Positional Specificity of a Phospholipase A Activity Induced by Wounding, Systemin, and Oligosaccharide Elicitors in Tomato Leaves

Correspondence to: Clarence A. Ryan, Email maertens@wsu.edu;, 509-335-7643 (fax)

	ABSTRACT

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Phospholipase A (PLA) activity, as measured by the accumulation of ¹⁴C-lysophosphatidylcholine in leaves of tomato plants, increased rapidly and systemically in response to wounding. The increase in PLA activity in the systemic unwounded leaves was biphasic in wild-type tomato plants, peaking at 15 min and again at 60 min, but the second peak of activity was absent in transgenic prosystemin antisense plants. Supplying young excised tomato plants with the polypeptide hormone systemin also caused ¹⁴C-lysophosphatidylcholine to increase to levels similar to those induced by wounding, but the increase in activity persisted for >2 hr. Antagonists of systemin blocked both the release of ¹⁴C-lysophosphatidylcholine and the accumulation of defense proteins in response to systemin. ¹⁴C-lysophosphatidylcholine levels did not increase in response to jasmonic acid. Chemical acylation of the lysophosphatidylcholine produced by wounding, systemin, and oligosaccharide elicitors followed by enzymatic hydrolysis with lipases of known specificities demostrated that the lysophosphatidylcholine is generated by a PLA with specificity for the sn-2 position.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

In tomato leaves, wounding by attacking herbivores induces the activation of >20 systemic wound-responsive genes both locally, that is, in wounded leaves, and systemically, that is, in distal unwounded leaves (Green and Ryan 1972 ; Constabel et al. 1995 ; Bergey et al. 1996 , Bergey et al. 1999 ). The signals generated by wounding include systemin, an 18–amino acid polypeptide from tomato leaves (Ryan and Pearce 1998 ), and oligogalaturonide (OGA) fragments from plant cell walls (Bishop et al. 1981 ; Hahn et al. 1981 ; Bergey et al. 1999 ). Systemin is produced from a larger precursor protein called prosystemin (McGurl et al. 1992 ) and has been shown to be an essential component of the systemic defense signaling pathway in response to herbivore attacks (Orozco-Cardenas et al. 1993 ; Narvaez-Vasquez et al. 1995 ). OGA elicitors are derived through the action of a wound-inducible polygalacturonase (Bergey et al. 1999 ) or by pectin-degrading enzymes of pathogenic origins (John et al. 1997 ).

The activation of defense genes by systemin and oligosaccharide elicitors is considered to be receptor mediated (Ebel 1998 ; Mendl et al. 1998 ; Scheer and Ryan 1999 ) and to involve the octadecanoid signaling pathway (Farmer and Ryan 1992 ; Doares et al. 1995 ). In this pathway, linolenic acid (LA), released from membrane phospholipids by a phospholipase, is converted to 12-oxophytodienoic acid (PDA) and jasmonic acid (JA), which are powerful inducers of defense gene transcription (Farmer and Ryan 1992 ; Blechert et al. 1995 ). The activation of the phospholipase may be preceded or mediated by phosphorylation events (Farmer et al. 1991 ; Seo et al. 1995 ; Stratmann and Ryan 1997 ) and by transient increases in cytosolic Ca²+ (Knight et al. 1991 ; Moyen et al. 1998 ) and ion fluxes (Thain et al. 1990 ; Felix et al. 1993 ; Felix and Boller 1995 ; Moyen and Johannes 1996 ; Schaller and Oecking 1999 ).

The involvement of a lipid-mediated signaling cascade in the response of plants to wounding by herbivory and pathogen attacks has been documented (reviewed in Munnik et al. 1998 ). In castor bean, wounding caused the association of a soluble phospholipase D (PLD) activity with cell membranes and the rapid release of phosphatidic acid, possibly as a result of transient increases of Ca²+ (Ryu and Wang 1996 ; Lee et al. 1997 ). Multiple PLD enzymes have been purified from plant cells and tissues (Dyer et al. 1994 ; Wissing et al. 1996 ), and several cDNAs and genes encoding PLD have been cloned from different plant species. These PLD-encoding genes and their products seem to be differentially regulated via transcriptional and post-translational mechanisms (reviewed in Wang 1999 ). A rapid and transient increase in the levels of LA and lysophospholipids (Conconi et al. 1996 ; Lee et al. 1997 ) also has been demonstrated in wounded tomato leaves, but the nature of the hydrolytic enzymes, that is, phospholipase A₁ (PLA₁), PLA₂, or PLB (Wang 1993 ), was not established. Increases in PLA and PLC activities also have been observed during plant–pathogen interactions (Masui and Kojima 1990 ; Sekizawa et al. 1990 ; Kawakita et al. 1993 ; Walton 1995 ) and after treatment of plant cells and tissues with elicitors from plant and pathogen origins (Legendre et al. 1993 ; Toyoda et al. 1993 ; Roy et al. 1995 ; Walton 1995 ; Chandra et al. 1996 ). More recently, EDS1, a gene required for R gene–mediated disease resistance in Arabidopsis, was found to be related to eukaryotic lipases (Falk et al. 1999 ).

