reversible protein phosphorylation regulates jasmonic acid-dependent and -independent wound signal...

The Plant Journal (1998) 13(2), 153–165

Reversible protein phosphorylation regulates jasmonic acid-dependent and -independent wound signal transductionpathways in Arabidopsis thaliana

Enrique Rojo, Elena Titarenko, Jose Leon, Susanne

Berger1, Guy Vancanneyt and Jose J. Sanchez-Serrano*

Centro Nacional de Biotecnologıa CSIC, Campus de

Cantoblanco UAM, Cta. Colmenar Viejo km. 15,500,

28049 Madrid, Spain, and1Institut fur Pflanzenbiochemie, Weinberg 3, 06120 Halle

(Saale), Germany

Summary

Plants responses to mechanical injury are complex and

include the induced expression of defence-related genes.

The phytohormone JA has been reported to mediate

some of these responses. To elucidate further the signal

transduction processes involved, the action of specific

agonists and antagonists of known signalling effectors

on the response of Arabidopsis thaliana plantlets to JA

and wounding was investigated. The identification and

characterization of a reversible protein phosphorylation

step in a transduction pathway leading to JA-induced

gene transcription is reported. This phosphorylation

event involved the opposing activities of a staurosporine-

sensitive protein kinase, negatively regulating the path-

way, and a protein phosphatase, most probably of type

2 A, which activated JA-responsive gene expression. JA

activation via this pathway was blocked in the A.

thaliana JA-insensitive mutants jin1, jin4 and coi1, and

by exogenous application of cycloheximide or auxins.

Wound-induced activation of JA-responsive genes was

also regulated by this protein phosphorylation step. An

alternative wound signalling pathway, independent of

JA, was also identified, leading to the transcriptional

activation of a different set of genes. This JA-independent

pathway was also regulated by a protein phosphorylation

switch, in which the protein kinase positively regulated

the pathway while the protein phosphatase negatively

regulated it. Moreover, a labile protein apparently

repressed the expression of these genes. One of the

genes analysed, JR3, had a complex pattern of expres-

sion, possibly because it was regulated via both of the

wound signalling pathways identified. According to the

function of an homologous gene, JR3 may be involved

in feedback inhibition of the JA response.

Received 9 May 1997; revised 2 September 1997; accepted 5 September

1997.

*For correspondence (fax 134 1585 4506).

© 1998 Blackwell Science Ltd 153

Introduction

Jasmonic acid (JA) is a cyclopentanone derivative of

linolenic acid that exhibits features of a plant hormone

(Sembdner and Parthier, 1993). A function of JA in plants

may be in regulating gene activation in response to

mechanical damage. Upon wounding, there is a rise in

endogenous levels of JA (Albrecht et al., 1993; Creelman

et al., 1992; Pena-Cortes et al., 1993), due to increased

de novo synthesis, which triggers the activation of

wound-inducible genes. Exogenously applied JA is able

to induce these wound-responsive genes in non-

damaged plants (Farmer et al., 1992; Hildmann et al.,

1992; Reinbothe et al., 1994). In addition, application of

exogenous JA also induces secondary metabolic path-

ways leading to phytoalexin production (Dittrich et al.,

1992). Elicitor-induced synthesis of phytoalexins may also

require de novo synthesis of JA (Gundlach et al., 1992),

thus establishing a link between defence responses to

injury and pathogen attack.

Novel roles for JA in the physiology of non-stressed

plants have been reported. For example, JA may be

involved in tuberization in potato, and is regarded as a

tuber-inducing factor (Koda et al., 1991; Pelacho and

Mingo-Castel, 1991). In addition, it may regulate nitrogen

partitioning in soybean, where it induces the accumulation

of vegetative storage proteins (VSP) in leaves. These

proteins act as transient nitrogen reservoirs in depoded

plants (Anderson et al., 1989; Mason and Mullet, 1990).

The recent characterization of Arabidopsis thaliana mutants

insensitive to or deficient in the production of JA has also

suggested that it is involved in pollen formation (Feys

et al., 1994; McConn and Browse, 1996).

While gene activation upon wounding requires an

increase in the endogenous JA levels due to induced de

novo synthesis, it is not established whether the effects

the hormone exerts on plant development also require

organ- or stage-specific increases of JA levels. Basal JA

levels in the different organs may play roles in plant

physiology, and differences between basal and wound-

induced JA may underlie the distinct effects this hormone

exerts in apparently unrelated processes. In this regard,

co-suppression in transgenic A. thaliana of a lipoxygenase

gene, encoding an enzyme involved in JA synthesis,

indicates that wound-induced and basal JA may be synthe-

sized through different pathways (Bell et al., 1995). More-

over, expression of the flaxseed allene oxide synthase (also

involved in JA synthesis) in transgenic potato plants results

154 Enrique Rojo et al.

in JA levels comparable to those attained upon

wounding. However, these elevated levels do not

activate the JA-responsive, wound-inducible genes (Harms

et al., 1995). Taken together, these data suggest the pres-

ence of separate JA pools that are differently perceived,

and lead to the activation of distinct physiological pro-

cesses, most probably through the induction of separate

sets of genes.

Very little is known about how JA is perceived, and of

the mechanisms which may allow cells to discriminate the

origin of JA and which specifically activate defined sets of

genes in response. Several A. thaliana mutants altered in

sensitivity to JA have been identified based on phenotypic

screenings for normal growth in the presence of JA. Among

them, three non-allelic mutants, jin1, jin4 (Berger et al.,

1996) and coi1 (Feys et al., 1994), were identified by their

ability to root in JA-containing medium in which wild-

type plants exhibited a markedly reduced root growth. In

addition, JA induction of VSP genes in these mutants was

impaired or much weaker than in wild-type plants. The

availability of JA-insensitive mutants in A. thaliana will

certainly yield highly valuable information on the

components involved in JA signalling, and on their roles

in wound-induced gene expression. However, characteriza-

tion of the mutated genes and their functions in the

signalling pathways often proves to be tedious. In addition,

screening procedures may be biased towards preferential

isolation of components of an inducible pathway, if

separate JA pools do exist.

Pharmacological approaches have proved useful in the

characterization of components of signal transduction

pathways in eukaryotes. These experiments have con-

firmed that plant intracellular signalling proceeds via the

same basic components and motifs as their counterparts

in other eukaryotic systems (Bush, 1995; Ma, 1994; Smith

and Walker, 1996). Due to its versatility in modulating the

duration of a response to a stimulus and its ability to

amplify the signals powerfully, reversible protein phos-

phorylation is a common switching mechanism in signal

transduction pathways, including hormone perception in

plants (ethylene, Kieber et al., 1993; Raz and Fluhr, 1993;

gibberellin, Kuo et al., 1996; ABA, Leung et al., 1994; Meyer

et al., 1994; cytokinin, Kakimoto, 1996). The availability of

specific inhibitors of protein kinases and phosphatases

(MacKintosh and MacKintosh, 1994) has allowed the

elucidatation of the roles of their target enzymes in signal

transduction pathways of lower eukaryotes, and in animal

and plant systems.

We have taken advantage of these pharmacological

tools to characterize wound signalling pathways in A.

thaliana. In particular, we have investigated the involve-

ment of reversible protein phosphorylation in modulating

JA signalling.

© Blackwell Science Ltd, The Plant Journal, (1998), 13, 153–165

Figure 1. Effect of JA, staurosporine and okadaic acid on the expression of

JR genes.

Ten-day-old plantlets of Arabidopsis thaliana ecotype Landsberg erecta

were treated for 1 h with (1) or without (–) 1 µM staurosporine (STA) or

100 nM okadaic acid (OKA). Subsequently, 50 µM JA or an equivalent amount

of the solvent (Control) was added, and plantlets collected 6 h later. Total

RNA was isolated and used in Northern-type analysis. Blots were hybridized

with 32P-labelled probes for JA-responsive genes 1, 2 and 3 (JR1, JR2 and

JR3), vegetative storage protein gene (VSP), hydroperoxide lyase (HPL)

and chalcone synthase (CHS). Hybridization with a 18S rDNA probe (18S)

was used as control for RNA transfer to the blot.

Results

Reversible protein phosphorylation is involved in

JA-induced gene expression

The involvement of protein phosphorylation in the trans-

duction of the JA signal was assessed by analysing the

effect of protein kinase and phosphatase inhibitors on

JA-induced gene expression in A. thaliana. To this end,

plantlets were treated with JA in the presence or absence

of staurosporine and/or okadaic acid, and the expression

of several JA-responsive (JR) genes was analysed. These

genes included some previously described to respond to

JA in A. thaliana and other plant species (vegetative storage

protein, VSP, Berger et al., 1995; chalcone synthase, CHS,

Dittrich et al., 1992), and others we have isolated recently

by differential mRNA display (JR1, JR2, JR3) or obtained

from the EST project (hydroperoxide lyase, HPL).

As shown in Figure 1, treatment of A. thaliana plantlets

with 50 µM JA induced the accumulation of transcripts from

these genes. Thus, under these conditions, JA strongly

Protein phosphorylation and wound signalling 155

activated the expression of a large set of genes (Titarenko

et al., 1997). In the case of HPL, the appearence of two

hybridizing mRNA bands of different size was observed

upon JA treatment.

Staurosporine, a broad range serine-threonine protein

kinase inhibitor (MacKintosh and MacKintosh, 1994;

Tamaoki et al., 1986), could mimic the effect of JA by

inducing the same set of genes (Figure 1). However, the

levels of expression attained upon treatment with 1 µM

staurosporine were lower than those observed after treat-

ment with 50 µM JA. Moreover, increasing the stauro-

sporine concentration did not result in higher levels of JR

transcripts (results not shown). Under these conditions

staurosporine treatment had a slight effect on JR1 expres-

sion and none on VSP expression. Interestingly, JR1 and

VSP transcripts accumulated to a larger extent when JA

and staurosporine were applied simultaneously compared

with incubation with JA alone. This overinduction was not

observed for the other genes analysed in the conditions

used in this experiment, but it was clearly detectable at

lower concentrations of JA (see below). These data suggest

that a protein kinase activity negatively regulates the

expression of JR genes.

To elucidate whether a protein phosphatase was involved

in JA perception as well, we examined the effects of protein

phosphatase inhibitors on the induction of JR genes by JA

and/or staurosporine. The polyether compound okadaic

acid (Bialojan and Takai, 1988; Cohen et al., 1990) is a

potent inhibitor of protein serine-threonine phosphatase

types 1 (PP1) and 2A (PP2A) at submicromolar concentra-

tions, whereas type 2B is only inhibited at high micromolar

concentrations and type 2C activity is not affected. Treat-

ment with 100 nM okadaic acid abolished JA-induced

expression of all but one (JR3) of the genes investigated,

indicating that a PP1 or PP2A activity is involved in trans-

duction of the JA signal. In addition, okadaic acid blocked

the induction of these genes caused by staurosporine,

suggesting that JA and staurosporine activate JR genes

through a common pathway that requires the activity of

this okadaic acid-sensitive protein phosphatase.

As seen in Figure 1, the expression of JR3 was induced

by treatment with JA and/or staurosporine, as were the

rest of the JR genes. However, in plantlets treated with JA

and/or staurosporine in the presence of okadaic acid, JR3

was still expressed, in contrast to the other JR genes. Since

JR3 expression was induced upon treatment with okadaic

acid alone, it was not possible to determine whether the

protein phosphatase activity previously identified was also

required for JA-induced expression of JR3.

We tested the possibility that the inhibitors used were

altering the timing of JA-induced gene activation, and not

the level of expression. As shown in Figure 2, increases in

the steady-state mRNA levels of JR genes were detectable

after 90 min of JA treatment and, for most genes, maximal


Figure 2. Time–course of JA-induced mRNA accumulation in the presence

or absence of staurosporine and okadaic acid.

Ten-day-old plantlets of Arabidopsis thaliana ecotype Landsberg erecta

were treated for 1 h with 1 µM staurosporine (STA) or 100 nM okadaic acid

(OKA), or not treated with any of these inhibitors. Subsequently, 50 µM JA

was added, and plantlets were collected at the indicated times (in hours).

Total RNA gel blots were hybridized with the radioactive probes as described

in Figure 1.

accumulation was observed after 6 h. However, treatment

with JA and staurosporine could clearly increase accumula-

tion of JR1, VSP and CHS transcripts over the maximal

levels attained after JA treatment, suggesting that

staurosporine treatment affects the amount of transcript

accumulated but does not alter the timing of maximal

induction. Similarly, the inhibitory effect of okadaic

acid on JA-induced mRNA accumulation was observed by

90 min after treating with JA, and remained for at least

48 h, indicating that okadaic acid acts by blocking, and

not merely delaying, transduction of the JA signal. For

subsequent experiments, we took samples at 6 h after

treatments, when maximal expression of the genes was

observed.

An active protein phosphatase is required for JA-induced

gene expression

To gain further insight into the type of protein phospha-

tase activity involved in JA induction of gene expression,

a dose-dependence experiment was performed with the

inhibitors okadaic acid and calyculin A. These compounds

are structurally unrelated, and display inhibitory activity

towards PP1 and PP2A. While okadaic acid shows a

stronger inhibitory activity towards PP2A (concentration

for 50% inhibition (IC50) 5 0.1–1 nM for PP2A and 10–15 nM

for PP1; Cohen et al., 1990), calyculin A is reported to

be equally effective against both types (IC50 5 0.5–2 nM;

Ishihara et al., 1989).

As shown in Figure 3, as little as 20 nM okadaic acid (a


Figure 3. Effect of various concentrations of okadaic acid and calyculin A

on JR gene expression.

Arabidopsis thaliana plantlets were treated for 1 h in different

concentrations of okadaic acid (OKA) or calyculin A (CAL). Subsequently,

JA 50 µM or an equivalent amount of solvent (not indicated) was added,

and 6 h later the plantlets were collected. Total RNA gel blots were

hybridized with the probes as described in Figure 1.

concentration close to its in vitro IC50 for purified PP1)

was able to inhibit JA-induced mRNA accumulation signi-

ficantly. At similar concentrations, calyculin A was also

able to inhibit JA-induction of responsive genes. Both

calyculin A and okadaic acid induced JR3 expression in

the absence of JA. These results indicate that the effects

observed are due to the inhibitory activity towards protein

phosphatases of the compounds tested, further supporting

the involvement of a protein phosphatase in mediating JA-

induced gene expression.

The relatively low concentration of okadaic acid required

to block JA-induced gene activation points to a PP2A acting

in this pathway. To characterize further the type of protein

phosphatase involved, we studied the inhibitory effect of

okadaic acid and tautomycin (IC50 5 10 nM for type 2A and

5 nM for type 1; MacKintosh and Klumpp, 1990) on the

phosphatase activity present in plant extracts (tautomycin,

up to 5 µM, was not effective on intact plantlets; results not

shown). For that purpose a standard activity assay for

protein serine-threonine phosphatase types 1 and 2A was

used.

Crude extracts from A. thaliana plantlets have a readily

detectable protein phosphatase activity. Dose–response

curves for the inhibition by okadaic acid and tautomycin

of the phosphatase activity present in plant extracts are

shown in Figure 4. By using the two inhibitors with different


Figure 4. Dose–response curves of protein phosphatase inhibition by

okadaic acid and tautomycin.

Protein extracts from 10-day-old A. thaliana plantlets were assayed for PP1/

PP2A activity in the presence of different okadaic acid (OKA) and tautomycin

(TAU) concentrations as described in the Experiemental procedures. Values

are presented as the mean from three independent experiments. Vertical

bars indicate the standard deviation. Deviations below 1% are not included.

affinities for PP1 and PP2A, we could detect the relative

contribution of these protein phosphatase activities to the

total content present in A. thaliana. 23.4 6 1.2% of the total

protein phosphatase activity was inhibited by 5 nM okadaic

acid. A similar percentage was still active in the presence

of ™ 7 nM tautomycin, and is therefore likely to correspond

to the PP2A fraction. The relative activities of PP1 and

PP2A fractions in A. thaliana plantlets were similar to the

values obtained in other plant systems (MacKintosh and

Cohen, 1989; MacKintosh et al., 1991). The results shown

in Figure 4 indicate that, in these conditions, the in vitro

IC50 for okadaic acid inhibition of PP1 fraction from A.

thaliana extracts is close to 50 nM.

We measured the remaining protein phosphatase activity

present in plantlets incubated in vivo with 1 µM okadaic

acid. Protein phosphatase activity in extracts from treated

plantlets was 85.7 6 4.7% of that of untreated controls.

This rate of inhibition corresponded to an okadaic acid

concentration of 1 nM, as determined in vitro. This was

further confirmed by measuring okadaic acid and

tautomycin inhibition in crude extracts from treated and

untreated plantlets (data not shown). Taken together, these

data indicate that only the PP2A fraction is inhibited at the

okadaic acid concentrations used for in vivo treatments.


Figure 5. Staurosporine-mediated induction of JR gene expression in JA-

insensitive mutants.

Plantlets of coi1, jin1 and jin4 and their respective wild-type (WT)

backgrounds grown as described in the Experimental procedures, were

incubated in 1 µM staurosporine (STA) (1) or an equivalent amount of

solvent (–) for 6 h and collected. Total RNA gel blots were hybridized with

JR1, JR2, VSP, HPL and JR3.

A staurosporine-sensitive protein kinase negatively

regulates the activation of gene expression through a

JA-transduction pathway

The results presented suggest that a staurosporine-

sensitive protein kinase is involved in a signalling path-

way leading to JA-induced gene expression. To obtain

further support for this assumption, we took advantage of

the JA-insensitive mutants identified in A. thaliana to

assess whether or not alterations in the response to JA

concurrently affected the response to staurosporine.

As shown in Figure 5, staurosporine-induced expression

of JR genes was reduced to different degrees in the JA-

insensitive mutants. When transmission of the JA signal

was completely blocked, as in the coi1 mutants, stauro-

sporine could not induce the expression of these genes.

However, when the transmission was only partially

blocked, as in the leaky jin1 and jin4 mutants, staurosporine

treatment induced JR gene expression, although the levels

attained were lower than in the respective wild-type back-

grounds. It has to be noted that while jin1 and jin4,

and the corresponding wild-type plantlets, were grown in

continuous light, coi1 plantlets were grown under day–

night cycles. This difference accounts for the absence of

HPL transcript in the staurosporine-treated control for coi1.

CHS expression was also affected, as it was highly

expressed in the non-induced controls grown under con-

stant light (data not shown). In addition, prior to

staurosporine treatment coi1 mutants were selected as

described in the Experimental procedures. This selection

procedure resulted in a larger accumulation of VSP and

JR1 transcripts in staurosporine-treated wild-type plantlets

(compare with Figure 1).

It has been reported that in the jin4 mutants, VSP


Figure 6. JA and staurosporine interaction on JR gene expression.

Arabidopsis thaliana plantlets were incubated in the presence (1) or

absence (–) of 1 µM staurosporine (STA) and increasing concentrations of

JA (as indicated in µM) for 6 h. Total RNA gel blots were hybridized with

the probes as described in Figure 1.

expression is less responsive to wounding and methyl-JA

induction than in the jin1 mutant (Berger et al., 1996). As

shown in Figure 5, jin4 plants were also less sensitive to

the staurosporine-mediated activation of the JR genes.

Activation of JR genes by either JA or staurosporine was

thus similarly affected by these mutations, suggesting that

it is mediated through a common transduction pathway.

It is interesting to note that the induction of JR3 by

staurosporine was also blocked in the JA-insensitive

mutants, indicating that it shares at least in part the same

JA signalling pathway as the rest of the JR genes.

To gain further insight on how a staurosporine-sensitive

protein kinase regulates JA responses, we investigated the

effect of staurosporine on the activation of gene expression

at different JA concentrations. As shown in Figure 6, JR2

and CHS expression was induced by JA concentrations as

low as 100 nM and was strongly induced by treatment with

staurosporine. In contrast, expression of VSP required

concentrations of JA over 25 µM for induction, and no

activation was detected with staurosporine alone. Thus,


Figure 7. Protein phosphatase activity is required before the step regulated

by staurosporine in the JA signalling pathway.

(a) Arabidopsis thaliana plantlets were subjected to the following

treatments: control plants (lane 1), 50 µM JA (lane 2), simultaneous

incubation in 50 µM JA, 1 µM okadaic acid and 1 µM staurosporine (lane 3),

5 min preincubation in 50 µM JA, subsequently adding 1 µM okadaic acid

and 1 µM staurosporine (lane 4) or 1 µM okadaic acid (lane 5).

(b) Arabidopsis thaliana plantlets were subjected to the following

treatments: control plants (lane 1), 1 µM staurosporine (lane 2), 50 µM JA

(lane 3), 50 µM JA and 20 nM okadaic acid (lane 4), or 50 µM JA, 20 nM

okadaic acid and 1 µM staurosporine (lane 5).

Plantlets were harvested 6 h after the initiation of the treatment, and total

RNA was isolated. Northern blots were hybridized with JR1, JR2 and

VSP probes.

the sensitivity of the genes to staurosporine treatment

correlates with their sensitivity to JA treatment, confirming

that staurosporine activates the genes via a JA transduc-

tion pathway.

When plants were incubated with JA in the presence of

staurosporine, a co-operative effect of both stimuli on the

activation of JR gene expression was observed (Figure 6).

At low JA concentrations, staurosporine strongly enhanced

the JA-induced activation of all JR genes (see for instance

the effect on VSP expression at 1 µM JA). However, JR2,

HPL, CHS and JR3 expression reached a plateau at high

JA concentrations, and incubation with staurosporine

could only enhance this effect slightly. On the other hand,

JA-mediated expression of JR1 and VSP was strongly

enhanced by staurosporine even after the response to JA

was saturated. Thus, the level of expression attained by

treatment with both JA and staurosporine could not be

reached by treatment with JA alone. This indicates that

staurosporine does not induce JA synthesis, but rather

acts on a component, probably a protein kinase, of a JA

transduction pathway. Increasing staurosporine con-

centration did not result in higher levels of JR transcripts

(data not shown), indicating that the protein kinase was

completely inhibited at 1 µM and that the effect of JA was

not mediated by the inactivation of that protein kinase.

The reduction in mRNA levels observed at 1 mM JA,

especially in the case of HPL and CHS, may be a toxic

effect due to the high concentration of the hormone.


The dephosphorylation step affected by okadaic acid is

upstream of the phosphorylation step affected by

staurosporine in the JA-signalling pathway

We have obtained evidence for the presence of both a

protein kinase and a protein phosphatase acting with

opposite effects on a JA transduction pathway. We there-

fore sought to answer where this protein phosphatase

activity was located in the transduction pathway relative

to the step modulated by the protein kinase.

An experiment to address this point is presented in

Figure 7(a). No induction of the JR genes was observed in

plantlets incubated with 50 µM JA in the presence of 1 µM

okadaic acid and 1 µM staurosporine (shown for JR1, JR2

and VSP in Figure 7a, lane 3). This indicates that the protein

phosphatase activity was required for activation by JA and/

or staurosporine. However, if the JA signalling was allowed

to proceed for a short period of time (5 min treatment with

JA alone, in Figure 7a, lane 4), high induction of the genes

was attained upon subsequent incubation with staurospor-

ine and okadaic acid, which was not observed without

staurosporine in the incubation medium. Staurosporine

was therefore able to enhance the expression of JR genes

even in the presence of okadaic acid once the JA signal

had been transmitted for a short time lapse. Thus, the

protein phosphatase activity required for the transduction

of the JA signal probably acts prior to the protein kinase

inhibited by staurosporine, and not downstream of it. An

experiment that lends further support for this model is

shown in Figure 7(b). When plantlets were treated with JA

in the presence of 20 nM okadaic acid, a very weak activa-

tion of JR genes was observed (Figure 7b, lane 4), indicating

that the JA signal was being transduced in part. However,

if staurosporine was also included (lane 5), the signal was

amplified and JR genes were strongly activated.

The okadaic acid-sensitive protein phosphatase activity

thus appeared to be required for JA-induced accumulation

of a positive regulator of this transduction pathway, most

probably a dephosphorylated protein, which was main-

tained in an active state by inhibiting a staurosporine-

sensitive protein kinase. The effect of staurosporine on

gene induction was thus dependent on the JA-induced

accumulation of that activated protein component of the

pathway. Once this dephosphorylated protein had been

produced in sufficient amounts, staurosporine treatment

did not require the protein phosphatase activity to induce

JR gene expression.

Cycloheximide and auxins inhibit the JA-mediated

transcriptional activation of JR genes

We analysed the effect of other putative regulators of

signal transduction pathways on the activation of the

different JR genes upon JA treatment. De novo protein


Figure 8. Effects of cycloheximide and auxins on JA-induced expression of

JR genes.

(a) Arabidopsis thaliana plantlets were incubated for 1 h in the presence

(1) or absence (–) of 50 µM cycloheximide (CHX). 50 µM JA (1) or an

equivalent amount of solvent was subsequently added, and plants were

collected 6 h later.

(b) Arabidopsis thaliana plantlets were incubated for 1 h in the presence

(1) or absence (–) of 10 µM NAA. 50 µM JA (1) or an equivalent amount of

solvent was subsequently added, and plants were collected 6 h later.

RNA gel blots were hybridized with the probes described in Figure 1.

synthesis is required for the transduction of numerous

signals in plants, including JA in potato (Pena Cortes

et al., 1992). This reflects, in some cases, the need for the

synthesis of an inducible component or the renewal of a

labile component of the transduction pathway.

We wanted to ascertain whether protein synthesis is also

required for the transduction of the JA signal in A. thaliana.

To this end, the effect of cycloheximide on JR gene activa-

tion was examined. As shown in Figure 8(a), cycloheximide

blocked JA-mediated induction of all JR genes analysed,

with, perhaps, the exception of JR3. Moreover, cyclo-

heximide blocked staurosporine induction of JR genes

(data not shown), further supporting the involvement of a

staurosporine-sensitive protein kinase in JA signalling. As

JR3 was induced by treatment with cycloheximide alone,

it was not possible to distinguish if de novo protein

synthesis was also required for JR3 activation by JA.

Auxins have been shown to downregulate wound-

induced expression of the JA-responsive pin2 promoter–

CAT gene fusions in tobacco (Kernan and Thornburg, 1989)

and to repress methyl-JA and wound-induced VSP and

lipoxygenase A gene expression in soybean (DeWald et al.,

1994). We examined whether auxin treatment could alter

the response to JA in A. thaliana. As shown in Figure 8(b),

treatment with 10 µM NAA reduced JA-induced accumula-


tion of all JR transcripts, except JR3. This effect was clear

in VSP and HPL, but it has been reproducibly observed for

other JR genes. Treatment with another auxin (2,4-D) gave

similar results (data not shown).

Thus, JA-induced activation of JR1, JR2, VSP, HPL and

CHS was apparently mediated through a common JA

transduction pathway sensitive to auxin treatment and

requiring de novo protein synthesis. The data obtained

with JA-insensitive mutants indicated that JA induction of

JR3 shares a common signalling pathway with other JR

genes up to the step regulated by staurosporine. As

auxins do not induce JR3 expression on their own, they

probably affect a later step in this pathway, separating

auxin-sensitive and -insensitive branches. Alternatively JR3

could be induced through this common JA transduction

pathway and, in addition, through an independent, auxin-

insensitive one.

A wound induction pathway, independent of JA

synthesis, shows regulation by staurosporine, okadaic

acid and cycloheximide opposite to the JA-dependent

one

JA has been reported to be involved in plant responses to

mechanical damage. Upon wounding, JA biosynthesis is

induced and the resulting higher endogenous JA level

triggers activation of wound-induced genes (reviewed in

Pena-Cortes et al., 1995). All A. thaliana JR genes tested

were induced upon wounding (Titarenko et al., 1997), sug-

gesting that wound-induced synthesis of JA mediates the

subsequent upregulation of JR gene expression.

The wound response, however, includes other aspects

not mediated by JA. We have isolated, by differential

mRNA display, two genes, WR3 and choline kinase (CK),

that are induced by mechanical wounding but not by JA

treatment. The wound induction of these genes was not

blocked in the JA-insensitive mutants or by treatment with

inhibitors of JA synthesis (Titarenko et al., 1997). Thus,

wound signalling branches through at least two pathways,

one dependent on JA synthesis, and the other independent.

We analysed the effect of staurosporine, okadaic acid

and cycloheximide on wound-induced gene expression.

As shown in Figure 9, the induction of WR3 and CK

upon wounding was partially blocked by incubation with

staurosporine and amplified in the presence of okadaic

acid. Treatment with okadaic acid alone was sufficient to

activate these two genes, even at concentrations as low

as 20 nM (results not shown). This suggests that a protein

phosphatase, probably type 2A, acts as a negative regulator

in this JA-independent wound signalling pathway, and that

a protein kinase activity is needed for transduction of the

signal. The effect of okadaic acid and staurosporine on the

wound induction of JR genes was similar to the effect

these inhibitors had on their response to JA. This indicates


Figure 9. Effects of staurosporine and okadaic acid on wound-induced

gene expression.

Arabidopsis thaliana plantlets were incubated for 1 h in the presence (1)

or absence (–) of 1 µM staurosporine (STA) or 1 µM okadaic acid (OKA), and

subsequently wounded (W) as described above or treated with 50 µM JA

(JA). Control plants (C) were treated with an equivalent amount of the JA

solvent. Plantlets were collected after incubation for 6 h.

RNA gel blots were hybridized with the probes described in Figure 1 and

in addition with the wound-responsive gene 3 (WR3) and choline kinase

(CK) probes.

that indeed their activation by wounding is mediated

through a JA transduction pathway.

Inhibition of protein synthesis led to activation of WR3

and CK, and apparently did not affect their wound-induced

activation (Figure 10). This suggests that a labile

component of this pathway downregulated their expres-

sion in non-wounded plants. In contrast, the wound-

induced activation of JR genes, except JR3, was prevented

by inhibiting protein synthesis (shown in Figure 10 for JR2).

Treatment with okadaic acid or cycloheximide activated

the expression of JR3, in the same manner as WR3 and

CK, and did not affect its wound induction. Moreover,

treatment with inhibitors of JA biosynthesis did not inhibit

the wound-induced activation of JR3, WR3 and CK (J. Leon

et al., submitted). Thus, the wound-induced expression of

JR3 may be mediated through a JA-dependent pathway

and also through a pathway independent of JA synthesis.


Figure 10. Effect of cycloheximide on JR2, JR3, WR3, and CK expression.

Arabidopsis thaliana plantlets were incubated for 1 h in the presence (1)

or absence (–) of 50 µM cycloheximide (CHX), and subsequently they were

(1), or were not (–), wounded (W). Plantlets were collected 4 h later.

RNA gel blots were hybridized with JR2, JR3, WR3 and CK probes, and the

18S rDNA probe as a control for RNA transfer to the blot.

This pathway is probably common to the induction path-

way of WR3 and CK.

Discussion

We have undertaken a pharmacological approach to

identify components of JA-dependent and independent

wound signalling pathways in A. thaliana. In many cases,

cell suspension cultures have been used in such

approaches (Christie and Jenkins, 1996; Felix et al., 1994;

MacKintosh et al., 1994). However, their efficacy in studying

specific hormone-induced responses is hindered by the

possibility of interfering effects due to other hormones

required to maintain culture growth. Indeed, in cell suspen-

sion cultures of A. thaliana we could not detect JA-induced

activation of JR gene expression, except a weak induction

of JR3 (data not shown). We therefore used A. thaliana

plantlets grown in liquid medium, due to the reproduci-

bility of growth conditions and the ease of application of

the compounds tested, which were readily absorbed by

the plants as judged from the effects they had on gene

expression.

Protein phosphatase and kinase inhibitors reveal

phosphorylation events in the transduction of a JA signal

The use of specific inhibitors of protein kinases and

phosphatases has proved useful in the identification of


reversible phosphorylation of proteins as a mechanism

for signal transduction of plant hormones (reviewed in

Smith and Walker, 1996). We have shown that this

regulatory mechanism takes part in the transduction of

a JA signal in A. thaliana.

Okadaic acid and calyculin A, two structurally unrelated

protein phosphatase inhibitors, suppress JA-induced

expresssion of JR genes. This suggests that transmission

of this JA signal in A. thaliana requires a PP1 or

PP2A activity. Similar experiments performed in potato

(Dammann et al., 1997) and tomato (E. Rojo , unpublished

results) showed that okadaic acid also blocks JA-

inducible expression of proteinase inhibitor II (pin2),

cathepsin D inhibitor, leucine aminopeptidase, and

threonine deaminase genes. This indicates that a protein

phosphatase is a component of a JA transduction

pathway conserved in different plant species. This protein

phosphatase is likely to be of type 2A. In our experiments,

the IC50 for okadaic acid inhibition of PP1 activity in

extracts of A. thaliana was ™ 50 nM. The in vivo IC50 is

probably higher due to higher concentrations of the

enzymes and reduced permeability and stability of okadaic

acid in cells. Nevertheless, concentrations as low as

20 nM okadaic acid strongly reduced the JA-induced

activation of JR genes. Specific effects of okadaic

acid concentrations below 30 nM on A. thaliana root

development have been ascribed previously to inhibition

of PP2A activity (Smith et al., 1994). Moreover, we

observed that in extracts from plantlets incubated in vivo

with 1 µM okadaic acid, a concentration exceeding that

needed to abolish JA-induced activation of JR genes,

protein phosphatase activity was inhibited to a degree

attained in vitro at 1 nM okadaic acid, a concentration at

which only PP2A activity is inhibited. A similar approach

has been used previously to provide evidence for the

involvement of a PP2A in the regulation of cdc2 gene

activity in NIH 3T3 cells (Jaramillo-Babb et al., 1996).

However, since the inhibition by okadaic acid may be

reversible (Bialojan and Takai, 1988), the values obtained

in diluted extracts may not exactly match the situation

in vivo.

Our data strongly suggest the involvement of a protein

kinase activity in negatively regulating this JA-signalling

pathway. The protein kinase inhibitor staurosporine activ-

ated the same set of genes as JA itself. This staurosporine-

mediated activation of JR genes required a protein

phosphatase activity and was impaired in the JA-

insensitive mutants coi1, jin1 and jin4, to the same

degree as the JA-mediated activation. Moreover, the

sensitivity of different JR genes to JA or staurosporine

treatment was similar. Staurosporine may therefore inhibit

a protein kinase, and thus activate JR genes through the

same signalling pathway as JA. In experiments conducted

in tomato plantlets, staurosporine activated the expres-


sion of pin2 and the other JA-inducible genes (E. Rojo,

unpublished results), indicating that this may be another

general component of the JA transduction pathway in

different plant species.

We have analysed how JA and staurosporine interact

to activate the expression of JR genes. Maximal activation

can only be attained by incubation with staurosporine

and JA, and not by either compound alone. This indicates

that JA does not activate the pathway by inhibiting the

staurosporine-sensitive kinase, and that the effects of

staurosporine are not due to induction of JA biosynthesis.

Rather, staurosporine and JA act co-operatively on a

step downstream in the JA-signalling pathway.

Our results give some clues to how a staurosporine-

sensitive protein kinase and a protein phosphatase may

interplay in JA signal transduction. When this JA signalling

pathway is blocked initially by okadaic acid, no induction

of target genes is observed. However, if the signal is

transmitted for a short period of time, staurosporine

treatment is able to maintain the pathway and induce

gene expression even if a PP2A activity is completely

inhibited. These results indicate that treatment with JA

triggers, via a PP2A, the transduction cascade and

induces the accumulation of an activated component of

the pathway that can be maintained in the active state by

the staurosporine-mediated inhibition of a protein kinase.

Taken together, these data suggest the presence of a

protein phosphorylation–dephosphorylation switch that

transduces a JA signal when the protein is dephos-

phorylated. The accumulation of the dephosphorylated

target protein probably occurs by a JA-induced activation

of a protein phosphatase, rather than by inducing de novo

synthesis of the target protein.

Treatment with staurosporine induces the expression of

some JR genes. This gene activation does not occur if

plantlets are treated, simultaneously, with staurosporine

and okadaic acid, suggesting that basal levels of all

components of this molecular switch are present under

non-inducing conditions. Staurosporine-induced activation

of JR genes is impaired in the JA-insensitive mutants

tested. However, combined treatment with JA and stauro-

sporine results in similar levels of JR gene expression in

wild-type plants and in the jin1 and jin4 mutants (data not

shown). A possible interpretation of these results is that

the weaker activation of JR genes upon staurosporine

treatment of jin1 and jin4 plantlets is due a reduced

perception of endogenous JA levels in these mutants,

which is largely overcome by the simultaneous application

of exogenous JA. Thus, jin1 and jin4 mutations would

affect components of the signalling pathway prior to the

phosphorylation switch, and not downstream to it as

staurosporine restores wild-type levels of induction in spite

of a reduced transmission of the JA signal.

Because of the more pleiotropic effects of the mutation,


coi1 has been suggested to be located before jin1 and jin4

in the JA signalling pathway (Berger et al., 1996). Thus,

mutations in any of these three loci, affecting steps

probably situated between the perception of JA and the

identified phosphorylation switch, block the staurosporine-

mediated activation of JR genes in non-inducing condi-

tions. This suggests that the presence of basal protein

phosphatase activity and/or its target protein depends on

perception of basal JA levels.

Identification of a JA-independent wound induction

pathway that shows opposite regulation to the JA-

dependent one

There is overwhelming evidence indicating that JA plays

a role in plant responses to wounding. A model that

integrates our current knowledge (Bergey et al., 1996;

Farmer and Ryan, 1992; Pena-Cortes et al., 1995; Seo et al.,

1995) suggests that upon wounding a cascade of events,

involving elements such as systemin, ABA and a MAP

kinase, leads to de novo synthesis of JA that, in turn,

regulates gene expression by a previously unknown trans-

duction pathway that may involve ethylene (O’Donnell

et al., 1996). We have shown that the A. thaliana JR genes

can also be regulated by mechanical damage of the plant

(Titarenko et al., 1997), suggesting that their wound induc-

tion is mediated by JA.

However, there is also evidence indicating that wound

responses include JA-independent effects. We have

isolated two genes from A. thaliana, WR3 and CK, that

respond to mechanical injury but not to JA treatment.

Furthermore, inhibitors of JA synthesis or mutations in

the JA transduction pathway do not affect their induction

(Titarenko et al., 1997; Leon et al., submitted), suggesting

that their activation upon wounding does not involve JA

signalling.

The wound induction of JR genes is blocked by treatment

with okadaic acid and enhanced by staurosporine, most

probably as a result of their action on the same phospho-

rylation switch that regulates JA signalling. In contrast, the

wound-inducible expression of WR3 and CK is reduced in

the presence of staurosporine, and enhanced by treatment

with okadaic acid. This indicates that a protein phos-

phorylation switch may also regulate transduction of the

wound signal through this pathway, although in this case

the signal is forwarded by the target protein in the phos-

phorylated state.

The activation of JR genes by wounding requires de

novo protein synthesis for transmission of the signal,

whereas the expression of WR3 and CK is supressed by a

labile protein. A candidate is the protein phosphatase

acting on the phosphorylation switch. Thus, wound-

induced activation of genes through the JA-dependent

and -independent pathways is regulated by protein


Figure 11. A model for wound signalling in Arabidopsis thaliana.

Wound-responsive genes activated through a JA-dependent pathway are

shown on the top set. Genes activated through the JA-independent pathway

are shown on the bottom set. Putative components of both signalling

pathways are shown. Arrows indicate positive action; blunted lines indicate

negative regulation. For positioning the point of action of COI1, JIN1, and

JIN4 we assume in this model that the presence of basal levels of the

protein phosphatase activity, or its target protein, required for

staurosporine-induced JR gene expression depends on JA perception.

W, wounding; PP2A, protein phosphatase type 2A; PK, protein kinase; OKA,

okadaic acid; STA, staurosporine.

phosphorylation and requires protein synthesis in opposite

ways, further supporting the divergence of the wound

signal through, at least, these two pathways. A scheme

depicting the two wound signalling pathways and the sites

of action of the compounds tested is shown in Figure 11.

Other compounds affecting putative components of

these pathways, such as G proteins, PLA2 and Ca21, also

have opposite effects on transduction through them (J.

Leon et al., submitted). Thus, conditions that block one of

the pathways induce the other, suggesting that cross-talk

between them may exist, as observed for other signalling

pathways involving lipid second messengers (Liscovitch

and Cantley, 1994).

A working model for transduction of the wound signal in

which JR may modulate wound-induced gene expression

The JR3 gene is highly similar to ILR1 (Bartel and Fink,

1995), which codes for an enzyme shown to hydrolize

amino acid conjugates of indole-3 acetic acid (IAA) to active

IAA. JR3 expression is induced by wounding and by

exogenous application of JA or staurosporine, and this

induction is reduced in the JA-insensitive mutants coi1,

jin1 and jin4. This indicates that JR3 is activated through

a JA transduction pathway common to the rest of the JR

genes. However, we have obtained data that indicate that

JR3, as well as WR3 and CK, can be induced by wounding

even if de novo synthesis of JA is prevented (J. Leon et al.,

submitted). Moreover, inhibition of either protein synthesis

or protein phosphatase activity is sufficient to activate JR3,


CK and WR3 expression. Taken together, the evidence

suggests that JR3 could be activated by wounding through

two different pathways, one common to the other JA-

inducible genes and the other independent of JA, common

to CK and WR3.

This dual regulation of JR3 expression may provide a

way to modulate the response to mechanical damage. It

has been shown that the concentration of endogenous

auxins declines upon wounding (Thornburg and Li, 1991).

The time required for recovering the initial auxin levels

may thus define the duration of the response to a wound

stimulus. Consistent with this, application of exogenous

auxins reduced the JA-induced expression of JR genes,

except that of JR3, in A. thaliana. If JR3 has a similar

enzyme activity to ILR1, releasing active auxins from the

pool of inactive conjugates, its JA-induced expression

could play a role in feedback inhibition of the JA response

and in restoring the hormonal balance in the plant. An

injury, however, would also induce JR3 expression via a

JA-independent signalling pathway. It is interesting to note

that, in contrast to CK and WR3, the expression of JR3

induced by wounding, okadaic acid and cycloheximide is

blocked in the coi1 mutant (E. Rojo, unpublished data),

indicating that the activation of JR3 through this alternative

wound signalling pathway requires perception of JA. This

requirement would provide the fine tuning in the regulation

of JR3 expression, as it would be switched off once JA

levels return to basal values. Furthermore, the time–course

of expression of JR3 upon wounding (Titarenko et al., 1997)

mirrors the wound-induced increase in the levels of JA in

A. thaliana (Laudert et al., 1996). Thus, JR3 expression

seems to be tightly linked to the presence of levels of JA

above basal values, consistent with its proposed function

in attenuating the JA-mediated response and maintaining

the JA-to-auxin ratio in the plant.

The characterization of reversible protein phosphoryla-

tion switches has allowed the definition of two partially

divergent signalling pathways involved in wound-induced

gene expression. The wealth of sequence information and

molecular genetic tools available in A. thaliana will provide

direct access to the identified components of wound signal-

ling pathways. This will enable their use in reverse genetic

approaches to test the proposed model in an effort comple-

mentary to standard mutational analyses.

Experimental procedures

Plant material and treatments

Seeds of Arabidopsis thaliana ecotype Landsberg erecta (Lehle

Seed Co., Tucson, AZ) were surface sterilized and sown in 24-well

(20 seeds well–1) tissue culture clusters (Costar Corp., Cambridge,

MA), containing 1 ml well–1 of sterile MS medium (Murashige and

Skoog salts; ICN Hubber, Barcelona, Spain) supplemented with

0.5% sucrose, and grown with shaking (150 r.p.m.) in a culture


room under 16-h day (26°C)/8-h night (22°C) diurnal cycles. Fresh

medium, 500 µl well–1, was added 8 days after sowing and experi-

ments were conducted 2 days later.

Jin1, jin4 mutants and their respective wild-type backgrounds

(Columbia ecotype for jin1 and Wassilewskija ecotype in the case

of jin4) were grown (12 seeds well–1) under constant light and

otherwise similar conditions to the Landsberg erecta ecotype

plantlets.

Coi1 mutant seeds were grown for selection on agar plates

containing Murashige and Skoog medium supplemented with 2%

sucrose and 10 µM methyl-JA, under 16-h day (26°C)/8-h night

(22°C) diurnal cycles. Plants were selected on the 6th day post-

germination, transferred to microplates (3 plants well–1) and grown

in the same conditions as the Landsberg erecta ecotype for 7

additional days. Seeds from the corresponding wild-type back-

ground (Columbia) were grown similarly, except that agar plates

did not contain JA.

Prior to treatment initiation, the medium remaining in the wells

was removed and 1 ml well–1 of fresh medium was added. The

different compounds tested were diluted directly in the wells to

their final concentration from stock solutions prepared as follows:

JA (mixed isomers; Apex Organics Ltd, Devon, UK) was diluted

in N,N dimethyl-formamide to obtain 50 mM stock solutions;

okadaic acid, staurosporine and cycloheximide (Sigma-Aldrich

Quimica, Madrid, Spain) were diluted in DMSO to obtain 1 mM,

1 mM and 50 mM stock solutions, respectively.

Wounding was performed by thoroughly crushing the plantlets

with a forceps and subsequent incubation in liquid medium for

the indicated times.

All experiments were independently performed at least three

times, yielding highly reproducible results.

RNA extractions and Northern hybridization

Total RNA from A. thaliana plantlets was isolated and

separated through 1,5% agarose/formaldehyde gels (10 µg lane21)

as described elsewhere (Logemann et al., 1987). RNAs were sub-

sequently blotted onto Hybond-N membranes (Amersham Corp.)

following standard procedures (Sambrook et al., 1989). Equal RNA

loading was assayed by visualizing the ethidium bromide-stained

ribosomal RNA content (not shown), and hybridization of the blots

with a cauliflower 18S ribosomal DNA probe. The inserts of clones

to be used as probes were 32P labelled by the rediprime labelling

system (Amersham Corp.) and hybridized in 0.25 M phosphate

buffer, 0.25 M NaCl, 1 mM EDTA, 7% SDS, 10% polyethylene glycol

6000, 40% formamide, 0.2 mg ml–1 denatured salmon sperm DNA,

at 42°C. Filters were washed in 33SSC, 0.5% SDS at 65°C (13SSC:

150 mM NaCl, 15 mM sodium citrate) and exposed for autoradio-

graphy.

For hybridizations with the different probes, replica blots were

used and hybridized with at most three probes each. Previous

labelling was stripped by washing twice with 0.5% SDS in distilled

water at 65°C for 30 min. The complete removal of the radioactive

probe was assessed by autoradiography.

PP1/PP2A activity assay

Plantlets grown for 10 days in liquid culture in microplates were

frozen in liquid N2, ground to a fine powder and homogenized for

90 sec in 0.7 ml of ice-cold 50 mM Tris–HCl (pH 7.0 at 25°C), 0.1 mM

EDTA, 0.1 mM EGTA, 0.5% Triton, 1 mM PMSF, 1 µg ml–1 leupeptine,

20 µg ml–1 pepstatin and 0.2% (v/v) 2-mercaptoethanol. Extracts

were centrifuged at 15 000 g (4°C) for 10 min, and the super-


natant was recovered and assayed for PP1/PP2A activity using a

protein phosphatase assay system (BRL-Life Technologies, Inc.,

Madrid, Spain). Protein concentration was determined using the

protein assay reagent (Bio-Rad Laboratories SA, Madrid, Spain)

and equal amounts of protein from the different extracts were

diluted 1:150 in phosphatase assay buffer (provided in the assay

system) and incubated for 15 min at 30°C with glycogen phos-

phorylase A, freshly labelled with [γ-32P] ATP (3000 Ci mmol–1) as

described in the protocol provided by the manufacturer. Reactions

were performed within the linear range of PP1/PP2A activities

(data not shown). Okadaic acid and tautomycin (1 mM stock

solution in DMSO) were preincubated with the extracts for 10 min

before adding phosphorylase A and allowing the reaction to

proceed.

Acknowledgements

The excellent technical assistance from Tomas Cascon is

gratefully acknowledged. We also wish to thank Ines Poveda

and Angel Sanz for the photographic work. Many thanks to Drs

Isabel Merida, John Mundy and Julio Salinas for their comments

on the manuscript. The hydroperoxide lyase (stock number

94 J16T7) and vegetative storage protein (stock number

108B11T7) clones were obtained from the Arabidopsis Biological

Resource Center at the Ohio State University. The cauliflower

18S ribosomal DNA and the Arabidopsis coi1 seeds were kindly

provided by Drs Julio Salinas and John G. Turner, respectively.

This work was funded in part by the European Communities’

BIOTECH Programme, as part of the Project of Technological

Priority 1993–96. Financial support was also provided by the

Spanish Comision Interministerial de Ciencia y Tecnologıa grants

BIO93–0678-CO2–02, BIO94–1502-CE, and BIO96–0532-CO2–01.

G. Vancanneyt and J. Leon were recipients of a postdoctoral

fellowship and contract, respectively, from the Spanish Ministerio

de Educacion y Ciencia.

References

Albrecht, T., Kehlen, A., Stahl, K., Knofel, H.D., Sembdner, G. and

Weiler, E.W. (1993) Quantification of rapid, transient increases in

jasmonic acid in wounded plants using a monoclonal antibody.

Planta, 191, 86–94.

Anderson, J.M., Spilatro, S.R., Klauer, S.F. and Franceschi, V.R.

(1989) Jasmonic acid-dependent increase in the level of

vegetative storage proteins in soybean. Plant Sci. 62, 45–52.

Bartel, B. and Fink, G.R. (1995) ILR1, an amidohydrolase that

releases active indole-acetic acid from conjugates. Science 268,

1745–1748.

Bell, E., Creelman, R.A. and Mullet, J.E. (1995) A chloroplast

lipoxygenase is required for wound-induced jasmonic acid

accumulation in Arabidopsis. Proc. Natl Acad. Sci. USA, 92,

8675–8679.

Berger, S., Bell, E. and Mullet, J.E. (1996) Two methyl jasmonate-

insensitive mutants show altered expression of AtVsp in

response to methyl jasmonate and wounding. Plant Physiol.

111, 523–531.

Berger, S., Bell, E., Sadka, A. and Mullet, J.E. (1995) Arabidopsis

thaliana Atvsp is homologous to soybean VspA and VspB,

genes encoding vegetative storage protein acid phosphatases,

and is regulated similarly by methyl jasmonate, wounding,

sugars, light and phosphate. Plant Mol. Biol. 27, 933–942.

Bergey, D.R., Howe, G.A. and Ryan, C.A. (1996) Polypeptide


signaling for plant defensive genes exhibits analogies to defence

signaling in animals. Proc. Natl Acad. Sci. USA, 93, 12053–12058.

Bialojan, C. and Takai, A. (1988) Inhibitory effect of a marine-

sponge toxin, okadaic acid, on protein phosphatases. Biochem.

J. 256, 283–290.

Bush, D.S. (1995) Calcium regulation in plant cells and its role in

signaling. Annu. Rev. Plant Physiol. Plant Mol. Biol. 46, 95–122.

Christie, J.M. and Jenkins, G.I. (1996) Distinct UV-B and UV-A/

blue light signal transduction pathways induce chalcone

synthase gene expression in Arabidopsis cells. Plant Cell, 8,

1555–1567.

Cohen, P., Holmes, C.F.B. and Tsukitani, Y. (1990) Okadaic acid: a

new probe for the study of cellular regulation. Trends Biochem.

Sci. 15, 98–102.

Creelman, R.A., Tierney, M.L. and Mullet, J.E. (1992) Jasmonic acid/

methyl jasmonate accumulate in wounded soybean hypocotyls

and modulate wound gene expression. Proc. Natl Acad. Sci.

USA, 89, 4938–4941.

Dammann, C., Rojo, E. and Sanchez-Serrano, J.J. (1997) Abscisic

acid and jasmonic acid activate wound-inducible genes in potato

through separate, organ-specific signal transduction pathways.

Plant J. 11, 773–782.

DeWald, D.B., Sadka, A. and Mullet, J.E. (1994) Sucrose modulation

of soybean Vsp gene expression is inhibited by auxin. Plant

Physiol. 104, 439–444.

Dittrich, H., Kutchan, T. and Zenk, M.H. (1992) The jasmonate

precursor 12-oxo-phytodienoic acid, induces phytoalexin

synthesis in Petroselinum hortense cell cultures. FEBS Lett. 309,

33–36.

Farmer, E.E. and Ryan, C.A. (1992) Octadecanoic precursors of

jasmonic acid activate the synthesis of wound-inducible

proteinase inhibitors. Plant Cell, 4, 129–134.

Farmer, E.E., Johnson, R.R. and Ryan, C.A. (1992) Regulation of

expression of proteinase inhibitor genes by methyl jasmonate

and jasmonic acid. Plant Physiol. 98, 995–1002.

Felix, G., Regenass, M., Spanu, P. and Boller, T. (1994) The protein

phosphatase inhibitor calyculin A mimics elicitor action in

plant cells and induces rapid hyperphosphorylation of specific

proteins as revealed by pulse labeling with [33P]phosphate.

Proc. Natl Acad. Sci. USA, 91, 952–956.

Feys, B.J.F., Benedetti, C.E., Penfold, C.N. and Turner, J.G. (1994)

Arabidopsis mutants selected for resistance to the phytotoxin

coronatine are male sterile, insensitive to methyl-jasmonate,

and resistant to a bacterial pathogen. Plant Cell, 6, 751–759.

Gundlach, H., Muller, M.J., Kutchan, T.M. and Zenk, M.H. (1992)

Jasmonic acid is a signal transducer in elicitor-induced plant

cell cultures. Proc. Natl Acad. Sci. USA, 89, 2389–2393.

Harms, K., Atzorn, R., Brash, A., Kuhn, H., Wasternack, C.,

Willmitzer, L. and Pena-Cortes, H. (1995) Expression of a flax

allene oxide synthase cDNA leads to increased endogenous

jasmonic acid (JA) levels in transgenic potato plants but not to

a corresponding activation of JA-responding genes. Plant Cell,

7, 1645–1654.

Hildmann, T., Ebneth, M., Pena-Cortes, H., Sanchez-Serrano, J.J.,

Willmitzer, L. and Prat, S. (1992) General roles of abscisic acid

and jasmonic acid in gene activation as a result of mechanical

damage. Plant Cell, 4, 1157–1170.

Ishihara, H., Martin, B.L., Brautigan, D.L. et al. (1989) Calyculin A

and okadaic acid, inhibitors of protein phosphatases activity.

Biochem. Biophys. Res. Commun. 159, 871–877.

Jaramillo-Babb, V.L., Sugarman, J.L., Scavetta, R., Wang, S.J.,

Berndt, N., Born, T.L., Glass, C.K. and Schoenthal, A.H. (1996)

Positive regulation of cdc2 gene activity by protein phosphatase

type 2A. J. Biol. Chem. 271, 5988–5992.


Kakimoto, T. (1996) CKI1, a histidine kinase homolog implicated

in cytokinin signal transduction. Science, 274, 982–985.

Kernan, A. and Thornburg, R.W. (1989) Auxin levels regulate

the expression of a wound-inducible proteinase inhibitor II-

chloramphenicol acetyl transferase gene fusion in vitro and

in vivo. Plant Physiol. 91, 73–78.

Kieber, J.J., Rothenberg, M., Roman, G., Feldmann, K.A. and

Ecker, J.R. (1993) CTR1, a negative regulator of the ethylene

response pathway in Arabidopsis, encodes a member of the

Raf family of protein kinases. Cell, 72, 427–441.

Koda, Y., Kikuta, Y., Tazaki, H., Tsujino, Y., Sakamura, S. and

Yoshihara, T. (1991) Potato tuber-inducing activities of jasmonic

acid and related compounds. Phytochem. 30, 1435–1438.

Kuo, A., Cappelluti, S., Cervantes-Cervantes, M., Rodriguez, M.

and Bush, D.S. (1996) