A requirement for calcium and protein phosphatase in the jasmonate-induced increase in tobacco leaf acid phosphatase specific activity

Journal of Experimental Botany, Vol. 50, No. 337, pp. 1331–1341, August 1999

A requirement for calcium and protein phosphatase inthe jasmonate-induced increase in tobacco leaf acidphosphatase specific activity

Paul Kenton, Luis A.J. Mur and John Draper1

Institute of Biological Sciences, University of Wales—Aberystwyth, Aberystwyth, UK

Received 8 September 1998; Accepted 6 April 1999

Abstract insect damage in a number species (Creelman et al., 1992;Albrecht et al., 1993; Pena-Cortes et al., 1993; Blechert

Jasmonic acid (JA) is an established wound signal andet al., 1995; Doares et al., 1995; Sano et al., 1996) and

also plays a role in plant–pathogen interactions. several wounding-related genes are induced by exogenousApplication of JA to tobacco leaf explants, tobacco

JA or its precursors (Mason and Mullet, 1990; Farmerseedlings or to intact leaves via the petiole resulted

et al., 1992; Bell and Mullet, 1993; Ellard-Ivey andin an increase in the specific activity of acid phosphat- Douglas, 1996). Recent data suggest that JA is a memberase (AP) and a reduction in overall protein content. of a family of related biologically active cyclopentanonesSimilar changes in AP activity were observed in which have been termed the ‘jasmonate signal complex’wounded tissue and in tissue undergoing a hypersens- (Farmer et al., 1998). This complex includes 12-oxo-itive response (HR) following infiltration with avirulent phytodienoic acid (PDA) which is a precursor of JA.bacteria. The AP activity increase was restricted to Evidence is accumulating that PDA may, in fact, be thewounded tissue and HR lesions and was absent from principal jasmonate signal since it accumulates to higherunwounded or uninfiltrated tissue on the same leaf. levels than JA following wounding and, unlike JA, isThe JA response (AP increase and protein loss secreted from elicited cell suspension cultures (Parchmanncombined) was investigated pharmacologically. et al., 1997).Co-incubation with EGTA, ruthenium red, LaCl

3and JA also plays a role in plant–pathogen interactions.

(±)-verapamil blocked the JA response suggesting a Thus, elicitor-treated suspension cell cultures exhibitrequirement for Ca2+ mobilization. Similarly, okadaic increases in JA (Gundlach et al., 1992; Blechert et al.,acid, cantharidin and microcystin LR abrogated the 1995; Nojiri et al., 1996). However, the role of JAresponse to JA implicating a protein phosphatase in following in planta pathogen challenge is unclear. In thethe JA signal transduction mechanism(s). No evidence barley–powdery mildew interaction the incompatiblewas found for kinase involvement as a mediator of JA response is not associated with an increase in endogenoussignalling in this system. JA nor with expression of a JA-inducible protein (JIP),

JIP23 (Kogel et al., 1995; Hause et al., 1996). In contrast,Key words: Jasmonate, signalling, wounding, patho- infection of Arabidopsis with Alternatira brassicicolagenesis. induces both local and systemic JA accumulation

(Penninckx et al., 1996). In addition, a HR-specific risein (–)-JA in Pseudomonas syringae pathovar phaseolicolaIntroduction(P. s. pv. phaseolicola)-infected tobacco has been observed

JA, a derivative of linolenic acid (LA) synthesized via ( Kenton et al., 1999). Recently it has been shown thatthe octadecanoid pathway (Vick and Zimmerman, 1984; Arabidopsis mutants compromised in their ability to syn-Farmer and Ryan, 1992; Sembdner and Parthier, 1993; thesize or respond to JA are rendered susceptible to soilFarmer, 1994), is an established local signal in plant fungi of the genus Pythium (Staswick et al., 1998; Vijayan

et al., 1998).wound responses. JA levels rise following wounding or

1 To whom correspondence should be addressed. Fax: +44 1970 622350. E-mail: [email protected]

© Oxford University Press 1999

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LaCl3, and ruthenium red were obtained from Sigma (Poole,The JA- and wound-inducible soybean vegetative stor-UK).age proteins (VSP) VspA and VspB and the Arabidopsis

VSP Atvsp show homology to AP (Mason and Mullet, Plants1990; Berger et al., 1995). Several reports have noted an Unless stated otherwise, experiments were conducted on matureincrease in phosphatase activity following different treat- greenhouse-grown tobacco (cultivar SR1)ments. Thus, AP activity rises in de-podded soybean

Treatmentsplants, the increase occurring over several weeks (DeWaldet al., 1992; Staswick et al., 1994). The increase involves Leaf disc experiments were conducted using 1.7 cm diameter

leaf cores floated on 2 ml water containing the compound(s)both VSP and a low abundance 51 kDa enzyme (DeWaldbeing tested. In all cases where reagents were solubilized in anet al., 1992; Staswick et al., 1994). In addition, it hasorganic carrier (i.e. ethanol or DMSO), carrier concentrationbeen noted that there was an increase in histochemical was kept constant for all samples including controls. Unless

staining for AP following lysosomal disruption during stated in figure legends, incubations were conducted for 3 d atthe HR of potato infected with Phytophthora infestans 24 °C with an 18 h photoperiod. Experiments using ionomycin

were carried out in 20 mM MOPS buffer (pH 7.0) rather than(Jordan and DeVay, 1990). A large number of plant APswater in order to stabilize the Ca2+–ionomycin complex.have been described, many with little identifiable substrate

specificity. It has been suggested that these unspecialized Pathogens and infectionsenzymes are involved in phosphate scavenging (reviewed

Pathogen culture and infection techniques were carried out asby Duff et al., 1994). Thus, release of vacuolar AP during previously described (Mur et al., 1996)senescence or HR may result in dephosphorylation of a

AP assaywide range of phosphorylated compounds with the sub-sequent release and export of Pi from dying tissues. Leaf discs or cotyledons, stored at −70 °C until required, were

extracted in 400 ml of ice-cold 25 mM Tris-HCl (pH 7.5), 1 mMLittle is known about the downstream signalling eventsEDTA, 5 mM dithiothreitol, 0.2 mM PMSF (extraction buffer;activated by JA. Recent data obtained in potato implic-Staswick et al., 1994) in a 1.5 ml Eppendorf tube using a micro-ates an okadaic acid-sensitive protein phosphatase (PP) homogenizer (Biomedix, Pinner, UK). The extracts were

in the transduction of the JA signal to leaf pin2 centrifuged for 1 min at 11 600 g. Protein was determined by(Dammann et al., 1997). In contrast, this pathway does the method of Bradford (Bradford, 1976). Where possible,

extracts were diluted to 0.05–0.1 mg ml−1 in extraction buffer,not appear to operate in potato roots where JA activateshowever, in some cases, extracts had to be assayed atexpression of a root-specific pin2. In Arabidopsis, JA andconcentrations of less than 0.05 mg ml−1. 50 ml of extract werewounding induce expression of a number of genes via a incubated with 50 ml of p-nitrophenyl phosphate ( pNPP—

PP-dependent pathway. However, Arabidopsis also pos- 1 mg ml−1, Sigma, Poole, UK) in 0.1 M NaCl, 5 mM MgCl2.sesses a separate JA-independent wounding pathway regu- After 5–10 min the assay was stopped by the addition of 100 mlof 6 M NaOH and the absorbance read at 405 nm using alated by a kinase (Rojo et al., 1998).Dynatech MR5000 plate reader. Since some extracts developedIn the present report it is shown that tobacco leaf APa strong yellow colour on addition of NaOH, a pNPP-freespecific activity is elevated and extractable protein content incubation was included as background for each incubation;

reduced following exogenous JA application and, in this value was subtracted from the value obtained with pNPP.wounded and infected tissue where endogenous JA accu- A standard curve was prepared using p-nitrophenol. The rate

of change of absorbance was linear with time for incubationsmulation occurs (Kenton et al., 1999), it is further shownof up to at least 12 min for both control- and JA-treatedthat the JA-induced increase in AP specific activity issamples. The unit definition used is: 1 unit AP=1 mmol pNPPblocked by inhibitors of PP and Ca2+ mobilization. hydrolysed min−1. Although the tobacco leaf AP activity has apH optimum of 5.5, assays were conducted at pH 7.5 becauseof high background (i.e. pNPP-free) development under acidconditions.

Materials and methodsSDS-PAGEReagentsElectrophoresis was carried out as previously described ( WarnerStaurosporine (soluble in DMSO) and mastoparan wereet al., 1993) using a BioRad ‘Mini-Protean II’ system.obtained from Alexis Corp. (Nottingham, UK). C2 ceramide,

C16 ceramide, cypermethrin, (-)-indolactam V, microcystin LR,nifedipine, okadaic acid, okadaol, U73122, U73343 (soluble in Resultsethanol ), concanamycin, dephostatin, ionomycin, herbimycin,K252a (soluble in DMSO), ouabain, suramin, and neomycin JA induces an increase in AP specific activity and proteinsulphate were obtained from Calbiochem-Novabiochem

loss(Nottingham, UK). Lithium chloride was obtained from FisherScientific UK (Loughborough, UK). Cantharidin, (±)-JA, Exogenously applied JA induced a dose-dependentLA, manoalide, phorbol-12,13-dibutyrate, phorbol-12,13-dide-

increase in a readily assayable pNPP-hydrolysing specificcanoate, (±)-verapamil, wortmannin (soluble in ethanol ),activity in leaf explants (Fig. 1a). In crude extracts thisA23187, anisomycin, forskolin, genistein, quercetin (soluble in

DMSO), compound 48/80, dibutyryl-cAMP, dibutyryl c-GMP, activity (in both control and JA-treated tissue) exhibited


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Jasmonate and acid phosphatase 1333

Fig. 1. AP specific activity and protein content of JA-treated tobacco leaf tissue. (a) Leaf discs were incubated for 3 d on increasing concentrationsof JA. n=3. (b) Seedlings of two tobacco cultivars (Samsun and SR1) from which one cotyledon had been removed (D 0) were floated for 3 d oncarrier alone (Control ) or 200 mM JA (JA) after which time the remaining cotyledon was sampled (D 3). n=3 (5 seedlings per replicate). (c)Tobacco leaves were detached and fed with carrier (Control ) or 200 mM JA (JA) via the cut petiole. n=3. (d) Leaf discs were floated on 200 mMJA for the indicated time after which the discs were rinsed and transferred to water for the remainder of the 72 h period. n=3. (e) Leaf discs wereincubated on increasing concentrations of JA for 3 d. n=3. (f ) Protein extracted from leaf discs treated with carrier (C), 100 mM JA (J), 400 mMLA (L) or from discs taken from tissue undergoing HR induced by P. s. pv. syringae (I ) or surrounding uninfected tissue on the same leaf (S) wasseparated on SDS-PAGE. 7 mg protein per lane. Figures below each lane indicate AP activity and protein contents as a % of carrier control ( lanesJ and L) or uninfected tissue (I ). The gel was stained with Coomassie Brilliant Blue. The sizes of molecular weight markers (M ) are indicated onthe right and are (from the top) 66, 45, 36, 29, 24, 20, and 14.2 kDa. (g) Leaf discs were treated with carrier (control ), 100 mM JA (JA), 50 mMcyclohexamide (Cy) or 100 mM JA together with 50 mM cyclohexamide (JA+Cy). n=6. Means and standard errors (where larger than symbolsused) are shown except in (f ) which shows individual values.

a pH optimum of 5.5 and was sensitive to a similar range number of prominent constitutive bands were seen todecrease following JA treatment whilst an 18.5 kDa com-of inhibitors (e.g. molybdate and orthovanadate) as

soybean AP (DeWald et al., 1992; Staswick et al., 1994). ponent was enriched (Fig. 1f, compare lanes JA and C).This 18.5 kDa band showed no activity on phosphataseJA also induced an AP activity increase in whole tobacco

seedlings and petiole-fed leaves (Fig. 1b, c). Although the activity gels (data not shown).As predicted by the biosynthetic model for JA synthesisincrease in AP activity was not detectable for 2 d following

treatment (Fig. 1c), the presence of JA in the incubation (Vick and Zimmerman, 1984; Farmer and Ryan, 1992),LA also induced an increase in AP activity, protein lossmedium was only required for 6 h before tissue became

committed to the subsequent AP activity increase and the appearance of an 18.5 kDa component (Fig. 1f,compare lane LA with lanes C and JA). Arachidonic(Fig. 1d). The naturally occuring JA stereoisomer, (–)-JA,

also induced AP activity increases in leaf discs (data acid, an inducer of salicylate synthesis and resistance inpotato to Phytophthora infestans and Alternaria solaninot shown).

As well as inducing an AP specific activity increase, JA (Coquoz et al., 1995), failed to induce an AP increase orprotein loss (data not shown). A number of inducers ofstimulated a concomitant loss in overall protein content

(Fig. 1e). This combined AP activity increase and protein JIP synthesis in other species (Hildmann et al., 1992;Sembdner and Parthier, 1993), e.g. ABA, mannitol, suc-loss were taken together as a marker for the JA response

in all subsequent experiments. On SDS-PAGE gels a rose, and dessication (data not shown) induced protein


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loss without an attendant increase in AP activity. Despite and Ca2+ ionophores were tested in relation to the JAresponse. EGTA alone inhibited the JA response onlythe overall loss of protein, both protein loss and APslightly, however, in combination with either A23187 orincrease were sensitive to cyclohexamide suggesting aionomycin, substantially inhibited the JA responserequirement for protein synthesis (Fig. 1g).(Fig. 3a). However, neither Ca2+ alone nor Ca2+ witheither of the ionophores were capable of mimicking JA.AP specific activity is elevated in wounded and infectedNeither EGTA alone, nor EGTA in combination withtissueionophores had any marked effect on either AP activity

In accompanying studies increases in JA in tobacco which or protein levels in control (i.e. not treated with JA)had been mechanically wounded or which was undergoing explants. Increasing concentrations of EGTA in iono-HR induced by P. s. pv. phaseolicola were measured phore-containing incubation medium gave a dose-(Kenton et al., 1999). These JA increases were restricted dependent reversal of the JA response (Fig. 3b, c). Into wounded tissue or HR lesions and absent from sur- crude extracts AP in either control- or JA-treated tissuerounding unwounded or uninfected tissue on the same is insensitive to Ca2+, Mg2+, EDTA or EGTA (dataleaf. Wounding of tobacco induced a gradual increase in not shown).AP activity along with an accompanying loss of protein These data suggest that JA signalling requires Ca2+(Fig. 2a). This increase was restricted to tissue cores taken mobilization. To test this hypothesis further, a numberat, and including, wound sites and absent from of putative Ca2+ channel blockers were used to try tounwounded tissue on the same leaf (Fig. 2b). Similarly, intefere with induction of the JA response. RutheniumAP increase and protein loss correlated with sites of JA red, an inhibitor of mitochondrial channels (Campbell,synthesis in pathogen-challenged tobacco. Thus HR 1983), effectively blocked the JA response at 1 mMlesions developing following infection of tobacco with (Fig. 3d) whereas La3+, a plasma membrane channelP. s. pv. syringae and P. s. pv. phaseolicola exhibited blocker, and nifedipine, an L-type channel blocker, wereelevated AP activity and protein loss when compared only effective at higher concentrations of 0.5–1.0 mM andwith surrounding tissue (Fig. 2c). At 24–48 h post infil- 0.25–0.5 mM, respectively (Fig. 3e, f ). (±)-Verapamil,tration (p.i.), neither heat-treated bacteria, a HR-non- another L-type channel blocker, did not affect the JAinducing mutant of P. s. pv. phaseolicola (hrpL) nor buffer response over the concentration range tested (Fig. 3g).induced either an AP activity increase or protein loss. AP These data suggest that activation of one or more Ca2+increase occurred rapidly with P. s. pv. phaseolicola and channels is an integral part of the JA response.was apparent 12–18 h p.i. (Fig. 2d). In contrast, APactivity remains constant following hrpL infiltration until JA induction of AP increase and protein loss is dependent72 h p.i. at which point AP activity was observed to rise. on protein dephosphorylationProtein loss after P. s. pv. phaseolicola and hrpL infection

Since Ca2+ alone appeared to be incapable of mimickingfollowed essentially the same time-course as the APJA action, other components of a possibly bifurcatingactivity increase ( Kenton et al., 1999).signalling mechanism were sought. Protein phosphoryl-ation and dephosphorylation regulate a number of plantJA induction of AP increase and protein loss is dependentsignalling pathways (Chandra and Low, 1995; Ohta andon calcium mobilizationNakamura, 1995; Baskin and Wilson, 1997; Frohmeyer

The ability of JA to induce an AP increase associated et al., 1997; Hey et al., 1997). The PP1 and PP2a inhibitorwith protein loss provides a potentially useful tool for okadaic acid partially blocked JA induction of the APstudying JA signalling in tobacco. The dual response of activity increase but had only a small effect on reversingAP increase together with protein loss (henceforth termed protein loss (Fig. 4a). In contrast the inactive analoguethe ‘JA response’) also provides a useful cross-check to of okadaic acid, okadaol, did not affect the JA responseensure that any effects observed using modulators of (Fig. 4b). Methyl okadaate, another inactive okadaic acidsignalling pathways are not due simply to direct inhibition analogue, gave results similar to those obtained withof AP. Thus, it would be anticipated that agents which okadaol (data not shown). Microcystin LR and can-block JA signalling would reverse both the AP activity tharidin, both PP1 and PP2a inhibitors proved far moreincrease and protein loss and that activators of the JA effective in blocking the JA response particularly withsignalling pathway would induce an AP activity increase respect to protein loss (Fig. 4c, d). Neither okadaic acid,together with protein loss in the absence of JA. microcystin LR nor cantharidin were observed to inhibit

Calcium mobilization is a characteristic component of AP in crude extracts (data not shown). Cypermethrin, aa large number of plant cellular signalling pathways PP2b inhibitor, and dephostatin, a tyrosine PP inhibitor(Knight et al., 1996; Chandra and Low, 1997; Gelli et both failed to block the JA response (data not shown).al., 1997; Neuhaus et al., 1997). Several combinations of These data suggest that activation of a PP is also required

for JA signalling.a membrane-impermeable Ca2+ chelator (EGTA), Ca2+


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Fig. 2. AP activity in wounded and infected tobacco leaf tissue. (a) Leaves were crushed with forceps and samples taken at the times indicated.Samples (1.7 cm diameter discs) consisted of the wound site and immediately adjacent tissue. n=12. (b) Tobacco leaves were crushed with forcepsand 1.7 cm diameter discs taken so as to include the wound site and immediately adjacent tissue (Wound) or unwounded tissue on the same leaf(Unwounded). n=6. (c) Tobacco leaves were infiltrated with P. s. pv. syringae (P. s. pv. syringae), P. s. pv. syringae treated for 10 min at 100 °C(Heat), P. s. pv. phaseolicola (P. s. pv. phaseolicola), a hrpL mutant of P. s. pv. phaseolicola strain 143 (hrpL) or buffer (Buffer). All bacteria wereinfiltrated at a concentration of 2×108 ml−1. Tissue was sampled 24–48 h after infiltration when HR lesions were fully developed both from withinthe lesion/infiltrated area and surrounding asymptomatic/uninfiltrated tissue (Surround). n=3–9. (d) Tobacco leaf panels were infiltrated with2×108 ml−1 P. s. pv. phaseolicola (P. s. pv. phaseolicola) on one side of the mid-vein and 2×108 ml−1 of a hrpL mutant of P. s. pv. phaseolicola(hrpL) on the other side. Samples taken from the infiltrated area at the times indicated. n=6. Means and standard errors (where larger thansymbols used) are shown.

A range of other pharmacological reagents were tested regulatory role in the transduction of the JA signal.Herbimycin, a tyrosine kinase inhibitor, significantlyfor their ability either to block or mimic the JA response.

The data from these experiments are summarized in inhibited the JA response whilst having little effect onbasal AP activity or protein levels. However, this effectTable 1. No evidence was found for the involvement of

GTP-binding proteins or ATPases in the JA response. In was not reproduced when another tyrosine kinaseinhibitor, genistein, was used. Similarly, whilst neomycinaddition, serine/threonine kinases appear to play no


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Fig. 3. (a) Leaf discs were pre-incubated for 1 h with 5 mM EGTA (EGTA), 1 mM CaCl2 (Ca2+), 10 mM A23187, 10 mM ionomycin (Iono) orcombinations of these compounds as indicated. After 1 h carrier (Control ) or JA (final concentration 200 mM ) was added. Incubations with carrieror JA alone (i.e. without pre-incubation with any of the above compounds) were also carried out and used to calculate the results shown. n=6.(b,c) Leaf discs were pre-incubated for 1 h with 10 mM A23187 (b) or 10 mM ionomycin (c) together with EGTA at the concentrations shown afterwhich time JA (final concentration 200 mM) was added. n=3 (b) and 4 (c) (d–g) Leaf discs were pre-incubated for 1 h with ruthenium red (d),LaCl3 (e), nifedipine (f ) or (±)-verapamil (g) at the concentrations indicated after which time JA (final concentration 100 mM) was added. n=6–9(d), 6 (e–g). Means and standard errors shown.


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Fig. 4. Leaf discs were pre-incubated for 1 h with okadaic acid (a), okadaol (b), microcystin LR (c), cantharidin (d) at the concentrations indicatedafter which time JA (final concentration 100 mM) was added. n=8–11 (a), 9 (b), 3 (c), 6 (d). Means and standard errors shown.

sulphate, a PI-PLC inhibitor, inhibited the JA response, increase is a consequence (and therefore a measure) ofthe said loss. This may indeed be the case. However, it isLiCl (an inhibitor of PI turnover) and U73122 (an inhib-clear that the mechanism of protein loss following JAitor of mammalian PLC) were ineffective.treatment is different to that following treatment withABA, mannitol, sucrose, following dessication or after

Discussion application of H2O2 ( Kenton, unpublished observations).In none of these treatments, despite extensive proteinEffects of JA on AP and protein contentloss, was there any significant increase in AP specific

APs have been described in a number of plant species activity (in some cases specific activity declined).and some, for example, soybean and Arabidopsis VSPs In crude extracts and on activity gels it has not beenare JA-inducible or are enriched following JA treatment possible to detect any significant inhibitor sensitivity or(DeWald et al., 1992; Berger et al., 1995). Here it is size differences in the AP activities in control- ordemonstrated that JA is able to stimulate an increase in JA-treated tissue ( Kenton, unpublished data). Thus, it isthe specific activity of tobacco leaf AP at the same time likely that JA treatment leads to the specific enrichmentas overall protein content is declining. The significance of a pre-existing enzyme. The mechanism by which thisof this change is unclear; likewise clearly defined roles occurs is unknown, but could involve activation of AP,for AP have yet to be identified (Duff et al., 1994). either by post-translational modification, desequestrationNevertheless, this combination of AP activity change and or by failure to target this enzyme for proteolyticprotein loss, measured at the same time in a simple assay, degradation.may prove to be a valuable tool for investigating JA Nevertheless, an effect of JA at either the transcriptionalsignalling in tobacco. or translational level cannot be excluded. The ability of

The fact that the AP activity increase occurs together JA to induce transcriptional activation of a wide rangeof genes has been well documented (Wasternack andwith protein loss may lead to the conclusion that the AP


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Table 1. Effects of pharmacological agents on JA-induced AP activity increase and protein loss

Compound Concentration AP activity Protein AP activity Protein n Effects(% of JA alone) (% of JA alone) (% of control ) (% of control )

ATPasesConcanamycin 1 nM 84.5±5.6 112.7±7.4 nd nd 6 Inhibits vacuolar-type H+ ATPaseOuabain 50 mM 126.3±16.1 91.7±7.5 nd nd 6 Inhibits Na+/K+-dependent ATPases

GTP-Binding ProteinsCompound 48/80 100 mM nd nd 120.9±11.4 62.3±4.5 9 Activates GiMastoparan 0.1 mM nd nd 114.8±10.8 104.5±13.6 6 Activates Gi and GoSuramin 100 mM 111.3±6.2 106.0±4.7 nd nd 6 G protein uncoupler

KinasesAnisomycin 1 mM nd nd 87.5±8.6 103.3±5.3 6 Activates JNK2 and MAP kinasesDibutyryl-cAMP 0.63 mM nd nd 72.7±12.0 184.3±34.1 3 Membrane permeable PKA activatorDibutyryl-cGMP 10 mM nd nd 93.5±3.6 107.3±3.9 6 Membrane permeable PKG activatorForskolin 10 mM nd nd 108.7±11.8 93.7±12.4 3 Adenylate cyclase activatorGenistein 10 mM 95.3±5.6 111.0±11.5 nd nd 9 Tyrosine kinase inhibitorHerbimycin 5 mM 40.5±2.9 344.0±55.1 94.3±7.7 90.7±3.0 6 Tyrosine kinase inhibitor(–)-Indolactam V 50 mM nd nd 109.5±6.0 107.5±9.1 6 PKC activatorK252a 100 nM 97.0±11.4 122.5±17.1 nd nd 6 Serine/threonine kinase inhibitorPdBu 30 nM 104.3±5.4 105.0±4.3 87.2±6.3 118.7±10.5 6 PKC activatorPdDn 30 nM 105.3±5.6 118.7±10.5 97.3±5.5 118.5±13.6 6 PKC activatorQuercetin 10 mM 126.5±10.9 84.2±11.4 nd nd 6 PI kinase and PKC inhibitorStaurosporine 125 nM 93.0±8.6 104.3±9.3 nd nd 6 Serine/threonine kinase inhibitorWortmannin 5 nM 82.8±4.2 139.7±13.1 nd nd 6 PI kinase inhibitor

PhospholipaseLithium Chloride 1 mM 101.5±2.5 93.5±10.1 nd nd 6 Blocks PI turnoverManoalide 2.5 mM 129.8±14.6 70.2±9.1 nd nd 6 PLA2 and PLC inhibitorNeomycin Sulphate 50 mM 48.6±7.2 182.8±41.5 72.0±6.3 89.4±5.9 9 PI-PLC inhibitorU73122 20 mM 99.3±10.2 90.0±12.8 nd nd 6 Neutrophil and platelet PLC inhibitorU73343 20 mM 117.0±6.8 85.2±5.9 nd nd 6 Inactive U73122 analogue

All compounds were added 1 hour prior to addition of JA or control. Means and SE shown. nd—not tested.PdBu—phorbol-12,13-dibutyrate; PdDn—phorbol-12,13-didecanoate; PI—phosphatidylinositol; PKA—cAMP-dependent kinase; PKC—proteinkinase C; PKG—cGMP-dependent kinase; PLA2—phospholipase A2; PLC—phospholipase C.

Parthier, 1997) and a putative JA response element has HR correlate closely with sites of JA synthesis in tobacco,been identified in the soybean vspB promoter (Mason et i.e. at or close to the wound site or in the HR lesion, butal., 1993). In addition, JA can also negatively regulate not in unwounded or uninfiltrated tissue on the same leaftranscription of other genes, for example, rbcS which ( Kenton et al., 1999). Furthermore, the ability of the JAencodes Rubisco small subunit (Reinbothe et al., 1994). precursor LA to induce AP activity increase, protein lossJA is also able to exert an effect over translational rates, and the appearance of an 18.5 kDa band suggests thatpossibly via induction of ribosome-inactivating proteins endogenous JA synthesis can indeed mimic exogenously(RIPs), discriminating between JA-upregulated and applied JA.constitutive transcripts (Reinbothe et al., 1994). More Whilst the wound-induction of the JA response followsrecently it has been demonstrated that expression of the the same time-course as that exhibited by exogenous JAbarley JA-inducible protein JIP23 in tobacco results in a application (cf. Figs 1c, 2a), the increase in AP activityreduction in rbcS translation (Gorschen et al., 1997). is much smaller than that observed following JA treat-Thus, it is possible that expression of an AP, indistinguish- ment. This may be due to the heterogeneity of theable in our assay from the constitutive isoform(s), is wounded tissue which will contain dead tissue, cellsupregulated at the transcriptional level, or that existing directly adjacent to the wound site as well as essentiallyAP mRNA transcripts escape the influence of a unwounded cells. Thus the increase in activity and proteinJA-induced RIP-like mechanism. Whatever the mechan- loss may be restricted to a few cells of the area sampled.ism, activation of a pre-existing apparatus is unlikely to In contrast, in tissue undergoing HR the AP increase andbe sufficient since the translation of at least one protein protein loss are comparable to those observed followingwould appear to be required in order to effect the JA application. In this case it is the rapidity of theJA-induced changes. increase which differs either from wounding or exogenous

JA treatment (cf. Figs 1c, 2d). AP activity increasesCorrelation of AP and endogenous JA 12–18 h p.i. This may be as little as 3–6 h after the first

detectable increase in HR-specific JA ( Kenton et al.,The appearance of AP activity increases and protein lossin wounded tobacco and in tobacco leaf tissue undergoing 1999). In tobacco infected with avirulent bacteria, SA


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accumulation both precedes and occurs concurrently with are required remains to be determined and awaits theidentification of specific plant PP activators. The require-JA synthesis (Draper, 1997; Kenton et al., 1999). Since

SA has been shown to potentiate the pathogen-induced ment for PP activation in JA signalling in tobacco agreeswith recent data obtained in potato leaves and Arabidopsisoxidative burst and peroxide-sensitive gene expression

(Kauss and Jeblick, 1995; Shirasu et al., 1997; Mur (Dammann et al., 1997; Rojo et al., 1998) where it wasdemonstrated that JA-inducible gene induction waset al., 1996), it is possible that early SA accumulation

may potentiate subsequent JA-inducible responses. abrogated in the presence of okadaic acid.Although the present study provided no evidence thatAlternatively, potentiation may result from some other

avr-dependent mechanism, since, although AP activity kinase activity modulates (either positively or negatively)the JA response in tobacco, rapid wound-induction of awas increased following infiltration with the hrpL mutant

of P. s. pv. phaseolicola, this effect was only seen 72 h p.i. MAP kinase-related enzyme (WIPK ) has been demon-strated in tobacco (Seo et al., 1995). In addition, trans-genic tobacco plants that constitutively overexpressJA signalling requires Ca2+ mobilizationWIPK accumulate high levels of endogenous JA (Seo et

Mobilization of Ca2+ is a central theme in eukaryotical., 1999). Thus JA-dependent wound signalling in

intracellular signalling. In plants Ca2+ mobilization hastobacco appears to involve kinase-dependent JA accumu-

been shown to mediate responses to a number of stimulilation the effects of which are, in turn, transduced by a

including cold shock, red/far-red stimulation, pathogenPP and Ca2+.

challenge, and the pathogen-induced oxidative burstIn summary, these data demonstrate (1) that JA

(Knight et al., 1996; Chandra and Low, 1997; Gelli etinduces an increase in the specific activity of a tobacco

al., 1997; Neuhaus et al., 1997). The present study sug-leaf AP accompanied by protein loss and the appearance

gests that JA signalling may also be Ca2+-dependent. Theof an 18.5 kDa component, (2) that similar changes

sensitivity of the JA response to ruthenium red coupledcorrelate with sites of endogenous JA synthesis, and (3)

with the observation that EGTA is a far more effectivethat the combined effects of JA on AP and protein

inhibitor when used in combination with Ca2+ ionophorescontent can be utilized to investigate JA signalling. With

suggests that JA-induced Ca2+ mobilization is likely toregard to the latter point, measurement of AP/protein is

occur from intracellular stores rather than from thetechnically simple compared to Northern or Western

apoplast. Although La3+ is considered to be a plasmablotting and can be carried out on wild-type tobacco of

membrane channel blocker, high concentrations can exerta least two cultivars thus avoiding artefacts that may

a less site-specific effect on Ca2+ flux (Quinquampoix etarise from the analysis of heterologous promoter–reporter

al., 1990). Whilst (±)-verapamil and nifedipine are bothconstructs (Taylor, 1997).

considered L-type channel blockers these data are derivedlargely from animal studies. The results obtained heresuggest that one component of the JA-induced AcknowledgementsCa2+ mobilization involves a nifedipine-sensitive,

We wish to thank Dr Willem Broekaert (Leuven), Mr Robert(±)-verapamil-insensitive channel. Similarly the rca Ca2+ Darby and Professor Stefan Nahorski (Leicester), Dr Johnchannel from wheat roots is sensitive to inhibition by Turner (UEA), and Dr Claus Wasternack (Halle) for theirLa3+, ruthenium red, (±)-verapamil, but not nifedipine, advice, comments and discussion. We also wish to thank Dr

John Mansfield (Wye College, London) and Dr John Taylorindicating that (±)-verapamil and nifedipine distinguish(HRI, Wellesbourne) for their gifts of pathogens, and Mrbetween discrete classes of plant Ca2+ channels (PinerosGraham Benskin for growing and maintaining plants. PK andand Tester, 1997). The inability of Ca2+/ionophore com- LM were supported by grants from the BBSRC and MAFF,

binations to mimic JA suggests that Ca2+ mobilization respectively.represents one arm of a bifurcating or multibranchedsignalling network in which elevation of cytosolic Ca2+

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A requirement for calcium and protein phosphatase in the jasmonate-induced increase in tobacco leaf acid phosphatase specific activity