. 16, 601–611.[CrossRef][Web of Science]
Petkovi Ratajczak, R., and Wilkins, T.A. (2000). Energizing the tonoplast. Annu. Plant Rev. 5, 133–173. Roos, W. (2000). Ion mapping in plant cells: Methods and applications in signal transduction research. Planta 210, 347–370.[CrossRef][Web of Science][Medline] Roos, W. (2002). Confocal pH topography in plant cells: Shifts of proton distribution are involved in plant signalling. In Handbook of Plant Growth: pH as a Major Variable in Plant Growth, Z. Rengel, ed (New York: Marcel Dekker), pp. 73–106. Roos, W., Dordschbal, B., Steighardt, J., Hieke, M., Weiss, D., and Saalbach, G. (1999). A redox-dependent, G-protein-coupled phospholipase A of the plasma membrane is involved in the elicitation of alkaloid biosynthesis in Eschscholtzia californica. Biochim. Biophys. Acta 1448, 390–402.[Medline]
Roos, W., Evers, S., Hieke, M., Tschöpe, M., and Schumann, B. (1998). Shifts of intracellular pH distribution as a part of the signal mechanism leading to the elicitation of benzophenanthridine alkaloid biosynthesis. Plant Physiol. 118, 349–364.
Roos, W., Schulze, R., and Steighardt, J. (1997). Dynamic compartmentation of vacuolar amino acids in Penicillium cyclopium. J. Biol. Chem. 272, 15849–15855. Roy, S., Pouénat, M.-L., Caumont, C., Cariven, C., Prévost, M.-C., and Esquerré-Tugayé, M.-T. (1995). Phospholipase activity and phospholipid patterns in tobacco cells treated with fungal elicitor. Plant Sci. 107, 17–25.[CrossRef][Web of Science] Ryan, C.A. (2000). The systemin signaling pathway: Differential activation of plant defensive genes. Biochim. Biophys. Acta 1447, 112–121. Sakano, K. (2001). Metabolic regulation of pH in plant cells: Role of cytoplasmic pH in defense reaction and secondary metabolism. Int. Rev. Cytol. 206, 1–44.[Web of Science][Medline]
Schaller, A., and Oecking, C. (1999). Modulation of plasma membrane H+-ATPase activity differentially activates wound and pathogen defense responses in tomato plants. Plant Cell 11, 263–272. Scherer, G.F.E., Hecker, D., and Muller, J. (1993). Ca2+ ions and lysophospholipids activate phosphorylation of different proteins in plasma membranes and tonoplast. J. Plant Physiol. 142, 425–431.[Web of Science] Schiller, J., Arnhold, J., Benard, S., Müller, M., Reichl, S., and Arnold, K. (1999). Lipid analysis by matrix-assisted laser desorption and ionization mass spectrometry: A methodological approach. Anal. Biochem. 267, 46–56.[CrossRef][Web of Science][Medline] Schiller, J., Arnhold, J., Glander, H.-J., and Arnold, K. (2000). Lipid analysis of human spermatozoa and seminal plasma by MALDI-TOF mass spectrometry and NMR spectroscopy: Effects of freezing and thawing. Chem. Physiol. Lipids 106, 145–156.[CrossRef][Web of Science][Medline]
Scott, A.C., and Allen, N.S. (1999). Changes in cytosolic pH within Arabidopsis root columella cells play a key role in the early signaling pathway for root gravitropism. Plant Physiol. 121, 1291–1298.
Senda, K., Doke, N., and Kawakita, K. (1998). Effect of mastoparan on phospholipase A2 activity in potato tubers treated with fungal elicitor. Plant Cell Physiol. 39, 1080–1086. Simon-Plas, F., Rusterucci, C., Milat, M.L., Humbert, C., Montillet, J.L., and Blein, J.P. (1997). Active oxygen species production in tobacco cells elicited by cryptogein. Plant Cell Environ. 20, 1573–1579.[CrossRef] Testet, E., Bessoule, J.J., Mongrand, S., Guillot-Salomon, T., Cantrel, C., and Cassagne, C. (1996). Occurrence of an acyl-CoA:1-acylglycerophosphorylcholine acyltransferase in plant mitochondria. FEBS Lett. 399, 87–91.[Medline] Trewavas, A.J., and Malho, R. (1998). Ca2+ signalling in plant cells: The big network! Curr. Opin. Plant Biol. 1, 428–433.[CrossRef][Web of Science][Medline]
van der Luit, A.H., Piatti, T., van Doorn, A., Musgrave, A., Felix, G., Boller, T., and Munnik, T. (2000). Elicitation of suspension-cultured tomato cells triggers the formation of phosphatidic acid and diacylglycerol pyrophosphate. Plant Physiol. 123, 1507–1515. Wang, X. (2001). Plant phospholipases. Annu. Rev. Plant. Physiol. Plant. Mol. Biol. 52, 211–231.[CrossRef][Web of Science][Medline] Weiser, T., and Bentrup, F.-W. (1990). (+)-Tubocurarine is a potent inhibitor of cation channels in the vacuolar membrane of Chenopodium rubrum L. FEBS Lett. 277, 220–222.[Medline] Xing, T., Higgins, V.J., and Blumwald, E. (1996). Regulation of plant defense response to fungal pathogens: Two types of protein kinases in the reversible phosphorylation of the host plasma membrane H+-ATPase. Plant Cell 8, 555–564.[Abstract] Yoshida, S. (1991). Chilling-induced inactivation and its recovery of tonoplast H+-ATPase in mung bean cell suspension cultures. Plant Physiol. 95, 455–460. Zhen, R.-G., Baykov, A.A., Bakuleva, N.P., and Rea, P.A. (1994). Aminomethylenediphosphonate: A potent type-specific inhibitor of both plant and phototrophic bacterial H+-pyrophosphatases. Plant Physiol. 104, 153–159.[Abstract] This article has been cited by other articles:
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Abstract
INTRODUCTION
) or withdrawal from (
) the perfusion medium of each inhibitor.
pH) measured between the addition and removal of LPC (10 min). This shift is ratioed to the pH gradient between the vacuole and the perfusion medium at the time of LPC addition (
1 on elicitation with xylanase. Plant J. 26, 237–247.
