Phosphoinositide and Inositolpolyphosphate Signalling in Defense Responses of Arabidopsis thaliana Challenged by Mechanical Wounding
Department of Plant Biochemistry, Albrecht-von-Haller-Institute for Plant Sciences, Georg-August-University Göttingen, Justus-von-Liebig-Weg 11, 37077 Göttingen, Germany
1 To whom correspondence should be addressed. Heilmann: e-mail iheilma{at}uni-goettingen.de, fax +49 551 39 5749, tel. +49 551 39 5748. Feussner: e-mail ifeussn{at}uni-goettingen.de, fax +49 551 39 5749, tel. +49 551 39 5743
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Abstract |
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Various biochemical signals are implicated in Arabidopsis wound signalling, including jasmonic acid (JA), salicylic acid, auxin, and Ca2+. Here, we report on cross-talk of phytohormones with phosphoinositide signals not previously implicated in plant wound responses. Within 30 min of mechanical wounding of Arabidopsis rosette-leaves, the levels of the lipid-derived soluble inositolpolyphosphate, inositol 1,4,5-trisphosphate (InsP3), increased four to five-fold. Concomitantly, the precursor lipids, phosphatidylinositol 4,5-bisphosphate, phosphatidylinositol 4-phosphate and phosphatidylinositol transiently depleted, followed by re-synthesis after 30–60 min of stimulation. Increased InsP3 levels with wounding coincided with JA increases over the first hours of stimulation. In dde2-2-mutant plants deficient in JA biosynthesis, no InsP3 increase was observed upon wounding, indicating that JA was required for InsP3 formation, and InsP3 levels increased in wild-type plants challenged with sorbitol, increasing endogenous JA levels. In InsP 5-ptase plants with attenuated phosphoinositide signalling, the induction of wounding-inducible genes was diminished compared with wild-type plants, suggesting a role for phosphoinositide signalling in mediating plant wound responses. The gene-expression patterns suggest that phosphoinositides contribute to both JA-dependent and JA-independent aspects of wound signalling. Weight gain of Plutella xylostella caterpillars feeding on InsP 5-ptase plants was increased compared with that of caterpillars feeding on wild-type plants. The ecophysiological relevance of phosphoinositide signals in plant defense responses to herbivory is discussed in light of recent findings of inositolpolyphosphate involvement in phytohormone-receptor function.
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INTRODUCTION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATA |
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Plants in their natural habitats are exposed to frequent stress by herbivory, mechanical wounding, and/or subsequent pathogen attack. In order to cope with the detrimental consequences of physical injury, a system of active, wounding-induced defense mechanisms are in place (Leon et al., 2001; Liechti and Farmer, 2002; Kessler et al., 2004; Browse, 2005; Delker et al., 2006; Wasternack et al., 2006). Plant responses to wounding are diverse and include altered plant growth (Agrawal, 1998, 2000), intensified trichome formation (Traw and Bergelson, 2003), increased production of signalling molecules (O'Donnell et al., 2003), and the production of defense chemicals, such as protease inhibitors or other proteins decreasing insect-feeding performance (Ryan, 1992; McConn et al., 1997; Li et al., 2002; De Vos et al., 2005). The manifestation of the wounding response requires the induction of wound-responsive genes (Reymond et al., 2000, 2004) by integrated biochemical signals that can originate in different signalling pathways, for instance for genes induced dependently or independently of jasmonic acid (JA) (Leon et al., 1998). Biochemical signals implicated in wound signalling include JA and oxylipins, salicylic acid (SA), auxin (β-indolyl acetic acid, IAA), or Ca2+ (Kernan and Thornburg, 1989; Leon et al., 2001; O'Donnell et al., 2003). Cross-talk between some of the known players has been previously demonstrated, such as between JA and SA (Traw et al., 2003). Still, the current knowledge about the integration of wound-signalling pathways is limited, and links between signals and downstream effects are not well defined.
Transient increases in cytosolic Ca2+ levels have been demonstrated to occur in wounded Arabidopsis plants (Knight et al., 1993), and application of exogenous JA to plant cells results in transient influx of Ca2+ from the extracellular space (Sun et al., 2006). The effects of the pharmacological agent, (2,5-di-tert-butyl)-1,4-hydroquinone, on Ca2+ levels during wound signalling led Leon and coworkers to suggest that Ca2+ may be released from internal stores rather than originate from the surrounding medium, and that the phosphoinositide-derived second messenger, inositol 1,4,5-trisphosphate (InsP3), may act as an intermediate in wound signalling (Leon et al., 1998). InsP3 is one of the best-characterized effectors of Ca2+ release from internal stores in animal cells (Berridge, 2005), and Ca2+ release by InsP3 has also been demonstrated in plants (Alexandre and Lassalles, 1990).
Another role for inositolpolyphosphates in the mediation of plant stress responses is suggested by the recent identification of an inositolhexakisphosphate (InsP6) cofactor in the binding site of the IAA-receptor protein TIR1 (Tan et al., 2007). InsP6 can be formed from InsP3 by action of inositolpolyphosphate kinases, as summarized in Stevenson-Paulik et al. (2005), and it is possible that InsP3 serves as a precursor of InsP6 required for optimal receptor function in auxin signalling. Whereas a causative dependency between InsP3 and IAA was not established, it has previously been demonstrated that InsP3 is generated rapidly upon gravistimulation in plant tissues producing and responding to IAA during gravitropic curvature (Perera et al., 1999, 2001). As TIR1 is closely related in sequence to proteins required for JA perception (Serino and Deng, 2003; Schwechheimer and Calderon Villalobos, 2004; Thines et al., 2007), it is tempting to speculate whether binding of JA to the JAZ1–SCF–COI1 receptor complex (Thines et al., 2007) may also involve inositolpolyphosphates.