Several PLA activities have been partially purified from plant tissues (Hasson and Laties 1976 ; Galliard 1980 ; Kim et al. 1994 ; Senda et al. 1996 ). Interestingly, patatin, a major soluble storage protein in potato tubers, has important acyl hydrolase activity (Racusen 1984 ; Senda et al. 1996 ). However, it was only recently (Stahl et al. 1998 ) that novel types of low molecular weight soluble and membrane-associated PLA₂ enzymes have been fully identified in plants. These enzymes have both catalytical and structural properties related to those of the animal 14-kD secretory PLA₂ (Tischfield 1997 ; Stahl et al. 1998 ). Several isoforms of these plant secretory PLA₂s have been found in the same tissue, which seem to be encoded by a gene family (Stahl 1998 ; Stahl et al. 1998 ). Although the specific function and regulation of different PLA₂s still are not known, PLA activities have been associated with the defense response of plants against herbivory and pathogen attacks (as described above), with the plant response to auxins (Yi et al. 1996 ; Scherer and Arnold 1997 ; Paul et al. 1998 ), and in the removal of oxygenated and uncommon acyl groups from membranes, that is, membrane repair (Banas et al. 1992 ; Stahl et al. 1995 ). In animal systems, the release of signal-associated arachidonic acid in response to polypeptide elicitors, during the inflammatory defense response and the biosynthesis of eicosanoids, is mainly mediated by the activation of a different class of 85-kD cytosolic PLA₂ enzyme (Leslie 1997 ). However, no cytosolic PLA₂ similar to the animal enzyme has been purified, cloned, or sequenced from plant tissues.

The hydrolytic products of PLA activity, lysophospholipids, and free fatty acids also have been shown to modulate the activity of several membrane-associated enzymes, including NADH oxidase (Brightman et al. 1991 ), 1,3-ß-D-glucan synthase (Kauss and Jeblick 1986 ), protein kinases (Martiny-Baron and Scherer 1989 ; Scherer et al. 1993 ), ion channels (Chyb et al. 1999 ), and ATPases (Palmgren et al. 1988 ; Palmgren and Sommarin 1989 ; Scherer et al. 1993 ). Several of these enzymes have been implicated in different plant defense reactions, including the regulation of H⁺-ATPase activity during plant–pathogen interactions, elicitor treatments, and wounding (Vera-Estrella et al. 1994 ; Xing et al. 1996 ; Schaller and Oecking 1999 ).

In this study, we present evidence to support the involvement of PLA₂ activity in the early signal transduction events initiated by wounding, systemin, and oligosaccharide elicitors, leading to the synthesis and accumulation of defense proteins in tomato plants, with characteristics that resemble the activation of cytosolic PLA₂s during the defense response in animal systems.

	RESULTS

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Previous studies have shown that PLA activity increases in tomato leaves after wounding. Using young, excised ³²P-labeled tomato plants, Lee et al. 1997 found that the wound accumulation of radiolabeled lysophosphatidylcholine and lysophosphatidylethanolamine in tomato leaves peaked (approximately twofold over the controls) within 15 min after wounding in both the wounded and the unwounded leaves, and then declined after 30 min (Lee et al. 1997 ). On the other hand, using intact unlabeled tomato plants, Conconi et al. 1996 found that ~30 min after wounding, the levels of LA and lysophosphatidylcholine increased in the wounded leaves, whereas the levels of other polar lipids and especially those of the galactolipids, decreased (Conconi et al. 1996 ). The observed changes in the lipid composition of membranes might be the result of the action of nonspecific acyl hydrolases released from their storage compartments upon cell damage (Huang 1993 ), and they were not associated with systemic signaling of the defensive genes.

In this study, choline-containing lipids were efficiently labeled by applying ¹⁴C-choline directly to the leaf surfaces of intact tomato plants for 24 hr or by supplying excised tomato plants with a ¹⁴C-choline–containing buffer solution through their cut stems for ~6 to 8 hr. After labeling, ¹⁴C-phosphatidylcholine and ¹⁴C-lysophosphatidylcholine were the only radioactive lipids identified after thin-layer chromatography (TLC) separation, which previously has been reported for other plant systems (Scherer and Andre 1989 ; Scherer 1995 ; Paul et al. 1998 ). Phosphatidylcholine is a major membrane lipid and has been a preferred lipid substrate for the analysis of PLA activities in plants (Brown et al. 1987 ; Banas et al. 1992 ; Roy et al. 1995 ; Scherer 1995 ; Paul et al. 1998 ).

As shown in Figure 1, intact ¹⁴C-choline–labeled tomato plants accumulate ¹⁴C-lysophosphatidylcholine in the upper unwounded leaves within 15 min after wounding of the lower leaves, which is in agreement with the results of Lee et al. 1997 . Similarly, as shown in Figure 2A and Figure 2B, a time course of the accumulation of ¹⁴C-lysophosphatidylcholine in plants that had been excised at the base of the stems, ¹⁴C-choline labeled, and then wounded on the lower leaf demonstrates that ¹⁴C-lysophosphatidylcholine maximized in unwounded leaves of wild-type tomato plants at 15 min, decreased by 30 min, and increased again and peaked at ~60 min (Figure 2B). However, similarly treated transgenic prosystemin antisense plants, which have been impaired in their ability to systemically induce the accumulation of defense proteins in response to wounding (McGurl et al. 1992 ), showed the same initial peak of accumulation of ¹⁴C-lysophosphatidylcholine in the systemic leaf but notably lacked the later increase observed in the wild-type plants.

View larger version (14K): [in this window] [in a new window]   Figure 1. Systemic Wound Induction of PLA Activity in Upper, Unwounded Leaves of 2-Week-Old Intact Tomato Plants That Were Wounded on the Lower Leaves. 14C-choline (1.0 µCi) was distributed over the adaxial side of each of the two expanded leaves of four plants followed by incubation under light for 24 hr. The lower leaf then was wounded once with a hemostat; after 15 min, total lipids were extracted from the upper unwounded leaves. Approximately 5 x 105 dpm of total lipid extract from leaves of unwounded (control) and wounded plants was used for TLC analysis. Radioactive phosphatidylcholine (14C-PC) and lysophosphatidylcholine (14C-LysoPC) were visualized by using a PhosphorImager and Molecular Analyst software (Bio-Rad) and were identified by comigration with authentic lipid standards.

View larger version (20K): [in this window] [in a new window]   Figure 2. Time Course of the Release of 14C-Lysophosphatidylcholine in Leaves of Excised Tomato Plants in Response to Wounding. Two-week-old tomato plants were excised near the base of the stem and supplied with 14C-choline for 6 hr. The plants were transferred to phosphate buffer, incubated for an additional 1 to 2 hr, and wounded on the lower leaves. Approximately 2.5 x 105 dpm of total lipid extract of each sample was used for TLC analysis. (A) TLC autoradiogram (5 weeks of exposure) showing a 30-min time course of the accumulation of 14C-lysophosphatidylcholine (14C-LysoPC) in the upper, unwounded leaves of wild-type tomato plants. (B) A 2-hr time course of the release of 14C-lysophosphatidylcholine in the systemic unwounded leaves of wild-type (Wt; closed squares) and prosystemin antisense (AS; open circles) tomato plants after wounding as given in (A). The radioactivity in the 14C-lysophosphatidylcholine band was quantified and expressed as a percentage of the total radioactivity recovered from the TLC plates. PLA activity corresponds to the fold increase of 14C-lysophosphatidylcholine in wounded plants above that of the respective unwounded control for each time point. Each data point is the average of two independent experiments (±SD; n = 4).

Supplying excised plants with the polypeptide wound hormone systemin through the cut stems also induced an increase in levels of ¹⁴C-lysophosphatidylcholine within 5 min, but the levels continued to increase and remained elevated throughout the 2-hr duration of the experiment (Figure 3). Ala-17–systemin, an inactive analog of systemin in which Thr-17 is replaced with an Ala residue, is a potent competitive inhibitor of systemin in tomato plants (Pearce et al. 1993 ). Ala-17–systemin strongly inhibited the systemin-activated accumulation of both ¹⁴C-lysophosphatidylcholine and proteinase inhibitor I in a concentration-dependent manner (Figure 4).

View larger version (15K): [in this window] [in a new window]   Figure 3. Increases in Levels of 14C-Lysophosphatidylcholine in Leaves of Excised 2-Week-Old Tomato Plants in Response to Systemin. Plants were labeled with 14C-choline as given in Figure 2 and were supplied with phosphate buffer alone (control) or with 2.5 nM systemin. Quantification is as described in Figure 2B. Data are the average of two independent experiments (±SD; n = 4).

View larger version (17K): [in this window] [in a new window]   Figure 4. Inhibition of Systemin-Induced PLA Activity and Inhibitor I Accumulation in Leaves of Young Excised Tomato Plants by the Inactive Systemin Analog Ala-17–Systemin. Plants were supplied either with phosphate buffer alone or with increasing concentrations of Ala-17–systemin for 30 min and then supplied with 2.5 nM systemin. Inhibition of PLA activity (open circles) was determined after 15 min of incubation in light by using 14C-choline–labeled plants as described in Figure 2B. Inhibition of proteinase inhibitor I (Inh I) accumulation (closed squares) was assayed immunologically 24 hr later by using unlabeled plants similarly pretreated with unlabeled choline. Data are expressed as the percentage of inhibition of the PLA activity and inhibitor I accumulation observed in control plants treated with systemin alone. Each data point is the average of two independent experiments (±SD; n = 3).

Specific inhibitors of animal PLA₂ enzymes, manoalide (Lombardo and Dennis 1985 ), and arachidonyltrifluoromethyl ketone (AACOCF₃; Street et al. 1993 ), were assayed for their effects on the PLA activity and the activation of defense gene expression induced by systemin. When these inhibitors were supplied to young tomato plants through their cut stems for 1 hr before feeding systemin and then treated with systemin for 15 min, they both inhibited the systemin-inducible increase in ¹⁴C-lysophosphatidylcholine and proteinase inhibitor I in a concentration-dependent manner (Figure 5a and Figure 5b). The concentrations of manoalide and AACOCF₃ required for half maximal inhibition (IC₅₀) were ~0.5 and 50 µM, respectively. In the absence of systemin, each of the inhibitors also caused a small reduction in the basal levels of ¹⁴C-lysophosphatidylcholine and inhibitor I in the excised plants (data not shown).

View larger version (39K): [in this window] [in a new window]   Figure 5. Inhibition of Systemin-Induced PLA Activity and Inhibitor I Accumulation in Leaves of Young Excised Tomato Plants by Mammalian PLA2 Enzyme Inhibitors. Plants were supplied with either phosphate buffer alone or with increasing concentrations of inhibitors for 60 min and then supplied with 2.5 nM systemin. Inhibition of PLA activity (open circles) and inhibitor I (Inh I) accumulation (closed squares) was determined and expressed as in Figure 4 (±SD; n = 4). (A) Inhibition by manoalide. (B) Inhibition by AACOCF3.

OGAs derived from plant cell walls (Bishop et al. 1981 ) and chitosan, which is a mixture of glucosamine oligomers derived from fungal cell walls (Walker-Simmons et al. 1983 ), are elicitors of defensive genes in a variety of plant systems (Ryan and Farmer 1991 ; John et al. 1997 ). The inducing activities of both OGA and chitosan are considered to be receptor mediated (Felix et al. 1993 ; Ebel 1998 ) and, in tomato leaves, like systemin, they activate genes through the octadecanoid signaling pathway (Doares et al. 1995 ). Within 15 min, OGA and chitosan supplied to excised plants caused an increase in the levels of ¹⁴C-lysophosphatidylcholine in leaves to levels similar to those induced by systemin (Figure 6) and wounding (cf. Figure 2). In leaves of tomato plants supplied with JA, an increase in ¹⁴C-lysophosphatidylcholine levels was not detected (Figure 6), indicating that the increase in lysophosphatidylcholine was upstream from JA action in the signaling pathway (Farmer and Ryan 1992 ).

View larger version (13K): [in this window] [in a new window]   Figure 6. Effect of Systemin, Oligosaccharide Elicitors, and JA on the Levels of 14C-Lysophosphatidylcholine in Leaves of Excised Tomato Plants. Plants were labeled with 14C-choline as in Figure 2 and then were supplied with phosphate buffer alone (control) or with the following elicitors: 2.5 nM systemin; 0.5 mg/mL OGA (degree of polymerization = ~20); 125 µg/mL acid-treated chitosan; or 200 µM JA. After 15 min incubation under light, the leaves of each plant were excised and assayed for 14C-lysophosphatidylcholine as described in the text. PLA activity is expressed as the fold accumulation of 14C-lysophosphatidylcholine above that of the buffer control, which was assigned a value of 1.0 (±SD; n = 4).

To investigate the positional specificities of the wound-induced PLA activity, we extracted unlabeled lysophosphatidylcholine from unwounded leaves of tomato plants that had been wounded 15 min earlier. The lysophosphatidylcholine then was acylated with ¹⁴C–octanoic acid (¹⁴C-8:0) to produce ¹⁴C-8:0–labeled phosphatidylcholine, in which the ¹⁴C-fatty acid replaced the fatty acids in the lysophosphatidylcholine that had been released in vivo from cell membrane phosphatidylcholines after wounding. The ¹⁴C-8:0–labeled phosphatidylcholine then was used as a substrate for enzymes having specificities for either the sn-1 or the sn-2 positions of the phospholipids. The products of the reactions indicated that ¹⁴C-8:0 was released almost exclusively from the sn-2 position of the ¹⁴C-8:0–labeled phosphatidylcholine (Figure 7). In a similar way, lysophosphatidylcholine induced to accumulate in excised plants by systemin, OGA, and chitosan was extracted, acylated with ¹⁴C-8:0, and concurrently digested with the specific PLA enzymes, and the positional specificity of the PLA activity induced in the plant by the different treatments was determined and compared with that induced in wounded plants. The results shown in Table 1 confirm that ¹⁴C-8:0 was released mainly from the sn-2 position of the ¹⁴C-8:0–labeled phosphatidylcholine derived from leaves treated with systemin, OGA, and chitosan.

View larger version (75K): [in this window] [in a new window]   Figure 7. Positional Specificity of the Wound-Induced PLA Activity in Intact Tomato Plants. Total lipids were extracted from systemic unwounded leaves 15 min after wounding of the lower leaf of young unlabeled tomato plants and separated by TLC, and the wound-induced lysophosphatidylcholine (14C-LysoPC) was purified and acylated with 14C-8:0 to produce 14C-8:0–labeled phosphatidylcholine (14C-PC). 14C-8:0–labeled phosphatidylcholine was digested with enzymes, and the products were separated by TLC and visualized using a PhosphorImager (Bio-Rad). Lane 1, undigested 14C-8:0–labeled phosphatidylcholine; lane 2, 14C-8:0–labeled phosphatidylcholine digested with R. arrhizus lipase having an sn-1 specificity; lane 3, 14C-8:0–labeled phosphatidylcholine digested with PLA2 from bee venom; STD, 14C-8:0.

	DISCUSSION

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Wounding of plant leaves induces rapid and transient changes in the lipid composition of plant cell membranes (Conconi et al. 1996 ; Ryu and Wang 1996 ; Lee et al. 1997 ). These changes include a decrease in the content of polar lipids (Conconi et al. 1996 ; Ryu and Wang 1998 ) and the elevation of levels of lysolipids (Conconi et al. 1996 ; Lee et al. 1997 ), phosphatidic acid (Ryu and Wang 1996 ; Lee et al. 1997 ), and free fatty acids, predominantly LA and linoleic acids (Conconi et al. 1996 ; Ryu and Wang 1998 ). LA is the precursor of PDA and JA (Vick and Zimmerman 1984 ), which are potent signaling molecules inducing transcriptional activation of systemic wound-responsive genes in tomato leaves (Farmer and Ryan 1992 ; Bergey et al. 1996 ). Thus, the release of LA from membranes in response to intracellular signaling events is a key step in the activation of the defensive genes.

In this study of the systemic effects of wounding on PLA activity, measuring the release of ¹⁴C-lysophosphatidylcholine in unwounded leaves of ¹⁴C-choline–labeled tomato plants, we observed that the levels of ¹⁴C-lysophosphatidylcholine increase within 15 min after wounding in both intact and excised plants (Figure 1 and Figure 2), thereby confirming previous results of Conconi et al. 1996 and Lee et al. 1997 . In addition, we also observed that the kinetics for the systemic release or accumulation of ¹⁴C-lysophosphatidylcholine in the unwounded leaves was biphasic, peaked at 15 min, decreased by 30 min, and then increased again and maximized at ~1 hr (Figure 2B). We suggest that the first peak of this biphasic response is likely the result of an initial hydraulic signal caused by breaching the vascular system of the plant, as described by Malone 1996 . This effect would likely perturb membranes throughout the plant, triggering a transient wound response, analagous to the sensitive "touch" response of plants (Knight et al. 1991 ). The second increase in PLA activity can be ascribed to the systemic signal that is released at the wound site. This timing corresponds with the time course of the release of the wound signal, as has been previously described (Nelson et al. 1983 ; Narvaez-Vasquez et al. 1994 , Narvaez-Vasquez et al. 1995 ). This hypothesis is supported by the observation that transgenic prosystemin antisense plants (McGurl et al. 1992 ), in which synthesis of the systemin precursor protein is severely reduced, did not produce a second peak of accumulation of ¹⁴C-lysophosphatidylcholine in the systemic unwounded leaves (Figure 2B). Furthermore, when systemin was supplied through the cut stems of ¹⁴C-choline–labeled tomato plants, ¹⁴C-lysophosphatidylcholine was released to levels found after wounding. In contrast to wounding, the release of ¹⁴C-lysophosphatidylcholine induced by systemin continued for at least 2 hr (Figure 3). In these experiments, however, the plants were not wounded (excised plants were allowed to equilibrate during the 6-hr labeling period with ¹⁴C choline before initiating the systemin treatment). Thus, this rapid increase in the PLA activity can be ascribed exclusively to the action of systemin, because this increase is not observed in the control plants similarly treated but incubated in a buffer solution without systemin.

The biologically inactive systemin analog Ala-17–systemin is an antagonist of defense gene activation (Pearce et al. 1993 ) and is a potent inhibitor of a mitogen-activated protein kinase (MAPK) activity that is rapidly induced by systemin (Stratmann and Ryan 1997 ). Increasing concentrations of Ala-17–systemin strongly antagonized the systemin-induced increase in ¹⁴C-lysophosphatidylcholine from leaves of tomato plants (Figure 4). Thus, the characteristics of the activation of PLA by systemin correlate well with the characteristics of its activation of a MAPK and with defense gene induction, suggesting that all may be linked through a common pathway.

To investigate the possible role of PLA activation in the defense response induced by systemin, we used specific inhibitors of animal PLA₂ enzymes. Manoalide inhibited both the release of ¹⁴C-lysophosphatidylcholine and the accumulation of inhibitor I protein induced by systemin (Figure 5a). Manoalide is a potent inhibitor of animal PLA₂s (Lombardo and Dennis 1985 ) and an inhibitor of PLA₂-mediated, auxin-induced acidification and cell elongation in maize coleoptiles (Yi et al. 1996 ). However, manoalide also has been shown to block Ca²+ channels (Wheeler et al. 1987 ) and to inhibit phosphoinositide-specific phospholipase C (PLC) enzymes in animal systems (Bennett et al. 1986 ) so that its inhibition of the induction of defense genes by systemin may not be limited to its inhibition of PLA activity. However, when excised plants were pretreated with increasing concentrations of AACOCF₃, a highly specific inhibitor of the mammalian 85-kD cytosolic PLA₂ enzyme (Street et al. 1993 ; Bartoli et al. 1994 ), the systemin-induced PLA activity and inhibitor I accumulation both were inhibited in a concentration-dependent manner, suggesting that the systemin-inducible PLA enzyme might share some characteristics of the mammalian cytosolic PLA₂ enzyme. Aristolochid acid and chlorpromazine are other known inhibitors previously reported to block the activation of PLAs associated with auxin-mediated responses (Scherer and Arnold 1997 ) and the elicitation of the oxidative burst by elicitors in suspension-cultured soybean cells (Chandra et al. 1996 ). Neither one significantly affected PLA activation or the defense protein accumulation induced by systemin (data not shown). The causes of the differential effects of the various PLA inhibitors are not known, although they could be explained by the existence in plants of multiple and differentially regulated PLA enzymes (Stahl et al. 1998 ), as occurs in animal systems (Dennis 1997 ; Tischfield 1997 ).

Like systemin, OGA as well as chitosan oligomers can activate defensive genes via the octadecanoid signaling pathway (Doares et al. 1995 ). When these oligosaccharides were supplied to excised tomato plants, they caused an increase in the release of ¹⁴C-lysophosphatidylcholine within 15 min (Figure 6), suggesting that the early signaling events initiated by systemin, OGA, and chitosan converge on a common mechanism to release LA. This is consistent with previous data showing that a mutant tomato line compromised in the octadecanoid pathway (Howe et al. 1996 ) did not activate defense genes in response to any of the three signaling molecules (Doares et al. 1995 ). Treatment of tomato plants with JA did not result in ¹⁴C-lysophosphatidylcholine release, as expected, because JA behaves as an intracellular messenger molecule produced by the octadecanoid pathway downstream from LA. In a similar fashion, oligosaccharide fragments, but not JA, recently were shown to activate a MAPK in tomato plants (Stratmann and Ryan 1997 ), and the activation of a MAPK after wounding is required for the accumulation of JA and jasmonate-inducible proteinase inhibitors in tobacco plants (Seo et al. 1995 , Seo et al. 1999 ). On the contrary, Chandra et al. 1996 , using a fluorescent PLA substrate analog, did not find an increase in PLA activity after the treatment of suspension-cultured soybean cells with OGA but did find an increase when using other pathogen-derived elicitors. This result also could be explained by the presence of cell type–specific expression/regulation of multiple PLA isoforms in those cell cultures.

Only indirect evidence had been reported previously for the existence of PLA₂-specific enzymes associated with wounding or pathogen elicitor–stimulated responses in plants. In this study, we demonstrate that the lysophosphatidylcholine that accumulates systemically after wounding of tomato plants, or in response to systemin and the oligosaccharide elicitors OGA and chitosan, is generated by a PLA with specificity for the position sn-2, that is, a PLA₂ enzyme (Figure 7 and Table 1). This represents direct evidence that a PLA₂ participates in the intracellular signaling cascade that regulates defense gene expression in response to herbivore attacks.

In tomato plants, wounding and systemin caused a rapid, transient activation of a MAPK (Stratmann and Ryan 1997 ), with an activation kinetics similar to the one observed for PLA in this study. Both wounding and systemin also have been shown to cause a rapid and transient increase in the concentration of intracellular Ca²+ (Knight et al. 1991 ; Moyen et al. 1998 ). Accordingly, specific inhibitors of protein kinases and Ca²+ channel blockers can inhibit both the wound- and the systemin-induced MAPK and PLA activation and the accumulation of defense proteins (Seo et al. 1995 , Seo et al. 1999 ; Romeis et al. 1999 ; Schaller and Oecking 1999 ; J. Narváez-Vásquez and C.A. Ryan, unpublished results). The coincidence of the early activation of PLA₂ with the activation of a MAPK activity and Ca²+ in tomato plants suggests that the MAPK and PLA₂ activities may be coupled to the reception of the primary signals that converge on a common mechanism for the release of LA from plasma membranes, analogous to the release of arachidonic acid in animal systems (Bergey et al. 1996 ; Leslie 1997 ).

A growing body of evidence supports the existence of multiple lipid-mediated signaling pathways in plants (Munnik et al. 1998 ). The early accumulation of phosphatidic acid by the activation of a PLD enzyme known to occur after wounding (Ryu and Wang 1996 ; Lee et al. 1997 ) also suggests the concerted activation of several phospholipases in the mediation of extracellular signals across plant cell membranes. In this regard, a PLA₂ activity has been shown to be required for the phosphatidic acid–induced serum response element activation in animal fibroblast cells (Kim et al. 1998 ), possibly for the release of lysophosphatidic acid, which in turn might act as a second messenger in the activation of signaling cascades (Goetzl and An 1998 ). Furthermore, the treatment of carrot cell membranes with a snake venom PLA₂, but not with PLC or PLD enzymes, caused the release of a membrane-associated phosphatidylinositol kinase (Gross et al. 1992 ).

It is possible that at least one of the mechanisms of PLA action is through the regulatory effects of its hydrolytic products, lysophospholipids, and fatty acids on H⁺ transport and ion permeability of the plasma membrane (Palmgren et al. 1988 ; Palmgren and Sommarin 1989 ; Scherer et al. 1993 ; Chyb et al. 1999 ). Membrane depolarization and ion fluxes are among the earliest events observed in tomato cells after wounding or elicitor treatments (Thain et al. 1990 ; Felix et al. 1993 ; Felix and Boller 1995 ; Moyen and Johannes 1996 ; Schaller and Oecking 1999 ). On the other hand, linoleic acid and LA, the major fatty acids in plant membranes, are the substrates for lipoxygenase and the biosynthesis of oxylipins through the octadecanoid pathway (Vick and Zimmerman 1984 ). Both highly unsaturated fatty acids and their metabolites are able to elicit a defense reaction and other physiological responses in a variety of plant systems (Farmer and Ryan 1992 ; Blechert et al. 1995 ; Creelman and Mullet 1997 ).

The identification of a PLA₂-specific activity associated with the defense response in plants provides a further step to the isolation of the protein and gene, and the eventual understanding of the biochemistry of the intracellular information processing system leading to defense gene transcription. As the details of the signaling pathway of plants in response to wounding and pathogen attack are clarified, it may be possible to evaluate whether plant and animal signaling systems have derived from convergent or divergent ancestral signal transduction systems, providing new insights into the evolution of defense signaling in eukaryotes.

	METHODS

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Plant Materials
Wild-type tomato plants (Lycopersicon esculentum cv Castlemart) and transgenic plants transformed with a prosystemin antisense gene (McGurl et al. 1992 ) were grown from seeds for 14 to 16 days under 17 hr of light (300 µE m^-2 sec^-1) at 28°C and 7 hr of dark at 18°C. The plants had two expanding leaves and a small developing apical leaf.

¹⁴C-Choline Labeling of Tomato Leaves
Labeling of 2-week-old tomato plants was achieved by excising the plants at the base of the stem with a razor blade as previously described (Ryan 1974 ) and incubating the plants in 1.5 mL of phosphate buffer (15 mM NaH₂PO₄, pH 6.5) containing 4 µCi/mL of 50 µM ¹⁴C-choline (2.04 GBq mol^-1; Du Pont–New England Nuclear) for 6 hr under light (300 µE m^-2 sec^-1) at 25°C. After this labeling period, the plants had imbibed 0.5 to 1.0 mL of the solution. The plants then were transferred to a ¹⁴C-choline–free buffer solution and incubated for another 1 to 2 hr under similar conditions to allow the free ¹⁴C-choline to be incorporated into the membrane phospholipids. Leaf membranes of intact tomato plants also were labeled by applying 1.0 µCi of ¹⁴C-choline in 10 µL of 50% ethanol to the adaxial side of each of the two expanding leaves (2.0 µCi/plant) and incubating them for 24 hr under constant light at 28°C.

Wounding and Elicitor/Inhibitor Feeding Treatments
The lower leaves of ¹⁴C-choline–labeled and unlabeled control plants were wounded once across their midveins by using a hemostat, and the plants were incubated under light as described above. Elicitors and inhibitors were supplied in aqueous solutions to excised plants through their cut stems as reported previously (Stratmann and Ryan 1997 ). Plants were handled carefully to minimize touch activation of PLA. ¹⁴C-lysophosphatidylcholine in leaves of ¹⁴C-choline–labeled tomato was assayed before and after wounding or treatment with systemin, elicitors, or other substances. Levels of the wound-inducible and systemin-inducible defense proteins proteinase inhibitors I were quantified in leaf juice by radial immunodiffusion (Ryan 1967 ).

Lipid Extraction and Analysis
The extraction protocol for leaf lipids was a modification of Conconi et al. 1996 . At the indicated times after each treatment, the leaves of every two plants were excised together at the base of the petiole and immediately frozen in liquid N₂. Frozen leaves (0.5 to 1.0 g) were homogenized with a Polytron (Tekmar, Cincinnati, OH) in 6 mL of ice-cold chloroform:methanol:formic acid (10:10:1 [v/v]) and stored overnight at -20°C. Alternatively, leaves were ground under liquid nitrogen, and the powder was resuspended in 3 mL of prewarmed isopropyl alcohol containing 0.05% 2,6-di-tert-butyl-4-hydroxytoluene and incubated 15 to 20 min at 80°C in a water bath. Both methods resulted in similar recoveries of lipids. The tubes containing the lipids were chilled on ice, 3 mL of chloroform and 0.3 mL of formic acid were added, and the samples were incubated overnight at -20°C. Thereafter, the extraction protocol was as follows: samples were centrifuged at 3000g, and the pellets were reextracted with 3 mL of chloroform:methanol:water (5:5:1 [v/v]). The two supernatants were combined and washed with 3.5 mL of 0.2 M H₃PO₄ and 1 M KCl (Hajra 1974 ). The chloroform phase was washed again with 4 mL of 0.5 M KCl, dried under nitrogen, resuspended in a small volume of chloroform:methanol (2:1 [v/v]), and stored at -20°C.

Major lipid classes present in the extract were separated from lysophosphatidylcholine by thin-layer chromatography (TLC) on silica gel plates (250 µm; J.T. Baker Chemical Co.). Aliquots of each sample containing ~2 to 5 x 10⁵ dpm of radioactive lipids were loaded onto TLC plates and developed using two different solvents. First, the plates were developed in toluene:chloroform:methanol (85:15:5 [v/v]) to remove pigments and neutral lipids and then in chloroform:methanol:acetone:acetic acid:water (53:14:20:10:5 [v/v]), which allowed for a clear separation of phosphatidylcholine, lysophosphatidylcholine, and other major lipids (Scherer and Andre 1989 ). After chromatography, lipid bands were visualized by staining the plates with iodine vapor. Radioactive phosphatidylcholine and lysophosphatidylcholine were identified by cochromatography with authentic reference standards and the use of an Instant Imager (Packard, Meriden, CT), a PhosphorImager (Bio-Rad), and/or by x-ray autoradiography. Radioactive bands were scraped from the TLC plates, and the radioactivity was quantified by liquid scintillation counting. The radioactivity in the ¹⁴C-lysophosphatidylcholine band was expressed as a percentage of the total radioactivity recovered from the TLC plates. PLA activity was expressed as the fold increase of ¹⁴C-lysophosphatidylcholine in treated plants above that of the respective untreated control for each time point. The basal levels of ¹⁴C-lysophosphatidylcholine found in control plants ranged between 0.2 and 0.5% of the total ¹⁴C-choline–labeled lipid.

Identification of the Positional Specificities of the Wound- and Elicitor-Induced PLA Activities
A method was developed for distinguishing 1-acyl from 2-acyl lysophosphatidylcholine generated in biological systems (Florin-Christensen et al. 1999 ). In this method, the lysophosphatidylcholine is extracted and then acylated with a labeled fatty acid. This is followed by the enzymatic analysis of the resulting ¹⁴C-labeled phosphatidylcholine, by using commercially available phospholipase A₁ (PLA₁) or PLA₂ enzymes. Subsequent analysis by TLC determines the position of the radiolabeled fatty acid and thus the position of the fatty acid in the starting lysophosphatidylcholine (Florin-Christensen et al. 1999 ). Thus, unlabeled lysophosphatidylcholine was recovered from upper, unwounded leaves of intact or excised tomato plants wounded 15 min earlier on the lower leaves or from excised tomato plants treated with systemin (2.5 nM), OGA (0.5 mg/mL), or chitosan (125 µg/mL) for 15 min, as described above. After TLC separation, ¹⁴C-lysophosphatidylcholine was located by brief exposure to iodine vapors of authentic standards run on the same plates in parallel lanes, scraped from the TLC plates, extracted three times with 2 mL of chloroform:methanol (1:1), and thoroughly dried under nitrogen and then under high vacuum. Lysophosphatidylcholine then was dissolved in amylene-stabilized chloroform and quantified by determining the phosphate concentration, using the method of Ames 1966 . Purified lysophosphatidylcholine was chemically acylated using ¹⁴C–octanoic anhydride in the presence of dimethylaminopyridine, as described by Gupta et al. 1977 . The anhydride was prepared from ¹⁴C–octanoic acid (¹⁴C-8:0; 2.03 GBq mmol^-1; Du Pont–New England Nuclear) by reaction with dicyclohexylcarbodiimide, as described by Salinger and Lapidot 1966 , and diluted with unlabeled octanoic anhydride to a specific activity of 5 µCi/µmol. The acylating reactions were conducted in volumes of ~30 µL, and the molar proportions of lysophosphatidylcholine, dimethylaminopyridine, and ¹⁴C–octanoic anhydride used were 1:0.5:5. Routinely, 20 to 50 nmoles of lysophosphatidylcholine was used, and the reactions were conducted under N₂ (gas) in the dark and at 37°C. The acylation was allowed to proceed for 72 hr with constant stirring. The synthesized radiolabeled phosphatidylcholine products were purified by TLC, using cold soybean lysophosphatidylcholine and phosphatidylcholine as standards.

Enzymatic Digestion of Synthetic Phosphatidylcholine
Aliquots of synthetic ¹⁴C-8:0–labeled phosphatidylcholine (~5000 dpm) were mixed with 1.0 µmol of cold phosphatidylcholine from soybean (Sigma), dried under N₂, and resuspended by vortexing in 400 µL of 50 mM Tris-HCl, pH 8.1, 100 mM boric acid, 1 mM CaCl₂, and 0.05% Triton X-100. The mixture of ¹⁴C-8:0–labeled and unlabeled phosphatidylcholines was digested with either 10 units of PLA₂ (from bee venom; Sigma) or 6000 units of lipase (from Rhizopus arrhizus; Sigma) as a source of PLA₁ activity, and the reaction mixtures were incubated at 37°C for 0.5 and 3 hr, respectively. The reactions were terminated by lipid extraction by using the method of Bligh and Dyer 1959 . The lipid phase was analyzed by TLC, and the radioactive products were visualized and quantified as described above. As a control for the enzymatic reaction, the method was applied to two different lysophosphatidylcholines obtained from synthetic dioleoyl-phosphatidylcholine by PLA₂ and R. arrhizus lipase, as described (Florin-Christensen et al. 1999 ). The procedures used did not result in appreciable acyl migration, and the same result was observed when ¹⁴C–stearic acid (¹⁴C-18:0) was the fatty acid acylated to lysophosphatidylcholine instead of ¹⁴C-8:0 (Florin-Christensen et al. 1999 ).

	ACKNOWLEDGMENTS

This research was supported in part by Washington State University College of Agriculture and Home Economics (Project No. 1791), the National Science Foundation (Grant No. IBN 9601099), and the United States Department of Agriculture Competitive Grants Program (Grant No. 9801502). We thank Anders Carlsson, Martine Miquel, and John A. Browse for their technical advice and useful discussions. We also thank Amanda Vaughn for technical assistance and Sue Vogtman and Thom Koehler for growing and maintaining plants. J.F.-C. is an Organization of American States Research Fellow, on leave from the Institute of NeuroScience, National Research Council of Argentina.

Received June 18, 1999; accepted September 16, 1999.

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