Present addresses: BORB, New York University, Department of Biology, 100 Washington Square East, 1009 Silver Building, New York, NY 10003, USA; BtR, The Netherlands Cancer Institute, Plesmalaan 121, NL-1066 CX Amsterdam, the Netherlands; AML, Instituto de Investigaciones Biológicas, Facultad de Ciencas Exactas y Naturales, Universidad Nacional de Mar del Plata, CC 1245, 7600 Mar del Plata, Argentina.
Reassessing the role of phospholipase D in the Arabidopsis wounding response
Article first published online: 9 FEB 2009
© 2009 Blackwell Publishing Ltd
Plant, Cell & Environment
Volume 32, Issue 7, pages 837–850, July 2009
How to Cite
BARGMANN, B. O. R., LAXALT, A. M., RIET, B. T., TESTERINK, C., MERQUIOL, E., MOSBLECH, A., LEON-REYES, A., PIETERSE, C. M. J., HARING, M. A., HEILMANN, I., BARTELS, D. and MUNNIK, T. (2009), Reassessing the role of phospholipase D in the Arabidopsis wounding response. Plant, Cell & Environment, 32: 837–850. doi: 10.1111/j.1365-3040.2009.01962.x
- Issue published online: 1 JUN 2009
- Article first published online: 9 FEB 2009
- Received 13 November 2008; received in revised form 14 January 2009; accepted for publication 14 January 2009
- jasmonic acid;
- phosphatidic acid;
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- Supporting Information
Plants respond to wounding by means of a multitude of reactions, with the purpose of stifling herbivore assault. Phospholipase D (PLD) has previously been implicated in the wounding response. Arabidopsis (Arabidopsis thaliana) AtPLDα1 has been proposed to be activated in intact cells, and the phosphatidic acid (PA) it produces to serve as a precursor for jasmonic acid (JA) synthesis and to be required for wounding-induced gene expression. Independently, PLD activity has been reported to have a bearing on wounding-induced MAPK activation. However, which PLD isoforms are activated, where this activity takes place (in the wounded or non-wounded cells) and what exactly the consequences are is a question that has not been comprehensively addressed. Here, we show that PLD activity during the wounding response is restricted to the ruptured cells using 32Pi-labelled phospholipid analyses of Arabidopsis pld knock-out mutants and PLD-silenced tomato cell-suspension cultures. pldα1 knock-out lines have reduced wounding-induced PA production, and the remainder is completely eliminated in a pldα1/δ double knock-out line. Surprisingly, wounding-induced protein kinase activation, AtLOX2 gene expression and JA biosynthesis were not affected in these knock-out lines. Moreover, larvae of the Cabbage White butterfly (Pieris rapae) grew equally well on wild-type and the pld knock-out mutants.
myelin basic protein
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- Supporting Information
Insect herbivory is a consequential stress in higher plants that leads to a loss of nutrients and photosynthetic capacity and, as a result, reduced seed production. Plants respond to the wounding and counter-attack with direct and indirect defensive strategies (Wasternack et al. 2006). For example, proteins that interfere with the digestion of plant material in the insect gut are synthesized (Green & Ryan 1972; Orozco-Cárdenas, Narváez-Vásquez & Ryan 2001) and compounds that are toxic or repellant with respect to the herbivore also accumulate in local and systemic parts of the wounded plant (Baldwin 1998; Leitner, Boland & Mithöfer 2005). Additionally, wounded plants emit volatile compounds that attract natural enemies of herbivores (Sabelis, Janssen & Kant 2001; Ament et al. 2004). In such a way, plants can fend off an ongoing attack and prepare themselves for further assault.
Wounding-induced signalling molecules, such as JA and systemin, are produced upon wounding and elicit the above-mentioned responses throughout the plant. The plant's wounding response can be partitioned into three territories: (1) the ruptured cells; (2) the local-responding tissue; and (3) the systemic-responding tissue. The ruptured cells emit a non-cell-autonomous, primary signal that is perceived by intact cells in the local-responding tissue. Upon perception of the wounding signal, intact cells in the surrounding tissue respond with changes in gene expression, protein phosphorylation and metabolite production, leading to an induction of defensive strategies and amplification of the primary wounding signal by the production of secondary signals. Systemic plant tissues perceive the wounding signals and react with a wounding response (Schilmiller & Howe 2005).
PA production by PLD reportedly occurs in all three territories (Ryu & Wang 1996; Lee et al. 1997; Lee, Hirt & Lee 2001) and has been proposed to play important roles in the wounding response (Wang et al. 2000; Lee et al. 2001). PLD catalyses the hydrolysis of structural phospholipids, such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE), producing PA and a free headgroup. In the Arabidopsis (Arabidopsis thaliana) genome, there are 12 PLD genes, whereas animals only contain two PLDs and yeast even only one. The plant PLD family can be divided into six classes, α, β, γ, δ, ε and ζ, based on the enzymes' sequence homology and biochemical properties (Wang 2005). Functions have been suggested for PLD in various processes, including membrane degradation, vesicular transport and intracellular signalling (Wang 2005; Bargmann & Munnik 2006).
The activity of different PLD classes can be separated in vitro by varying the buffer in which the assay is performed and the lipid environment in which the substrate is presented (Qin & Wang 2002). Four kinds of in vitro PLD activity have been distinguished in this way, depending on their pH, [Ca2+], oleate and phosphatidylinositolbisphosphate (PIP2) requirements. The α-class PLDs require an acidic pH and millimolar calcium concentrations but do not require inclusion of PIP2 in their lipid substrate preparation. In contrast, β-/γ-class PLDs require neutral pH, micromolar calcium concentrations and PIP2. The δ-class PLDs are active at high micromolar to low millimolar calcium concentrations and are stimulated by the inclusion of oleic acid (or TX-100) in the substrate preparation (Qin, Wang & Wang 2002). Lastly, the ζ-class PLDs require a neutral pH and PIP2 but do not require calcium (Qin & Wang 2002).
Although PLD activity has been implicated in the wounding response (Ryu & Wang 1996; Lee et al. 1997, 2001; Wang et al. 2000), it is not evident which isoforms are involved nor is it apparent where it takes place, that is, in ruptured or intact cells, locally or systemically. Earlier, Wang et al. (2000) demonstrated that Arabidopsis plants expressing an antisense AtPLDα1 construct exhibited reduced PA production in wounded leaves, signifying that this isoform is responsible for a part of the PLD activity. The authors proposed that this activity takes place in the intact, responding cells, although this question was never experimentally addressed (Wang et al. 2000). Which isoform accounts for the observed residual PLD activity also remains unclear. Similarly, it remains to be shown which PLD isoform is responsible for the systemic PLD activity reported by Lee et al. (1997, 2001).
PLD has been proposed to play several roles in the wounding response. Antisense AtPLDα1 plant lines have been reported to have reduced wounding-induced JA production and impeded wounding-induced gene expression (Wang et al. 2000). These authors put forward a model in which AtPLDα1-derived PA is a precursor for JA biosynthesis. In addition, Lee et al. (2001) used PLD activity inhibition in soybean (Glycine max) seedlings and a cell-suspension wounding model and suggested that PLD activity lies upstream of MAPK signalling.
The aim of this study is to scrutinize the location of wound-activated PLD activity and to assess its role in the downstream wounding response. To this end, two plant systems were employed: Arabidopsis pld knock-out mutants and PLD-silenced tomato (Solanum lycopersicon) cell-suspension cultures. These model systems were used to examine in vivo PLD activity, protein kinase activity, gene expression, JA biosynthesis and herbivore performance.
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- Supporting Information
Arabidopsis thaliana var. Col-0 T-DNA insertion lines were obtained from the SALK Institute. pldα1 (SALK_067533) and pldδ (SALK_023247) were crossed to obtain the double mutant. The following primers were used to verify genomic insertions:
SALK_067533F 5′-GACGATGAATACATTATCATTG G-3′
SALK_LeftBorderA 5′-TGGTTCACGTAGTGGGCCAT CG-3′
SALK_LeftBorderA was used in combination with SALK_067533R and SALK_023247F. Col-5 (accession number N1644; Nottingham Arabidopsis Stock Centre, University of Nottingham, UK) and coi1-16 were obtained from Maarten Koornneef and John Turner, respectively. For routine plant growth, seeds were sown on soil and vernalized at 4 °C for 2 d. For analysis of systemic PA formation, seeds were sown on rockwool cubes. Ordinarily, plants were grown in a growth chamber under a 12 h light/12 h dark regime, with a 23 °C/18 °C cycle and 70% humidity. For the herbivore performance assay, plants were grown under a 9 h light/15 h dark regime. Mas7 (Peninsula Laboratories, Belmont, CA, USA) stock solution of 100 mm was made in water and stored in aliquots at −20 °C.
Suspension-cultured cells (Solanum lycopersicon Mill.; line Msk8; Felix et al. 1991) were grown at 24 °C in the dark shaking at 125 r.p.m. in Murashige and Skoog medium supplemented with 3% (w/v) sucrose, 5.4 µm 1-naphthaleneacetic acid, 1 µm 6-benzyladenine and vitamins (pH was adjusted to 5.7 with 1 m KOH) as described by Felix et al. (1991) and used 4–6 d after weekly subculturing.
For the LePLDα1-RNAi construct, an inverted repeat specific for LePLDa1 was generated targeting the gene's 3′ UTR. PCR amplification of LePLDa1 cDNA was performed with the oligonucleotides: 1_5′-CGGGATCCCCA TCGATCAGTCAATTAAAGCATCTC-3′ (reverse) with BamHI and ClaI restriction sites, 2_5′-CCGGAATTCCC CCGACACCAAGG-3′ (forward) with an EcoRI restriction site and 3_5′-CCGGAATTCCATCCAGAAAGTG AGG-3′ (forward) with an EcoRI restriction site. PCR products resulting from primer combinations 1-2 and 1-3 were ligated in a 1-2/3-1 orientation into pGreen1K, which was modified to contain the 35S-Tnos cassette from pMON999. Cell-suspension culture transformation was achieved as described by Bargmann et al. (2006).
In vivo phospholipid analysis
Suspension-cultured cells (100 µL aliquots in 2 mL Eppendorf tubes, Eppendorf, Germany) were labelled in growth medium supplemented with 10 µCi 32PO43− (carrier free) for 3 h. Wounding was induced by freezing cells in liquid nitrogen and thawing. When indicated, incubations were performed in the presence of 0.5% (v/v) n-butanol. Treatments were stopped by adding 5% perchloric acid (final concentration), and lipids were extracted as described before (van der Luit et al. 2000). Leaf discs (5 mm Ø) were labelled by incubation with 10 µCi carrier-free 32PO43− on 100 µL 10 mm MES buffer [2-(N-Morpholino)ethane sulfonic acid] pH 5.7 (KOH) in a 2 mL microcentrifuge tube (Frank et al. 2000). Two-week-old Arabidopsis seedlings, grown on rockwool cubes, were labelled by pipetting 100 µL water containing 100 µCi carrier-free 32PO43− onto the rockwool and leaving them overnight under fluorescent light in a fume hood. Wounding was induced by freezing/thawing or a hemostat, as indicated. Treatments were stopped by incubation with 5% (v/v) perchloric acid. Plant material was transferred to a new tube containing 375 µL CHCl3/MeOH/HCl [50:100:1 (v/v)] shaken vigorously for 10 min. A two-phase system was induced by the addition of 375 µL CHCl3 and 200 µL 0.9% (w/v) NaCl. The remainder of the extraction was performed as described before (van der Luit et al. 2000). Lipids were separated on thin-layer chromatography (TLC) plates using the organic upper phase of an ethyl acetate mixture, ethyl acetate/iso-octane/formic acid/water [12:2:3:10 (v/v); Munnik et al. 1998], or using an alkaline solvent system, CHCl3/MeOH/25% NH4OH/H2O [90:70:4:16 (v/v); Munnik, Irvine & Musgrave 1994], when indicated. Radio-labelled phospholipids were quantified by phosphoimaging (Molecular Dynamics, Sunnyvale, CA, USA).
RNA and protein blot analysis
RNA blot analysis was performed as described by Bargmann et al. (2006). Protein extraction buffer [9.5 m urea, 0.1 m Tris-HCl pH 6.8, 2% (w/v) sodium dodecyl sulphate (SDS) and 2% (v/v) β-mecraptoethanol] was added to an equal volume of ground leaf tissue, vortexed and centrifuged in an Eppendorf centrifuge for 10 min at 1000 g. Samples were separated by 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) gel electrophoresis, blotted on nitrocellulose and incubated overnight with polyclonal anti-LePLDα1 antibody (rabbit; Eurogentech, Liege, Belgium). Antibodies were generated using the final 12 amino acids of LePLDα1: N-TKSDYLPPNLTT-C. Peroxidase activity of horseradish peroxidase-coupled goat anti-rabbit immunoglobulin G antibody (Pierce, Rockford, IL, USA) was detected by enhanced chemiluminescence (Amersham, Buckinghamshire, UK). A duplicate gel was stained with Coomassie Brilliant Blue as a loading control.
In vitro PLDα activity assay
PLDα activity was assayed by using a combined protocol of Pappan, Zheng & Wang (1997) and Ella et al. (1994). Briefly, 10 µg of total protein extract was incubated with 250 µm BODIPY-PC as a substrate in a buffer containing 50 mm MES pH 6.5, 80 mm NaCl, 0.5 mm SDS and 10 mm CaCl2 and 1% (v/v) n-butanol, for 1 h at 30 °C. Cabbage PLD (1 U; type V; Sigma-Aldrich, Steinheim, Germany) was used as a positive control. Lipids were extracted as described earlier and separated by ethyl acetate. TLC BODIPY-lipids were visualized by fluoroimaging.
In-gel kinase assay
Proteins were extracted from ground plant and cell-suspension material using 1 vol of extraction buffer [50 mm Tris-HCl pH 7.5, 5 mm ethylenediaminetetraacetic acid (EDTA), 5 mm ethylene glycol tetraacetic acid (EGTA), 2 mm dithiothreitol (DTT), 25 mm soduimfluoride (NaF), 1 mm Na3VO4, 50 mmβ-glycerophosphate, 1× complete protease inhibitor cocktail] and a 15 min 10 000 g centrifugation. Samples were assayed for protein content and 10 µg protein was loaded onto a 10% SDS–PAGE gel containing 4 µg mL−1 myelin basic protein (MBP) (Upstate, Lake Placid, NY, USA). The gel was washed three times for 30 min with wash buffer [25 mm Tris-HCl pH 7.5, 500 µm DTT, 100 µm Na3VO4, 5 mm NaF, 500 µg mL−1 bovine serum albumin, 0.1% (v/v) Triton X-100] and renatured overnight in renaturation buffer (25 mm Tris-HCl pH 7.5, 1 mm DTT, 100 µm Na3VO4, 5 mm NaF). The gel was washed three times for 30 min in reaction buffer (25 mm Tris-HCl pH 7.5, 1 mm DTT, 100 µm Na3VO4) and incubated in reaction buffer supplemented with 25 µm cold dATP and 50 µCi 32P-labelled γ-ATP for 1 h. The reaction was stopped, and the gel was washed six times for 30 min with stop buffer [1% (w/v) Na2H2P2O7, 5% (v/v) trichloric acid]. The gel was dried, and the signal was visualized by phosphoimaging.
cDNA was synthesized as described in Ament et al. (2004) and used as a template for amplification of AtPLDδ (with primers: AtPLDδRT-F 5′-CGAGACCTTCCCAGATGT TG-3′ and dT18) and AtTUA4 (with primers: AtTUA4RT-F 5′-CCAGCCACCAACAGTTGTTC-3′ and AtTUA4RT-Rev 5′-CACAAGACGAGATTATAGAGA-3′). PCR products were separated by gel electrophoresis, blotted onto nitrocellulose and hybridized with 32P-labelled AtPLDδ and AtTUA4 probes. The signal was visualized by phosphoimaging.
Oxylipins were extracted, derivatized and analysed as previously described by Stumpe et al. (2005). Pentafluorobenzyl esters were analysed by gas chromatography/mass spectrometry using the following ions and retention times for quantification: m/z 215 (D6-JA; Rf = 14.11, 14.46 min), 209 (JA; Rf = 14.15, 14.51 min), 237 (OPC-4; Rf = 16.76, 16.98 min), 265 (OPC-6; Rf = 18.84, 19.08 min), 293 (OPC-8; Rf = 20.72, 20.95 min), 296 (D5-oPDA; Rf = 20.8, 21.18, 21.52 min), 291 (oPDA; Rf = 20.84, 21.22, 21.56 min) and 263 (dinor-oPDA; Rf = 18.94, 19.39, 19.75 min).
Herbivore performance assay
The assay was performed as described earlier (de Vos et al. 2006). Briefly, 6-week-old Arabidopsis plants were transferred to modified magenta boxes. One P. rapae caterpillar in larval stage L2 was placed on each plant and allowed to feed for 96 h. Caterpillar weight was determined at t = 0 and at t = 96 h, and the relative weight increase of multiple larvae during this period was averaged.
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- Supporting Information
AtPLDα1 is activated after wounding
As shown in Fig. 1a, mechanical wounding of leaf discs induced a rapid and transient increase of PA. Interestingly, wounded leaf discs from pldα1 knock-out lines produced significantly less PA (Fig. 1a; two-tailed paired t-test, P < 0.05 at 10 and 20 min). The kinetics of the transient PA response in wild-type and pldα1 knock-out lines were similar. These results correlate well with the earlier study of antisense AtPLDα1 plants (Wang et al. 2000), demonstrating that AtPLDα1 is activated upon wounding and that there is likely another PLD activated under these circumstances. Our data also shows that an overnight incubation of the leaf discs left them responsive despite earlier wounding by excision from the plant.
AtPLDα1 is activated in ruptured cells
It is unclear whether the wounding-induced AtPLDα1 activity originates from the ruptured cells or the intact cells. The correlation between the in vitro α-class PLD enzymatic requirements and the conditions found in the plant apoplast and vacuole, namely an acidic pH (pH ∼6.3; Gao et al. 2004) and millimolar calcium concentrations (>10−3 M; Björkman & Cleland 1991; Cabañero et al. 2006) indicate that AtPLDα1 could become active upon disruption of cellular compartmentalization. To address this question, analyses of solely dead or living cells would be required.
The response in dead cells can be assessed by rupturing every cell in the leaf disc. This can be achieved by snap freezing the leaf discs in liquid nitrogen and subsequent thawing; the formation of ice crystals causes mechanical damage, disrupting cellular compartmentalization. Analysis of leaf discs that had been snap frozen and thawed revealed dramatically increased PA levels (Fig. 1b). The increase in PA in the pldα1 mutant was consistently lower than in wild type (Fig. 1b). Analysis of electrolyte leakage showed that cells had been ruptured equally in both the wild-type and mutant leaf discs (Fig. 1c). These data indicate that AtPLDα1 becomes active upon loss of cell membrane integrity and that at least part of the increase in PA measured in wounded leaf discs is caused by AtPLDα1 activity in the ruptured cells.
Having established that AtPLDα1 is active in ruptured cells, our next efforts were directed towards assessing PLD activity in the remaining territories in the wounding response, namely, the local and systemic tissues. To assess AtPLDα1 activity in the intact tissue of a wounded leaf disc, half of a leaf disc was wounded and the wounded and uninjured halves were dissected and analysed separately.
As shown in Fig. 2, PA levels were not increased in unwounded halves of wounded leaf discs, whereas wounded halves showed an increase that was the same as in non-dissected wounded leaf discs. Treatment in the presence of n-butanol, allowing exclusive visualization of PLD activity via PLD-catalysed transphosphatidylation (Munnik et al. 1995), showed a PBut accumulation that mirrored that of PA (Fig. 2b). These results suggest that the increase in PA in wounded Arabidopsis leaf discs is not produced in intact tissue but only in the ruptured cells.
PLD is activated locally
PLD has also been implicated in the systemic wounding response. Lee et al. (1997, 2001) reported increased systemic PA levels in seedlings within minutes after wounding. Although Wang et al. (2000) investigated local and systemic JA levels and gene expression in wounded Arabidopsis plants, their report did not present data concerning systemic PA levels. This hiatus prompted us to investigate whether PLD activity increases systemically in Arabidopsis and, if so, whether the AtPLDα1 isoform is involved. When the first true leaf of seedlings was mechanically wounded and PA levels were followed for 20 min in both the wounded and in the second, systemic, true leaf, PA production in the wounded leaf was rapid and substantial (Fig. 2c). In contrast, no statistically significant increase in PA was detected in the systemic leaf; instead, levels remained basal throughout the 20 min following treatment (two-tailed paired t-test, P < 0.05). These results indicate that there is no systemic PA response in wounded Arabidopsis plants within the first 20 min.
PLDα1 activity in a cell-suspension wounding model
PLD and PA involvement in the wounding response has been previously studied utilizing a cell-suspension wounding model (Lee et al. 2001). The authors showed that addition of ruptured cells to a soybean cell-suspension activated a wounding-induced MAPK (Lee et al. 2001). Moreover, they showed that the addition of PA to the cell-suspension culture could induce MAPK activation. These results were interpreted to suggest that PLD activity in the intact, responding cells was upstream of the observed MAPK activation. However, neither PLD activity nor PA levels were measured in this report. Nonetheless, a cell-suspension wounding model would be well suited for differentially labelling and analysing the PA levels in ruptured and intact cells independently.
To investigate the involvement of tomato PLDα1 (LePLDα1) in the wounding response, an RNAi construct targeting the gene's 3′ UTR was used to knock down LePLDα1 in a tomato cell-suspension culture (Msk8). Five independently transformed cell-suspension culture lines were obtained, as well as an empty-vector control line. RNA blot analysis showed that three of the five lines carrying the RNAi construct displayed a negligible mRNA transcript level (Fig. 3a). This finding was confirmed by protein blot analysis (Fig. 3b). An assay for in vitro activity demonstrated that the silenced cell-suspension cultures displayed a greatly reduced PLDα activity (Fig. 3c). Together, these results show that we were able to successfully knock down LePLDα1 in tomato cell-suspension cultures.
Increased PLD activity upon disruption of compartmentalization has been noted before (Roughan, Slack & Holland 1978; Wang 2005), yet it is unknown which PLDs are involved. As mentioned earlier, it seems likely that the α-class PLDs becomes active when apoplastic conditions are encountered. In general, cell-suspension cultures are grown in media with conditions that mimic the apoplast. In this case, cells were cultured in Murashige and Skoog medium with a pH of 5.7 and a final Ca2+ concentration of 2 mm (see ‘Methods’). In order to achieve a loss of cell membrane integrity, cells were snap frozen and thawed. Vitality staining with fluorescein diacetate revealed that this treatment led to a 100% cell death (data not shown). When cells were treated in this way, a remarkable PA production could be observed (Fig. 3d). Concomitantly, the levels of the structural phospholipids PC, PE and phosphatidylglyercol (PG) decreased dramatically, whereas levels of phosphatidylinositol (PI) remained relatively stable. An increase in lysophophatidic acid (LPA) could be seen following the PA increase (Fig. 3d). These data suggest that the structural phospholipids PC, PE and PG (and not PI) are rapidly and massively converted to PA by PLD upon loss of cellular membrane integrity. Phosphatidylinositolmonophosphate (PIP) and PIP2 levels also decreased, possibly because of the action of phospholipase C, which, in combination with diacylglycerol kinase, may also contribute to the increased PA levels (Mosblech et al. 2008).
The PA and LPA increases in LePLDα1-silenced cell-suspension cultures were significantly reduced when compared with those in control lines (Fig. 3d). Concurrently, the decreases in PC, PE and PG were also slower. These results indicate that the PA production observed upon loss of cell membrane integrity is in part derived from LePLDα1 activity, and that this isoform uses PC, PE and PG as substrates, but not PI.
In order to investigate whether it was indeed the extracellular pH and Ca2+ concentrations that induced the PLD activity after loss of cell membrane integrity, cells were lysed in the presence of 50 mm Tris-HCl (pH 7.5) and/or 10 mm EGTA to buffer protons and Ca2+ ions, respectively. Buffering the growth medium and chelating the free Ca2+ ions both decreased the lysis-induced PA production individually, and almost completely blocked this activity when applied in combination (Fig. 4). These results show that PLD activity upon loss of cell membrane integrity in a tomato cell-suspension culture is dependent on the extracellular low pH and high [Ca2+].
A wounding model system analogous to the one employed by Lee et al. (2001) was set up to examine PLD activity in the responding cells. Cells that had been ruptured (snap frozen and thawed 5 min) were added to a cell-suspension culture in a 1:9 ratio, and the induced protein kinase activity towards MBP was analysed by in-gel kinase assays (Fig. 5a). A band of approximately 48 kDa was detected, which displayed a fast and transient activation in response to the treatment, with a maximum activity at 5 min that declined again after 20 min. Analysis solely of the ruptured cells showed that this MBP kinase did not originate from these cells (Fig. 5a). This protein kinase activity is similar to the 48 kDa MBP kinase found by Stratmann & Ryan (1997) in wounded tomato leaves. Once more, a dramatic increase in PLD activity could be observed in the snap frozen and thawed cells (Fig. 5b), and an LePLDα1-silenced line exhibited reduced PLD activity. Interestingly, no increase in PA levels could be detected when intact cells were treated with unlabelled ruptured cells (Fig. 5c). In contrast, cells treated with mas7, a strong elicitor of PLD responses (Munnik et al. 1996, 1998; Van Himbergen et al. 1999; van der Luit et al. 2000), displayed a clear PA response within 20 min (Fig. 5c). These results further indicate that wounding-induced PLD activation is restricted to the ruptured cells and does not occur in the intact, responding cells.
The Arabidopsis pldα1/δ double mutant and the wounding response
Although the Arabidopsis pldα1 knock-out line exhibited a reduced PA response to wounding and loss of membrane integrity, there was still a considerable production of PA in this line (Figs 1a–d & 2). This indicates that there is likely another PLD isoform active in the ruptured cells. AtPLDδ seemed a good candidate because, like AtPLDα1, it is active in relatively high Ca2+ concentrations (Qin et al. 2002) and AtPLDδ is relatively highly expressed (Li et al. 2006). pldα1/δ double knock-out mutants were generated and verified by genomic PCR, protein blot analysis and RT-PCR (Fig. 6). No obvious growth or developmental phenotype was observed under standard greenhouse and growth chamber conditions in either single or double mutants.
Leaf discs from wild-type and pld knock-out lines were wounded or snap frozen and thawed in the presence of n-butanol in order to assess the effect of individual and combined pld knock-outs on the induction of PA and PBut production. In agreement with earlier results (Figs 1 and 2), the PA and PBut production in the pldα1 mutant line was consistently lower than in the wild-type line (Fig. 7). In contrast, PA and PBut increases in the pldδ knock-out line were closer to wild-type levels (Fig. 7). However, in the double mutant, no increase of wounding- and cell lysis-induced PA and PBut was observed (Fig. 7). When followed for longer periods of time, up to 80 min, the double mutant still did not display a notable increase in PA production when snap frozen and thawed (Supporting Information Fig. S1). These results indicate that AtPLDδ is responsible for the residual PLD activity seen upon wounding and loss of membrane integrity in the pldα1 knock-out line and suggest that AtPLDα1 can compensate for the loss of AtPLDδ in the pldδ mutant. Lastly, these results indicate that these two PLD isoforms together account for all the discernible activity seen in response to these treatments.
Rapid and transient MAPK activation in response to wounding has also been found in Arabidopsis (Ichimura et al. 2000). AtMPK4 and AtMPK6 are 47 and 44 kDa wounding-activated MBP kinases, respectively. MBP kinases of corresponding sizes were activated in Arabidopsis leaves in response to mechanical wounding (Fig. 8a). Surprisingly, the MBP kinase activation seen in the pldα1/δ double mutant was identical to the activation seen in wounded wild-type leaves (Fig. 8a). In order to examine conditions comparable with the assays for wounding-induced PLD activity (Figs 1, 2 & 7), protein extracts from wounded leaf discs were analysed for MBP kinase activity. Again, the same activation as in whole leaves could be observed in both wild-type and mutant leaf discs, with the exception that in this case the wounding-induced activity declined again within the assay period (Fig. 8a). These results show that, although the wounding-induced PA increase is completely abolished in this mutant, the wounding-induced MAPK activation is not affected in the pldα1/δ double mutant.
AtLOX2, encoding a lipoxygenase involved in JA synthesis, is transcriptionally up-regulated in response to wounding in Arabidopsis (Bell & Mullet 1993). In an Arabidopsis line expressing the antisense AtPLDα1, the wounding-induced expression of AtLOX2 was reported to be adversely affected (Wang et al. 2000). We therefore decided to follow the wounding-induced AtLOX2 expression in the pld knock-out lines (Fig. 8b). In contrast to expectations, there was no evident difference in the induction of AtLOX2 expression between wild-type and mutant lines. This finding shows that wounding-induced AtLOX2 expression is not affected in pldα1 nor in the pldδ knock-out mutants or pldα1/δ double knock-out mutant.
Similarly, wounding-induced JA synthesis has been reported to be negatively affected in antisense AtPLDα1 Arabidopsis plants (Wang et al. 2000). However, when we measured JA levels and those of its precursors oxophytodienoic acid (oPDA), dinor-oPDA, and the 3-oxo-2-(pent-20-enyl)-cyclopentane-1-octanoic acids OPC-8, OPC-6 and OPC-4 (Afitlhile et al. 2005) in the wounded pld mutants, no significant difference could be detected between wild type and the lines missing either one or both AtPLDα1 and AtPLDδ (Fig. 8c; two-tailed paired t-test, P < 0.05, data not shown). These results show that even though the wounding-induced PLD activity is completely lacking in the pldα1/δ double mutant, JA biosynthesis was not disturbed.
Plants respond to wounding with the production of proteins and chemicals that are toxic to potential future attackers or that affect herbivore appetite or digestion (Schilmiller & Howe 2005). Consequently, mutants with impaired basal defenses or an impaired wounding response are more nutritious to herbivores (Li et al. 2003). We investigated whether herbivore performance was affected on the Arabidopsis single and double pld knock-out lines. The weight gain of Cabbage White butterfly larvae (Pieris rapae) while feeding on wild-type and mutant Arabidopsis plants over a period of 4 d was measured and averaged (Fig. 8d). A glabrous Arabidopsis variety (Col-5) and the JA-insensitive coi1-16 mutant (Ellis & Turner 2002) were assayed as positive controls along with the wild type (Col-0) and the single and double pld knock-out mutants. Glabrous and JA-insensitive lines have previously been shown to have reduced resistance to P. rapae herbivory (Reymond et al. 2004). Whereas the caterpillar weight gain was significantly greater on the Col-5 and coi1-16 lines as compared with the performance on the wild-type plants, the weight gain achieved on pldα1, pldδ or pldα1/δ double knock-outs did not differ from that on wild type (Fig. 8d). These data indicate that the wounding response in the pld mutants is not affected in any such way that it influences herbivore performance.
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Where is PLD activity located upon wounding?
The fact that massive PLD activation can be observed in leaf discs and cell-suspension cultures in which cellular compartmentalization is lost by snap freezing and thawing (Figs 1–5 & 7), indicates that a large proportion of the PA increase seen in mechanically damaged leaf material (Figs 1, 2 & 7) is derived from ruptured cells. Analysis of the PA response in pldα1/δ double knock-out lines shows that virtually all the wounding-induced PLD activity can be ascribed to these two isoforms (Fig. 7). Purified, recombinant proteins of both of these PLDs have been analysed in vitro (Pappan & Wang 1999; Qin et al. 2002). These studies indicated that they are active at millimolar Ca2+ concentrations and, in the case of AtPLDα1, at acidic pH. Taken together, these data suggest that AtPLDα1 and AtPLDδ become active autonomously upon encountering apoplastic conditions, which resemble the conditions that are required in vitro.
Dissection of wounded Arabidopsis leaf discs (Fig. 2a,b) indicates that the PLD activity seen in this material is restricted to the ruptured cells. This finding is corroborated by the analysis of PLD activity in a tomato cell-suspension wounding model (Fig. 4). In this system, the activity is also massive in ruptured cells and not detectable in the intact cells. Additionally, no significant increase in systemic PA levels could be measured in mechanically wounded Arabidopsis plants (Fig. 2c). These results demonstrate that PLD activity upon wounding is restricted to the ruptured cells and not present in the intact, responding cells. These findings contradict earlier reports of wounding-induced PLD activation in intact tissues (Ryu & Wang 1996, 1998; Lee et al. 1997, 2001). This disparity can be explained by differences in lipid extraction methods, lipid analysis, plant species, examined tissues or circumstances of plant growth. Our laboratory has developed sensitive phospholipid analysis techniques that have been used to detect PA signalling events in numerous plant systems (Munnik et al.1995, 1996, 2000; Frank et al. 2000; den Hartog, Musgrave & Munnik 2001; den Hartog, Verhoef & Munnik 2003; Bargmann et al. 2006). Although it cannot be fully excluded that there is some undetectable PLD activity, we deduce that PLD activity in Arabidopsis wounding response is restricted to ruptured cells.
Does PLD activity play a role in the wounding response?
Results in earlier studies of PLD activity in the wounding response, using PLD inhibition and plant lines expressing antisense AtPLDα1 constructs, have been interpreted in a way that suggests that it is required for wild-type responses to wounding. Our analyses of four separate wounding response assays, however, show that the knock-out of AtPLDα1 and AtPLDδ does not influence the wounding response (Fig. 8), even though the pldα1/δ double mutant completely lacks wounding-induced PA accumulation (Fig. 7).
PLD activity in the wounding response has been proposed to be positioned upstream of MAPK activation (Wang et al. 2000; Lee et al. 2001). The fact that a wounding-induced MBP kinase response can be measured in intact cells that do not display a detectable PA response (Figs 2, 5 & 8a) suggests that PLD activity in these cells is not involved in the protein kinase activation. This conclusion is strengthened by the finding that wounding-induced protein kinase activation is not impaired in the pldα1/δ double knock-out mutant (Fig. 8a). Lee et al. (2001) based their hypothesis on results showing MAPK activation by application of exogenous PA and inhibition of wounding-induced MAPK activation by n-butanol. Application of exogenous PA has been shown to induce numerous responses, including the production of reactive oxygen species (Sang, Cui & Wang 2001; de Jong et al. 2004), cytoskeletal rearrangements (Lee, Park & Lee 2003; Huang et al. 2006) and cell death (Park et al. 2004). However, it does not necessarily mimic intracellular PLD activation. The specificity of PLD inhibition by primary alcohols is also disputable; it has been shown that alcohols induce changes in microtubule organization (Dhonukshe et al. 2003; Gardiner et al. 2003; Motes et al. 2005) that might well indirectly influence MAPK activation.
An Arabidopsis line expressing an antisense AtPLDα1 construct was reported to have perturbed wounding-induced gene expression (e.g. LOX2), as well as JA production (Wang et al. 2000). These authors suggested that the PA produced by AtPLDα1 upon wounding was a precursor for JA synthesis. In contrast, we found that AtLOX2 expression and the synthesis of JA and its peroxisomal precursors were not affected in wounded pldα1, pldδ or pldα1/δ knock-out mutants (Fig. 8b,c), indicating that these PLD isoforms are not involved in these wounding responses. This discrepancy is hard to explain; however, it could be accounted for by the fact that the former study made use of a silencing strategy, whereas we used T-DNA insertion lines. Wang et al. (2000) used an antisense line created previously (Fan, Zheng & Wang 1997) that expressed 785 bp of AtPLDα1 (bp 1446–2231 of the AtPLDα1 cDNA) in reverse orientation. This region shows high similarity to several other PLD isoforms in Arabidopsis and could conceivably be affecting their expression, as well as other, secondary silencing effects.
Pertaining to the lack of an effect on JA levels, DONGLE (DGL) and DEFECTIVE IN ANTHER DEHISCENCE (DAD1) are plastidic galactolipases that have been demonstrated to be necessary and sufficient for JA production (Hyun et al. 2008). These authors suggested that PLD was only involved in the wounding-induced expression of the genes encoding these lipases and not in the synthesis of JA precursors. A number of studies have indicated that JA originates from the plastidial oxylipin precursors 12-oPDA and dinor-oPDA (Wasternack et al. 2006). Certain JA precursors appear to be esterified to plastidial galactolipids, termed Arabidopsides, which are being discussed as another source of JA (Kourtchenko et al. 2007). In addition, cross-talk of oxylipins with phospholipids at the signalling levels has previously been proposed (Mosblech et al. 2008), in which the phospholipids do not act as biosynthetic precursors of JA.
Lastly, herbivore performance on the pld mutants was also not altered compared with that on wild type, as seen by the weight gain of Cabbage White butterfly caterpillars (Fig. 8d). These larvae did show enhanced performance on glabrous and JA-insensitive plant lines, demonstrating the efficacy of the assay. Taken together, these results show that no function can be ascribed to these two PLD isoforms in the wounding response, even though they are responsible for all the detectable PLD activity upon wounding.
All these results together demonstrate that the role of PLDs in the Arabidopsis wounding response needs to be reassessed. It cannot be fully excluded that PLDs have some kind of function in this response, but it can be stated that their role is not of great consequence and that the PLD-produced PA does not seem to be a precursor for JA synthesis as formerly proposed. The fact that PA is produced in massive amounts by ruptured cells at the wound site (Figs 1–5 & 7) and that it has been shown to elicit a number of responses in living cells (Lee et al. 2001, 2003; Sang et al. 2001; de Jong et al. 2004; Park et al. 2004; Huang et al. 2006) could indicate that this molecule might have some non-cell-autonomous signalling function. However, in the wounding response, this putative cell-to-cell PA signalling is apparently not required for a wild-type reaction as determined by kinase activation, induced AtLOX2 expression, JA production or herbivore resistance (Fig. 8).
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The authors would like to thank Dr Maarten Koornneef (Max Planck Institute, Cologne, Germany) for the Col-5 line, Dr John Turner (University of East Anglia, Norwich, UK) for the coi1-16 line, Dr Otto Miersch (Leibniz Institute for Plant Biochemistry, Halle, Germany) for the OPC-8, OPC-6 and OPC-4 standards, Kenneth Birnbaum (New York University, NY, USA) for the use of growth chambers and Dr Gert-Jan de Boer (Enza Zaden, Enkhuizen, the Netherlands) for helpful suggestions. The contribution of D.B. and E.M. was supported by the EU ROST project (QLK5-CT-2002-00841) and that of A.M. and I.H. by the Göttingen Graduate School for Neurosciences and Molecular Biosciences (GGNB). Research in Munnik's laboratory was supported by The Netherlands Organization for Scientific Research (NWO 813.06.0039, 863.04.004 and 864.05.001), the European Union (COST Action FA0605) and the Royal Netherlands Academy of Arts and Sciences (KNAW).
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- 2005) A defect in glyoxysomal fatty acid beta-oxidation reduces jasmonic acid accumulation in Arabidopsis. Plant Physiology and Biochemistry 43, 603–609. , , & (
- 2004) Jasmonic acid is a key regulator of spider mite-induced volatile terpenoid and methyl salicylate emission in tomato. Plant Physiology 135, 2025–2037. , , , & (
- 1998) Jasmonate-induced responses are costly but benefit plants under attack in native populations. Proceedings of the National Academy of Sciences of the United States of America 95, 8113–8118. (
- 2006) The role of phospholipase D in plant stress responses. Current Opinion in Plant Biology 9, 515–522. & (
- 2006) LePLDbeta1 activation and relocalization in suspension-cultured tomato cells treated with xylanase. The Plant Journal 45, 58–68. , , , , , , , & (
- 1993) Characterization of an Arabidopsis lipoxygenase gene responsive to methyl jasmonate and wounding. Plant Physiology 103, 1133–1137. & (
- 1991) The role of extracellular free-calcium gradients in gravitropic signalling in maize roots. Planta 185, 379–384. & (
- 2006) New evidence about the relationship between water channel activity and calcium in salinity-stressed pepper plants. Plant & Cell Physiology 46, 224–233. , , & (
- 2003) Phospholipase D activation correlates with microtubule reorganization in living plant cells. The Plant Cell 15, 2666–2679. , , , & (
- 1994) A fluorescent assay for agonist-activated phospholipase D in mammalian cell extracts. Analytical Biochemistry 218, 136–142. , , , , & (
- 2002) A conditionally fertile coi1 allele indicates cross-talk between plant hormone signalling pathways in Arabidopsis thaliana seeds and young seedlings. Planta 215, 549–556. & (
- 1997) Antisense suppression of phospholipase D alpha retards abscisic acid- and ethylene-promoted senescence of postharvest Arabidopsis leaves. The Plant Cell 9, 2183–2196. , & (
- 1991) Rapid changes of protein phosphorylation are involved in transduction of the elicitor signal in plant cells. Proceedings of the National Academy of Sciences of the United States of America 88, 8831–8834. , , & (
- 2000) Water deficit triggers phospholipase D activity in the resurrection plant Craterostigma plantagineum. The Plant Cell 12, 111–124. , , , & (
- 2004) Self-reporting Arabidopsis expressing pH and [Ca2+] indicators unveil ion dynamics in the cytoplasm and in the apoplast under abiotic stress. Plant Physiology 134, 898–908. , , , & (
- 2003) The effects of the phospholipase D-antagonist 1-butanol on seedling development and microtubule organisation in Arabidopsis. Plant & Cell Physiology 44, 687–696. , , & (
- 1972) Wound-induced proteinase inhibitor in plant leaves: a possible defense mechanism against insects. Science 175, 776–777. & (
- 2001) Nod factor-induced phosphatidic acid and diacylglycerol pyrophosphate formation: a role for phospholipase C and D in root hair deformation. The Plant Journal 25, 55–65. , & (
- 2003) Nod factor and elicitors activate different phospholipid signaling pathways in suspension-cultured alfalfa cells. Plant Physiology 132, 311–317. , & (
- 2006) Heterodimeric capping protein from Arabidopsis is regulated by phosphatidic acid. Molecular Biology of the Cell 17, 1946–1958. , , & (
- 2008) Cooperation and functional diversification of two closely related galactolipase genes for jasmonate biosynthesis. Developmental Cell 14, 183–192. , , , et al. (
- 2000) Various abiotic stresses rapidly activate Arabidopsis MAP kinases ATMPK4 and ATMPK6. The Plant Journal 24, 655–665. , , , & (
- 2004) Phosphatidic acid accumulation is an early response in the Cf-4/Avr4 interaction. The Plant Journal 39, 1–12. , , , , & (
- 2007) Oxo-phytodienoic acid-containing galactolipids in Arabidopsis: jasmonate signaling dependence. Plant Physiology 145, 1658–1669. , , , , , , , & (
- 1997) Systemic elevation of phosphatidic acid and lysophospholipid levels in wounded plants. The Plant Journal 12, 547–556. , , , , , & (
- 2001) Phosphatidic acid activates a wound-activated MAPK in Glycine max. The Plant Journal 26, 479–486. , & (
- 2003) Phosphatidic acid induces actin polymerization by activating protein kinases in soybean cells. Molecules and Cells 15, 313–319. , & (
- 2005) Direct and indirect defences induced by piercing-sucking and chewing herbivores in Medicago truncatula. The New Phytologist 167, 597–606. , & (
- 2003) The tomato suppressor of prosystemin-mediated responses2 gene encodes a fatty acid desaturase required for the biosynthesis of jasmonic acid and the production of a systemic wound signal for defense gene expression. The Plant Cell 15, 1646–1661. , , , , , , & (
- 2006) Double knockouts of phospholipases Dzeta1 and Dzeta2 in Arabidopsis affect root elongation during phosphate-limited growth but do not affect root hair patterning. Plant Physiology 140, 761–770. , , & (
- 2000) Elicitation of suspension-cultured tomato cells triggers the formation of phosphatidic acid and diacylglycerol pyrophosphate. Plant Physiology 123, 1507–1516. , , , , , & (
- 2008) Phosphoinositide and inositolpolyphosphate signalling in defense responses of Arabidopsis thaliana challenged by mechanical wounding. Molecular Plant 1, 249–261. , , , , & (
- 2005) Differential effects of two phospholipase D inhibitors, 1-butanol and N-acylethanolamine, on in vivo cytoskeletal organization and Arabidopsis seedling growth. Protoplasma 226, 109–123. , , , , & (
- 1994) Rapid turnover of phosphatidylinositol 3-phosphate in the green alga Chlamydomonas eugametos: signs of a phosphatidylinositide 3-kinase signalling pathway in lower plants? The Biochemical Journal 298, 269–273. , & (
- 1995) G protein activation stimulates phospholipase D signaling in plants. The Plant Cell 7, 2197–2210. , , & (
- 1996) Identification of diacylglycerol pyrophosphate as a novel metabolic product of phosphatidic acid during G-protein activation in plants. Journal of Biological Chemistry 271, 15708–15715. , , & (
- 1998) Detailed analysis of the turnover of polyphosphoinositides and phosphatidic acid upon activation of phospholipases C and D in Chlamydomonas cells treated with non-permeabilizing concentrations of mastoparan. Planta 207, 133–145. , , , , , & (
- 2000) Hyperosmotic stress stimulates phospholipase D activity and elevates the levels of phosphatidic acid and diacylglycerol pyrophosphate. The Plant Journal 22, 147–154. , , , , , & (
- 2001) Hydrogen peroxide acts as a second messenger for the induction of defense genes in tomato plants in response to wounding, systemin, and methyl jasmonate. The Plant Cell 13, 179–191. , & (
- 1999) Plant phospholipase Dalpha is an acidic phospholipase active at near-physiological Ca(2+) concentrations. Archives of Biochemistry and Biophysics 1439, 347–353. & (
- 1997) Identification and characterization of a novel plant phospholipase D that requires polyphosphoinositides and submicromolar calcium for activity in Arabidopsis. Journal of Biological Chemistry 272, 7048–7054. , & (
- 2004) Phosphatidic acid induces leaf cell death in Arabidopsis by activating the Rho-related small G protein GTPase-mediated pathway of reactive oxygen species generation. Plant Physiology 134, 129–136. , , , & (
- 2002) The Arabidopsis phospholipase D family. Characterization of a calcium-independent and phosphatidylcholine-selective PLD zeta 1 with distinct regulatory domains. Plant Physiology 128, 1057–1068. & (
- 2002) Kinetic analysis of Arabidopsis phospholipase Ddelta. Substrate preference and mechanism of activation by Ca2+ and phosphatidylinositol 4,5-biphosphate. Journal of Biological Chemistry 277, 49685–49690. , & (
- 2004) A conserved transcript pattern in response to a specialist and a generalist herbivore. The Plant Cell 16, 3132–3147. , , , , & (
- 1978) Generation of phospholipid artefacts during extraction of developing soybean seeds with methanolic solvents. Lipids 13, 497–503. , & (
- 1996) Activation of phospholipase D and the possible mechanism of activation in wound-induced lipid hydrolysis in castor bean leaves. Biochimica et Biophysica Acta 1303, 243–250. & (
- 1998) Increase in free linolenic and linoleic acids associated with phospholipase D-mediated hydrolysis of phospholipids in wounded castor bean leaves. Biochimica et Biophysica Acta 1393, 193–202. & (
- 2001) Ecology. The enemy of my enemy is my ally. Science 291, 2104–2105. , & (
- 2001) Phospholipase D and phosphatidic acid-mediated generation of superoxide in Arabidopsis. Plant Physiology 126, 1449–1458. , & (
- 2005) Systemic signaling in the wound response. Current Opinion in Plant Biology 8, 369–377. & (
- 1997) Myelin basic protein kinase activity in tomato leaves is induced systemically by wounding and increases in response to systemin and oligosaccharide elicitors. Proceedings of the National Academy of Sciences of the United States of America 94, 1085–1089. & (
- 2005) Lipid metabolism in arbuscular mycorrhizal roots of Medicago truncatula. Phytochemistry 66, 781–791. , , , , , , & (
- 1999) Mastoparan analogues activate phospholipase C- and phospholipase D activity in Chlamydomonas: a comparative study. Journal of Experimental Botany 50, 1735–1742. , , , , & (
- 2006) Herbivore-induced resistance against microbial pathogens in Arabidopsis. Plant Physiology 142, 352–363. , , , , , & (
- 2000) Involvement of phospholipase D in wound-induced accumulation of jasmonic acid in Arabidopsis. The Plant Cell 12, 2237–2246. , , , , & (
- 2005) Regulatory functions of phospholipase D and phosphatidic acid in plant growth, development and stress responses. Plant Physiology 139, 566–573. (
- 2006) The wound response in tomato – role of jasmonic acid. Journal of Plant Physiology 163, 297–306. , , , , , , , & (
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Figure S1. PLD activity upon loss of cell membrane integrity in the pldα1/δ double knock-out line. Leaf discs from wild-type (Col-0) and pldα1/δ double knock-out lines were labelled overnight floating on buffer containing 32Pi. Discs were snap frozen and thawed. Lipids were extracted at the indicated time points, separated by TLC and analysed by phosphoimaging. PA was quantified as percentage of total radio-labelled lipids and is presented in a histogram ± SD (n = 3). Asterisks indicate a significant difference between mutant and wild type (two-tailed paired t-test, P < 0.05).
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