Whereas transient changes in phosphoinositide levels and inositolpolyphosphates have been shown to occur during plant responses to a variety of environmental stresses (Stevenson et al., 2000), no systematic study of phosphoinositides and InsP3 signalling in response to wounding has been reported to date. InsP3 is formed by phospholipase C (PLC)-mediated hydrolysis of the lipid, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), yielding the water-soluble InsP3 head group and diacylglycerol (DAG) (Berridge, 1983). Plant PLCs with preferences for phosphoinositides belong to the PLC
subfamily, which is activated by Ca2+ and cannot, in contrast to many mammalian PLCs involved in phosphoinositide signalling, be activated by 
-subunits of heterotrimeric G-proteins (Mueller-Roeber and Pical, 2002). Previous data indicate that InsP3 production in plants is regulated by increased availability of the lipid precursor, PtdIns(4,5)P2 (Heilmann et al., 1999, 2001; Perera et al., 1999, 2002), and, in support of this concept, PtdIns(4,5)P2 formed upon stress has been shown to consist of particular molecular species clearly differing from constitutive PtdIns(4,5)P2 in their metabolic origin (König et al., 2007).
In plants, PtdIns(4,5)P2 is generated from phosphatidylinositol (PtdIns) by a series of lipid kinases (Mueller-Roeber and Pical, 2002), including stress-activated PIP kinases (Heilmann et al., 1999, 2001; Perera et al., 1999). Besides serving as the precursor lipid for InsP3 production, PtdIns(4,5)P2 is a regulatory factor by itself and controls a multitude of different physiological processes by acting as a lipid ligand to diverse target proteins that are altered in their activity, localization or other properties by this interaction (Toker, 1998; Stevenson et al., 2000). The biogenesis of phosphoinositide signals in stressed plants is, thus, well documented; however, the contact points linking plant phosphoinositide signalling upstream and downstream to better established phytohormone signals and to target gene expression, respectively, remain unresolved.
In order to investigate possible cross-talk between oxylipin and phosphoinositide signalling, JA and InsP3 signals in wild-type plants were compared with those of two genetically altered plant lines impaired either in the biosynthesis of oxylipins or in InsP3 signalling: The delayed dehiscence 2-2 (dde2-2) mutant (von Malek et al., 2002) carries a transposon insertion disrupting the single locus encoding allene oxide synthase (AOS). The plastidial enzyme AOS catalyzes a key step in the formation of cyclic oxylipins, including JA and its precursor, 12-oxo phytodienoic acid (OPDA), in the octadecanoid pathway, and of dinor-OPDA (dn-OPDA) in the hexadecanoid pathway (Liavonchanka and Feussner, 2006). As a result of AOS disruption, dde2-2 plants are devoid of JA or other AOS-derived oxylipin products, and JA-dependent wound induction of a number of genes is compromised (Park et al., 2002). In the second transgenic line used, expression of a human type I inositolpolyphosphate 5-phosphatase (InsP 5-ptase) results in suppression of InsP3 accumulation (Perera et al., 2002, 2006). Upstream lipid intermediates of the phosphoinositide pathway are also reduced by the ‘pull’ on the phosphoinositide pathway exerted by increased PLC hydrolysis of PtdIns(4,5)P2 (Perera et al., 2002, 2006; König et al., 2007).
As indicators for downstream effects of a disturbed phosphoinositide system, a number of wound-inducible genes was selected for analysis of their specific transcript levels by real-time RT-PCR: AOS (Park et al., 2002), VSP1, encoding the vegetative storage protein1 (Benedetti et al., 1995; Liu et al., 2005), both require JA for wound induction. In contrast, OPR1 and RNS1, encoding OPDA reductase and ribonuclease1, respectively, are both induced by wounding, independently of JA (Reymond et al., 2000). Also investigated were the transcription factor WRKY70 (Li et al., 2006) and the Kunitz-family (Bauw et al., 2006) protease inhibitor T18K17.7 encoded by the gene locus At1g73260. In addition to changes in transcript levels of wound-inducible genes, plant defensive capabilities can be assessed by monitoring caterpillar feeding performance. As mentioned above, plants produce substances that affect the development (Kessler et al., 2004; Chen et al., 2005; Liu et al., 2005) or digestion (Ryan, 1992; McConn et al., 1997; Li et al., 2002; De Vos et al., 2005) of herbivorous insects. In consequence, in comparison to feeding on wild-type-plants, caterpillars exhibit enhanced growth rates when feeding on plants compromised in wound-signalling, such as on dde2-2 plants (Ryan, 1992; McConn et al., 1997; Li et al., 2002; De Vos et al., 2005) that lack proper defense gene expression.
Here, we show that timing and kinetics of wounding-induced changes in phosphoinositides and InsP3 coincide with increases in JA—a well characterized player in Arabidopsis wound signalling. Analysis of JA and InsP3 levels in plants impaired in either JA production or in InsP3 accumulation suggests that InsP3 can act downstream of JA in the same signalling pathway. Monitoring of wounding-induced changes in transcript levels of downstream target genes indicates an involvement of phosphoinositide signals in the induction of response gene expression. Caterpillar feeding experiments suggest a role for phosphoinositide signals in the mediation of plant defenses to herbivory.
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RESULTS |
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InsP3 and Polyphosphoinositide Levels Change Transiently in Wounded Arabidopsis Leaves
In order to establish whether or not the phosphoinositide system was a part of the plant-wounding response, a systematic study of InsP3 and phosphoinositide changes in mechanically wounded Arabidopsis plants was initiated. When Arabidopsis rosette leaves were analyzed at different times after wounding, a transient increase in the soluble messenger, InsP3, was observed (Figure 1A). Within 30 min, InsP3 values increased from 2.9 ± 0.4 to 12.7 ± 3.0 nmol g–1 fresh weight. Following this intermittent decrease, InsP3 levels increased again after 4–6 h of stimulation (Figure 1A, compare Figure 2B, right panel). To investigate whether InsP3 production correlated with changes in the levels of phosphoinositide lipid precursors, lipids were extracted from wounded Arabidopsis leaves at the times indicated, the mixtures separated by thin-layer chromatography, and phosphoinositides isolated. GC-based quantification of fatty acids chemically transmethylated from the isolated phosphoinositides gives an indication of the amounts of the different phospholipid classes, while simultaneously revealing their fatty acid compositions (Figure 1B–1D (König et al., 2007)). The transient increase in the soluble messenger, InsP3 (Figure 1A) concurred with corresponding decreases in PtdIns, PtdIns4P, and PtdIns(4,5)P2, as indicated by the amounts of fatty acids associated with the individual lipids (Figure 1B–1D, total bars). The sum of fatty acids associated with PtdIns(4,5)P2 (Figure 1B, total bars) and PtdIns4P (Figure 1C, total bars) showed initial decreases at 15 min (60 ± 10 to 30 ± 8 pmol g–1 fresh weight and 190 ± 24 to 90 ± 9 pmol g–1 fresh weight, respectively); however, the levels of both lipids were increasing again after 30 min and had recovered to their original values after 60 min. After 4 and 5 h, PtdIns(4,5)P2 and PtdIns4P were increasing beyond the initial values, reaching levels of 156 ± 17 and 380 ± 24 pmol g–1 fresh weight, respectively, which was approximately two to three times higher than the respective starting values (Figure 1B and 1C). The sum of fatty acids associated with PtdIns dropped over the first 60 min from 8.9 ± 1.2 to 3.6 ± 0.6 nmol g–1 fresh weight of stimulation (Figure 1D). At 120 min, PtdIns levels had recovered to the original values and were stable thereafter for the remaining time of the experiment (Figure 1D, total bars). In order to document global lipid degradation with wounding, changes in the levels of the structural lipids, phosphatidylcholine (PtdCho) and monogalactosyldiacylglycerol (MGDG) were also determined (Figure 1E and 1F). Within 15 min of wound stimulation, the sum of fatty acids associated with PtdCho decreased from approximately 670 ± 100 to 25 ± 15 nmol g–1 fresh weight (Figure 1E). After 2–3 h of stimulation, PtdCho-levels had increased again approximately up to the original levels prior to stimulation. Fatty acids associated with MGDG decreased with wounding from approximately 1120 ± 180 to 250 ± 100 nmol g–1 fresh weight (Figure 1F); however, the decrease was slower and occurred only at times exceeding 1 h of stimulation, coinciding with the recovery of PtdCho. Before stimulation, PtdIns had a fatty acid composition of mainly 16:0, 18:2
9,12 and 18:39,12,15 (Figure 1D, bar segments in white, diagonal stripes and black, respectively; x:yz denotes a fatty acid with x carbons and y double bonds in position z, counting from the carboxyl end), whereas PtdIns(4,5)P2 and PtdIns4P were more saturated (Figure 1B and 1C), containing mostly 16:0, 18:0 and 18:19 (bar segments in white, diamonds and grey, respectively), with only little to no 18:29,12 or 18:39,12,15. The changes in total levels of PtdIns, PtdIns4P, and PtdIns(4,5)P2 induced after stimulation were accompanied by slightly reduced unsaturation of PtdIns (Figure 1D) and slight increases in unsaturation of PtdIns4P and PtdIns(4,5)P2 (Figure 1B and 1C).
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Increased InsP3 Levels Coincide with Wounding-Induced Increases in JA
To determine the relations of InsP3 signalling with other signalling pathways mediating the wound response under the particular conditions used, InsP3 levels were analyzed in parallel with JA levels, which are known to increase transiently upon wounding (Delker et al., 2006). Increases in JA from 0.5 to 2.5 nmol g–1 fresh weight were detected within the first 5 min after wounding, steadily increasing until 60 min (Figure 2A, circles). JA levels reached a first maximum of 16.5 ± 2.9 nmol g–1 fresh weight after 1 h (Figure 2A, circles) and, between 2 and 4 h, showed an additional increase to 19.5 ± 8 nmol g–1 fresh weight (Figure 2A, circles). Similar to the experiment on younger plants shown in Figure 1, InsP3 levels rose from 3.9 ± 0.3 to 14.5 ± 9 nmol g–1 fresh weight within 30 min of stimulation (Figure 2B, circles), reaching a first maximum of 19.6 ± 8 nmol g–1 fresh weight after 60 min, followed by a second increase to 22 ± 1 nmol g–1 fresh weight after 4–5 h (Figure 2B, circles). Increases in InsP3 after mechanical wounding of wild-type plants were, thus, preceded by increases in JA, and, beyond 30 min of stimulation, both compounds were elevated over the remaining period of study.
JA Formation is Necessary for the Production of Wounding-Induced InsP3 Signals
In order to test whether or not JA or other octadecanoids were required for the generation of InsP3 signals, InsP3 formation was monitored in wounded dde2-2 plants deficient in the biosynthesis of JA (Figure 2B, squares). The levels of JA in the dde2-2 plants before and after wounding were below the limit of detection (Figure 2A, squares), as were those of OPDA and dn-OPDA (data not shown), thus reflecting the expected outcome of AOS disruption. InsP3 levels in non-stimulated dde2-2 plants were at 3.5 nmol g–1 fresh weight, similar to those of wild-type plants (Figure 2B, compare circles and squares). In contrast to wild-type plants (Figure 2B, circles), InsP3 levels did not increase after wound stimulation in dde2-2 plants (Figure 2B, squares). A slight increase in InsP3 after 4–5 h of wound stimulation of dde2-2 plants was observed in only one out of three independent experiments. The data indicate that wounding-induced JA formation is required for the generation of wounding-induced InsP3 signals.
Possible reciprocal effects of InsP3 signaling on JA formation after wounding were investigated by monitoring InsP3 and JA levels in wild-type and in transgenic plants expressing the human InsP 5-ptase, which exhibit strongly attenuated InsP3 accumulation (Figure 2, triangles) and have reduced levels of PtdIns(4,5)P2 (Perera et al., 2006; König et al., 2007). In non-stimulated InsP 5-ptase plants, InsP3 levels of 2.8 ± 0.2 nmol g–1 fresh weight were detected (Figure 2B, triangles), indicating a slight reduction in InsP3 compared with the wild-type plants (Figure 2B, circles). In contrast to wild-type plants, InsP3 levels in InsP 5-ptase plants did not increase after wounding, and InsP3 levels stayed at or below basal levels observed in wild-type plants at all times (Figure 2B, triangles). Wounding of InsP 5-ptase plants resulted in JA signals over the first hour of stimulation, similar to those of wild-type plants, reaching a maximum of 18.7 ± 2 nmol g–1 fresh weight after 1 h (Figure 2A, triangles). At time points exceeding 1 h, JA levels appeared to drop prematurely in the InsP 5-ptase plants (Figure 2A, triangles), whereas, in wild-type plants, JA levels showed a further increase beyond 4 h of stimulation and were dropping only after that time (Figure 2A, circles and triangles).
Effects of Exogenous and Endogenous JA on InsP3 Production
Because the data so far suggested that JA may effect InsP3 production, the hypothesis was tested that exogenous methyl-JA may cause increased production of InsP3. When whole rosettes of 6 week old wild-type Arabidopsis plants were floated for different time periods on water or an aqueous solution containing 5 µM methyl-JA, minor but reproducible transient increases in InsP3 levels were observed in both sets of samples after around 30 min of stimulation (data not shown). The increases observed were likely due to the wounding stress received by cutting the rosettes from the roots and cannot be attributed to the methyl-JA treatment. In order to test the effects of endogenous JA release while avoiding concurrent wound stimulation, plants were grown in hydroponic culture and treated with aqueous 0.8 M sorbitol, which has been shown to result in the release of endogenous JA (Löbler and Lee, 1998; Weichert et al., 2000; Stenzel et al., 2003). With the sorbitol treatment, a transient increase in InsP3 from 4.3 ± 2.3 to 49.6 ± 7.8 nmol g–1 fresh weight was detected within 30 min of sorbitol application that was absent from water controls (Figure 3).
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Enhanced IAA Production after Wounding of Plants Attenuated in Phosphoinositide Signaling
Because the IAA receptor protein, TIR1, was found to contain InsP6 as a cofactor in its IAA-binding site (Tan et al., 2007), we aimed to test whether altered phosphoinositide metabolism in InsP 5-ptase plants would affect IAA production in response to wounding. IAA levels in response to wounding are illustrated in Figure 4 for wild-type plants (circles) and InsP 5-ptase plants (triangles). Basal IAA levels were at 160 ± 20 and 430 ± 200 pmol g–1 fresh weight for wild-type and InsP 5-ptase plants, respectively, indicating raised basal IAA levels in the plants with attenuated phosphoinositide production. IAA levels increased with wounding two to three-fold over the respective basal levels, and overall IAA levels were higher in InsP 5-ptase plants compared with wild-type, with distinct maxima between 5 and 15 min and again after 4–5 h after wounding (Figure 4). In addition to IAA levels, the levels of SA were also tested in wounded wild-type and InsP 5-ptase plants. SA levels of wild-type and InsP 5-ptase plants were in the range between 2 and 4 nmol g–1 fresh weight and did not significantly differ between lines (data not shown).
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Phosphoinositides Are Required for Wounding-Induced Gene Expression
Results from the biochemical analyses so far indicated a dependency between JA and phosphoinositide signals during signal-transduction events after wounding of Arabidopsis leaves (Figure 2) and, possibly, also a connection to IAA (Figure 4). In order to test whether downstream wounding responses were altered with disturbance of phosphoinositide signaling, transcript levels of known wound-inducible genes were monitored in time-course experiments on wild-type and InsP 5-ptase plants (Figure 5). Real-time RT-PCR analysis of wild-type plants confirmed that transcript levels for AOS, OPR1, RNS1, VSP1, WRKY70, and T18K17.7 were all wound-inducible (Figure 5, white bars). In InsP 5-ptase plants, wound induction of the genes tested was either transiently or overall reduced in comparison to that observed in wild-type plants (Figure 5, black bars vs white bars). Altered gene expression patterns in InsP 5-ptase plants included attenuated wound induction early after wounding (AOS), delayed wound induction (OPR1), or attenuated wound induction at later time points (RNS1, WRKY70, T18K17.7). Wound induction of VSP1 was only transiently reduced at early time points after wounding, and VSP1 transcript levels at later time points were not different from those of wild-type plants (Figure 5).
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InsP 5-ptase Plants with Attenuated Phosphoinositide Signaling Are Compromised in Defense against Caterpillar Herbivory
In order to test whether altered wounding response patterns with abrogation of phosphoinositide signaling were relevant for an active defense of Arabidopsis plants against insect herbivory, caterpillar growth performance was evaluated with feeding on wild-type plants, InsP 5-ptase plants, or dde2-2 plants. Plutella xylostella caterpillars of approximately equal developmental stages and weight were placed on leaves of 6 week old plants, and the increase in caterpillar weight was monitored over several days. Figure 6 illustrates the relative caterpillar weight increase; absolute values are summarized in Supplemental Table 1. Caterpillars feeding on InsP 5-ptase plants or on dde2-2 plants exhibited significantly increased mean growth rates of 47 ± 9 µg h–1 and 66 ± 6 µg h–1, respectively, in comparison to caterpillars feeding on wild-type plants, which exhibited a growth rate of only 21 ± 6 µg h–1. Leaf area damage from caterpillar herbivory was roughly equal in all cases (data not shown). Caterpillar weight increase with feeding on InsP 5-ptase plants was, thus, similar to that observed on dde2-2 plants, and both these data sets differed significantly from that observed with feeding on wild-type plants (Figure 6, Supplemental Table 1).
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1 mg were allowed to feed on wild-type, InsP 5-ptase, or dde2-2 plants for various time periods. Caterpillar weight was determined at the times indicated, and caterpillars were allowed to continue feeding. Data were normalized against caterpillar weights at time zero and are given as percent increase over the time zero value ± SD. Data are from three independent biological experiments and numbers correspond to the examination of 30–36 caterpillars for each plant line. White, feeding on wild-type; black, on InsP 5-ptase plants; checkers, on dde2-2 plants. Asterisks indicate significant increases in caterpillar growth with feeding on transgenic plants compared with feeding on wild-type plants, according to a student's t-test (*, p < 0.05; **, p < 0.01). |
DISCUSSION |
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The key observation underlying the experiments described is that wound stimulation of wild-type Arabidopsis leaves resulted in transient changes in phosphoinositide levels and caused concomitant increases in InsP3 that depend on JA formation. The results from the systematic analysis of phosphoinositide intermediates and InsP3 reported here are internally consistent and allow some conclusions regarding the regulation of wound-induced InsP3 production from intermediates of the phosphoinositide pathway. The first wounding-induced InsP3 increases occurred between 15 and 60 min after wounding and were accompanied by transient decreases in the precursor lipids, PtdIns, PtdIns4P, and PtdIns(4,5)P2 (Figure 1A and 1B). Because InsP3 is produced by hydrolysis of PtdIns(4,5)P2, the decrease in that lipid may be explained in part by transient depletion of the hydrolysable PtdIns(4,5)P2 pool. As the observed molar decrease in PtdIns(4,5)P2 does not account for the molar increase in InsP3, continuous re-synthesis of PtdIns(4,5)P2 by activation of PIP-kinase must be postulated, as has previously been described for different environmental stresses in a number of studies (Heilmann et al., 1999, 2001; Perera et al., 1999; Pical et al., 1999). In support of this concept, the transient PtdIns(4,5)P2 decrease was followed by an increase in PtdIns(4,5)P2 beyond the original levels (Figure 1B). A similar pattern is apparent for PtdIns4P generated by PI-kinase (Figure 1C).
Whereas PtdIns(4,5)P2 decreases are consistent with PLC-mediated hydrolysis for InsP3 production, previous studies have indicated that InsP3 production in plants is driven by increased production of PtdIns(4,5)P2, rather than by increased PLC activity (Heilmann et al., 1999, 2001; Perera et al., 1999, 2002). The observation that PtdIns(4,5)P2 levels transiently decreased, in contrast to reported PtdIns(4,5)P2 increases with other stresses (Heilmann et al., 1999, 2001; Perera et al., 1999; Pical et al., 1999), may be explained by large-scale lipid degradation in disintegrating damaged cells occurring simultaneously with cellular responses in non-damaged cells, because lipid levels apparent with whole-tissue analysis will be influenced by both aspects. In this context, it is important to note that the levels of all phosphoinositides including PtdIns decreased during the first 15 min of wounding, as well as the levels of the structural lipids, PtdCho (Figure 1E) and, at later time points, also those of MGDG (Figure 1F), suggesting that global lipid degradation may take place rapidly after wounding. Increased biosynthesis of PtdIns(4,5)P2 in non-damaged cells as that reported for plant responses to other stresses may not be evident at early time points before the background of large-scale lipid degradation. After 30 min of stimulation, however, activation of PI-kinases and PIP-kinases and mobilization of PtdIns for polyphosphoinositide synthesis in non-damaged cells becomes apparent, and the total levels of PtdIns4P and PtdIns(4,5)P2 have recovered to their original levels (Figure 1B and 1C). Thus, at time points beyond 30 min, when PtdCho levels are greatly reduced (Figure 1E), stress-induced phosphoinositide biosynthesis can clearly be distinguished as increases in PtdIns4P and PtdIns(4,5)P2 that follow the initial decline (Figure 1B and 1C).
In non-damaged cells, hydrolysis of PtdIns(4,5)P2 for InsP3 production must be accompanied by increased flux through the phosphoinositide pathway (König et al., 2007), likely contributing to transient decreases in PtdIns and the lipid intermediates, PtdIns4P and PtdIns(4,5)P2, during the first 15 min of stimulation (Figure 1). PtdIns from non-stressed plants was highly unsaturated, whereas PtdIns4P and PtdIns(4,5)P2 had different fatty acid compositions from PtdIns, and were more saturated (compare Figure 1B–1D, zero time points). The apparent incongruence of fatty acid patterns between PtdIns and polyphosphoinositides was previously observed in studies on the molecular composition of PtdIns, PtdIns4P, and PtdIns(4,5)P2 from both the animal (Augert et al., 1989) and plant fields (König et al., 2007), and it was proposed that unsaturated PtdIns accumulating in non-stressed plants is ‘primed’ for stress-inducible phosphorylation and distribution into a hydrolysable polyphosphoinositide pool required for InsP3 production (König et al., 2007). In the present study, application of the wounding stress resulted in transiently reduced unsaturation of PtdIns (Figure 1D) concomitant with increased unsaturation of PtdIns4P (Figure 1C) and PtdIns(4,5)P2 (Figure 1B), suggesting that, in the wounded plants, unsaturated PtdIns was turned over into polyphosphoinositides.
The data so far indicated that phosphoinositides and InsP3 signals are generated upon wounding in Arabidopsis leaves. Based on this information, it was the aim of this work to elucidate what biochemical messengers act upstream and downstream of phosphoinositides and InsP3 in wound signaling. Wounding-induced increases in JA and InsP3 in wild-type plants were compared with those in transgenic plants deficient in either JA biosynthesis or InsP3 accumulation (Figure 2). In dde2-2 plants deficient in AOS activity, which do not produce JA, OPDA, or dn-OPDA, wounding did not affect the levels of InsP3 (Figure 2B, squares). This result indicates that InsP3 acts in a linear signaling pathway downstream of wounding-induced AOS oxylipin products. In order to directly test the effects of JA on InsP3 production, methyl-JA was exogenously applied, but failed to affect increased InsP3 levels in Arabidopsis rosette leaves (data not shown). Differences between the modes of action of endogenous and exogenously applied JA have previously been proposed (Löbler and Lee, 1998; Stenzel et al., 2003), and it has been demonstrated that uptake of methyl-JA is not achieved in some plant species with exogenous application of the compound (Bucking et al., 2004). Our subsequent experiments demonstrated increased levels of InsP3 in Arabidopsis rosettes treated with sorbitol (Figure 3), suggesting that InsP3 may have been formed upon release of endogenous JA (Löbler and Lee, 1998; Stenzel et al., 2003), which is consistent with the data from mutant analyses shown in Figure 2.
A signaling component affected by both JA and InsP3 is Ca2+ (Leon et al., 1998; Berridge, 2005), and previous studies have suggested that InsP3 may take part in mediating wounding-induced increases in intracellular Ca2+ levels (Knight et al., 1993; Leon et al., 1998). While the lack of AOS activity prevented wounding-induced InsP3 formation early in the cascade (Figure 2B, squares), attenuated InsP3 accumulation reciprocally resulted in attenuation of the late JA increase observed in wild-type plants (Figure 2A, compare triangles and circles). It has been discussed that, in tomato, Ca2+ can trigger the release of fatty acid substrates for JA production from membrane lipids by activation of phospholipase A2 (Conconi et al., 1996; Lee et al., 1997; Narvaez-Vasquez et al., 1999). Similarly, eicosanoid biosynthesis can be triggered in mammalian cells by Ca2+-dependent activation of phospholipases, which release fatty acid substrates, and of a LOX enzyme, converting the fatty acids to hydroperoxy fatty acids (Lotzer et al., 2005). Unfortunately, nothing is known so far on the effect of Ca2+ on enzymes involved in JA biosynthesis like phospholipases, LOX, AOS, or allene oxide cyclase. In accordance with the mammalian models, Ca2+ signals mediated by sustained InsP3 increases may underlie the continuous JA formation at late time points after wounding. As InsP3 can, in contrast to Ca2+, cross plasmodesmatal connections between plant cells (Tucker and Boss, 1996), an InsP3 signal would be capable of eliciting and synchronizing Ca2+ increases and subsequent JA production in non-wounded cells of an affected tissue. Early JA release may, thus, occur in the wounded tissue, whereas late JA increases may reflect de novo JA biosynthesis in adjacent non-wounded tissues, triggered by InsP3 distribution. Although the precise identity and mode of release of the initial oxylipin signal are not certain, the dependency between JA and InsP3 clearly indicates cross-talk between oxylipins and phosphoinositides in the wounding response of Arabidopsis.
Downstream of the initial biochemical signals, physiological responses to wounding are manifested by the expression of wound-inducible genes (Leon et al., 2001). In InsP 5-ptase plants with reduced phosphoinositide and InsP3 levels, wounding-induced accumulation of transcripts for various genes was attenuated compared to that in wild-type (Figure 5), suggesting a role for InsP3 in wound-induced gene expression. Reduced wound induction in InsP 5-ptase plants was observed with genes both dependent on COI1 for induction (e.g. AOS or VSP1) and for some independent of COI1 (e.g. RNS1 or OPR1), suggesting that phosphoinositides may act both in dependence or independently of JA signals. Reduced AOS induction in InsP 5-ptase plants (Figure 5) correlates well with possibly decreased JA production at late time points (Figure 2A, triangles). While wounding-induced gene expression is overall attenuated in InsP 5-ptase plants compared with wild-type plants, different temporal patterns of attenuation can be observed. The observation that some genes are attenuated early (AOS) and some late (RNS1), while other genes show no attenuation but are delayed in wound induction, suggests a complex network of phosphoinositide-dependent mechanisms for the regulation of gene expression. This interpretation is consistent with the biochemical data suggesting that phosphoinositides may influence parallel phytohormone signaling pathways, including JA (Figure 2) and IAA (Figure 4). The clear attenuation in InsP 5-ptase plants of the expression of the WRKY70 transcription factor implicated in defense signaling (Li et al., 2006) suggests effects of phosphoinositides on the induction of cis-regulatory elements. An example for directly phosphoinositide-dependent regulation gene expression is presented by the mammalian transcription factor TUBBY, which is attached to the plasma membrane in the presence of PtdIns(4,5)P2 and moves to the nucleus upon PtdIns(4,5)P2 hydrolysis (Santagata et al., 2001). Future studies will be directed towards the identification of equivalent mechanisms in Arabidopsis.
In order to circumvent the complexity presented by the so far unresolved phosphoinositide-dependent gene regulation, we aimed to investigate a more immediate way to assess the role of phosphoinositide signals in the mediation of plant defense responses. Previous studies have indicated that wounding induces the production of defensins that affect caterpillar digestion of plant material and, indirectly, caterpillar development (Kessler et al., 2004; Chen et al., 2005; Liu et al., 2005). Oviposition and larval development of insects feeding on plants compromised in the JA-signaling pathway have previously been demonstrated to be increased and faster, respectively, than was observed with feeding on wild-type plants (Kessler et al., 2004; Chen et al., 2005). Thus, effects of attenuated phosphoinositide signaling on the defensive capabilities of Arabidopsis plants against herbivory were tested in caterpillar performance assays. Caterpillar development with feeding on InsP 5-ptase plants was significantly faster than that on wild-type plants and resembled that observed with feeding on dde2-2 plants (Figure 6, Supplemental Table 1), indicating that phosphoinositide signals are of similar importance for the defensive capability of Arabidopsis leaves against herbivory as JA. The outcome of the caterpillar feeding tests is consistent with the notion that phosphoinositides and InsP3 act in the same signaling cascade as JA, as was concluded based on biochemical data (Figure 2). The increased growth rate of caterpillars feeding on InsP 5-ptase plants compared to that of those feeding on wild type-plants (Supplemental Table 1) indicates ecophysiological relevance of phosphoinositide signals in the mediation of wound-induced defense mechanisms of plants.
While an involvement of inositolpolyphosphates and phosphoinositides in Arabidopsis defense signaling can be deduced from results presented here, the mechanisms of action remain to be elucidated. From recent studies, it is clear that inositolhexakisphosphate, InsP6, is present as a cofactor in the IAA-binding site of the IAA receptor, TIR1 (Tan et al., 2007), and may be involved in the function of TIR1 and IAA sensitivity. The results on TIR1 lead us to speculate that a role for InsP3 in plant signal transduction alternative to Ca2+ signaling may be that of a precursor for InsP6 production required for phytohormone receptor function. In support of this concept, it has previously been shown that InsP3 is generated in response to gravistimulation in maize and oat pulvini (Perera et al., 1999, 2001), and this InsP3 production is required for IAA-dependent asymmetric growth of the pulvini and gravitropic curvature (Perera et al., 2001). During gravitropic signaling, InsP3-production precedes IAA redistribution (Perera et al., 2001), consistent with a role for InsP3 in the generation of InsP6 and TIR1 receptor function. InsP 5-ptase plants used in this study have previously been demonstrated to exhibit reduced gravitropic curvature responses (Perera et al., 2006)— an observation in line with reduced sensitivity to IAA in the absence of inositolpolyphosphates. In response to wounding, we detected elevated IAA levels and increased IAA production in InsP 5-ptase plants over the levels in wild-type plants (Figure 4). Following the hypothesis that inositolpolyphosphates may be required for TIR1 functionality, a possible explanation for this observation would be compensatory overproduction of IAA as a result of compromised IAA sensitivity in the InsP 5-ptase plants. As the data presented in this study do not pertain to IAA- or JA-receptor functionality, the exact role of inositolpolyphosphates in plant signaling must remain speculative at this time. It is interesting to note that plants compromised in enzyme activities phosphorylating InsP3 to InsP6 show reduced growth (Stevenson-Paulik et al., 2005), possibly due to IAA insensitivity in the absence of InsP6. Whereas, so far, no immediate information is available about an association of inositolpolyphosphates with the JA-receptor complex, it may well be envisioned that it employs a mode of action similar to that of the closely related TIR1 and involves InsP6 in JA binding.
In summary, the data presented in this study provide evidence that InsP3 signals occur downstream of oxylipin signals early in the wound-signaling cascade. Artificial reduction of phosphoinositide levels in InsP 5-ptase plants resulted in attenuated wound induction of various defense-related genes, suggesting a role for phosphoinositides in the regulation of defense gene expression. Growth of herbivorous caterpillars was increased on plants with attenuated phosphoinositide signaling. The results presented establish an involvement of the phosphoinositide system in signal-transduction events leading to the induction of defense responses after mechanical wounding of Arabidopsis leaves. Future experiments will show what signaling pathways exhibit cross-talk with the phosphoinositide system and may help to further elucidate the complex signaling network mediating plant stress responses or plant development.
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Plant Growth and Stress Treatments
The following plant lines were used in the experiments described: wild-type, Arabidopsis thaliana (L.) ecotype columbia-0; dde2-2 (von Malek et al., 2002); InsP 5-ptase (Perera et al., 2006). Plants were grown on soil in growth chambers (York, Mannheim) at 22–25°C under a regime of 8 h exposure to 130–150 µmol photons m–1 s–1 and 16 h darkness and at approximately 60% humidity. Rosette leaves of 7–8 week old plants (6 weeks for plants used to obtain data shown in Figure 1) were mechanically wounded using forceps, as described (Stenzel et al., 2003). Whole rosettes were harvested at different time points and immediately frozen in liquid N2. To account for biological variation, rosettes of six to ten plants were pooled for each time point. External application of methyl-JA was carried out by carefully cutting whole rosettes of 6 week old plants and placing on 100 ml of H2O or H2O containing 5 µM methyl-JA (Sigma) in glass petri dishes, as described (Stenzel et al., 2003). For sorbitol treatments, Arabidopsis plants were grown under sterile conditions in sealed jars on 0.5% Murashige and Skoog medium including modified vitamins (Duchefa) containing 1% (w/w) sucrose and 0.25% (w/w) Gelrite (Roth). After 14 d, plants were transferred to hydroponic cultures in liquid media, as described (Randall and Bouma, 1973). Hydroponic cultures were exposed to 140 µmol photons m–2 s–1 of light in an 8-h light and 16-h dark regime and continuously aerated. Eight to 10 week old plants were treated by adding a final concentration of 0.8 M sorbitol to the hydroponic media. In all experiments, non-stimulated controls were harvested at random time points and were found not to differ significantly in metabolite content over the 6-h duration of an experiment (data not shown). Care was taken to minimize perturbation of plants prior to and during treatments. Plant material was stored at –80°C.
Analysis of InsP3 and Phosphoinositides
Plant material was ground under liquid nitrogen to a fine powder. InsP3 levels were determined from ground powder using the [3H]InsP3 receptor binding assay system (GE Healthcare), as previously described (Perera et al., 1999). Polyphosphoinositides were extracted from powdered plant material by using an acidic extraction protocol (Cho et al., 1992). Lipids were separated by thin-layer chromatography (TLC) on silica gel plates (Merck) using developing solvents for optimal resolution: for phosphoinositides and PtdOH, CHCl3:CH3OH:NH4OH:H2O (57:50:4:11(v/v/v/v)) (Perera et al., 2005); for PtdCho, acetone:toluol:water (91:30:7 (v/v/v)) (Hartel et al., 2000); for isolating phosphatidylinositol, CHCl3:methyl acetate:isopropanol:CH3OH:0.25% aqueous potassium chloride (25:25:25:10:9 (v/v/v/v/v)) (Christie, 2003). Lanes with lipid standards (5 µg) run in parallel to biological samples were cut and lipids visualized in aqueous 10% (w/w) CuSO4 (Sigma) containing 8% H3PO4 (Sigma) and subsequent heating to 180°C. Unstained lipids were located on the remaining parts of the TLC plates according to standard migration, were scraped, re-dissolved in their respective developing solvents and dried under N2 flow. Lipids were transmethylated (Hornung et al., 2002), fatty acid methyl esters dissolved in acetonitrile and analyzed using a GC6890 gas chromatograph with flame ionization detection (Agilent) fitted with a 30 x 250 µm DB-23 capillary column (Agilent). Helium flowed as a carrier gas at 1 ml min–1. Samples were injected at 220°C. After 1 min at 150°C, the oven temperature was raised to 200°C at a rate of 8°C min–1, then to 250°C at 25°C min–1, and then kept at 250°C for 6 min (König et al., 2007). Fatty acids were identified according to authentic standards and by their characteristic mass spectrometric fragmentation patterns (data not shown), and quantified according to internal tri-pentadecanoic acid standards of known concentration. Due to limiting material in samples representing isolated minor lipids, fatty acids of low abundance may be absent from fatty acid patterns.
Analysis of JA, IAA, and SA Contents
Plant material was extracted as described (Schmelz et al., 2004), but with some modifications. Frozen ground plant material (50 mg) was mixed with 1 ml diisopropylamine containing 100 ng of D6-JA (kindly provided by Dr O. Miersch, Halle), 50 ng of D5-IAA (Eurisotop), or 50 ng of D5-SA (Icon Genetics) as internal standards. The mixture was sonified for 15 min, 1 ml of chloroform was added followed by sonification for 15 min. For detection, compounds were converted to their pentafluorobenzyl esters according to Mueller and Brodschelm (1994) by adding 17 mg pentafluorbenzylbromide (Sigma) and incubating for 1 h at 60°C. After evaporation under streaming nitrogen, residues were dissolved in 1 ml diethyl ether and filtrated through filter paper. For complete recovery of the pentafluorobenzyl esters, the sample tube was washed with 1 ml n-hexane, which was also filtered and combined with the diethyl ether filtrate. The filtered solution was evaporated under a stream of nitrogen. The vapor phase extraction was carried out at 270°C for 5 min, with argon as a carrier gas. Vaporized substances absorbed by the SuperQ column (100 x 4.6 mm SDB-L Strata, pore size 260 Å; Phenomenex) were eluted from the SuperQ columns subsequently with 3 ml n-hexane and 3 ml ethyl acetate. The solution was concentrated with a rotating evaporator. The remainder was dissolved in 40 µl dichlormethane and subjected to gas chromatography coupled to mass spectrometry.
The analysis was carried out using a ThermoFinnigan (Austin, Texas, USA) Polaris Q mass selective detector connected to ThermoFinnigan Trace gas chromatograph equipped with a capillary Rtx-5MS column (15 x 0.25 mm, 0.25 µm coating thickness; Resteck, Bad Homburg). Helium was used as carrier gas at a flow rate of 1 ml min–1. The temperature gradient was 100°C for 1 min, 100–300°C at 8°C min–1 and 300°C for 5 min. The phytohormone derivates were detected by negative chemical ionization, with ammonia as ionization gas. For quantification, the diagnostic ions m/z 215 (D6-JA; Rf = 16.15, 16.51 min), 209 (JA; Rf = 16.21, 16.56 min), 179 (D5-IAA; Rf = 17.78 min), 174 (IAA; Rf = 17.82 min), 141(D5-SA; Rf = 11.15 min), and 137 (SA; Rf = 11.18 min) were used.
Determination of Specific Transcript Levels
The levels of specific transcripts in rosette leaves were determined by real-time RT-PCR analysis of cDNA reverse-transcribed from 1 µg of total RNA using 100 U Reverse Transcriptase H– (MBI Fermentas), according to the manufacturer's instructions. The resulting cDNA was diluted 1:10 and 1 µl was used as a PCR template in a 25-µl reaction containing 2.5 µl 10 PCR-buffer (Bioline), 2 mM MgCl2, 100 µM dNTPs, 2.5 µl QuantiTect Primer-Mix (Qiagen), 0.1-fold Sybr green, 10 nM fluorescein, and 0.25 U BioTaq (Bioline). Samples were denatured for 3 min at 95°C, followed by 40 cycles of 20 s denaturation at 95°C, 20 s of annealing at 55°C and 40 s of elongation at 72°C. Fluorescence was monitored during each annealing and denaturation phase. The program was concluded by 4 min of elongation at 72°C and 1 min of denaturation at 95°C. Renaturing of amplified DNA was followed by the assessment of melting parameters by increasing the temperature in 0.5°C increments while monitoring fluorescence. In addition to the individual QuantiTect primers used, a DNA fragment of the PP2A subunit of PDF2 (At1g13320) was used as an internal reference. Transcript levels were calculated according to Livak and Schmittgen (2001). It should be noted that basal transcript levels were not always equal in wild-type and InsP 5-ptase plants. For easier comparison, data given in Figure 5 were normalized against transcript levels detected in non-stressed plants (set as 1).
Caterpillar Performance Tests
Diamondback-moth (Plutella xylostella) caterpillars of approximately 1 mg each were collected 3 d after oviposition, and 10–12 individuals were placed on the leaf surface of 5 week old Arabidopsis plants (wild-type, InsP 5-ptase, or dde2-2). For the duration of the experiment, plants were separated in closed containers, allowing gas exchange and exposition to 80–100 µmol photons m–1 s–1 in a light–dark regime of 12 h light and 12 h darkness. After the times indicated, caterpillars were collected and weighed. After weighing, caterpillars were allowed to continue feeding on the respective plants, in order to monitor a continuous defensive response. Care was taken not to harm caterpillars during the weighing process; individuals damaged during collection were eliminated from subsequent statistical evaluation.
Accession Numbers
Sequences used in this study can be identified by the following gene locus identifiers: AOS, At5g42650; OPR1, At1g76680; VSP1, At5g24780; RNS1, At2g02990; T18K17.7, At1g73260; WRKY70, At3g56400.
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Supplementary Data are available at www.mplant.oxfordjournals.org
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Acknowledgements |
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We would like to thank Dr Gregg Howe (Michigan State University, E. Lansing, MI, USA) for helpful comments. The D6-JA standard was a gift from Dr Otto Miersch (Institute for Plant Biochemistry, Halle, Germany). Drs Ellen Hornung and Michael Stumpe (Georg-August-University Göttingen, Germany) donated cDNA clones for probe amplification. The transgenic Arabidopsis plants expressing the human type I inositolpolyphosphate 5-phosphatase were kindly provided by Dr Imara Perera (North Carolina State University, Raleigh, NC, USA). We are grateful to Dr Christiane Gatz (Georg-August-University Göttingen, Germany) for the opportunity to perform the real-time RT-PCR analyses and especially Ms Katrin Gärtner for her kind support with these measurements. The diamondback moths were a gift from Dr Stefan Vidal (Georg-August-University Göttingen, Germany); we thank Ms Dorothea Mennerich for help rearing the caterpillars. This work was funded by the German Research Foundation (Emmy Noether grant He3424/1-3 to I.H.).
No conflict of interest declared.
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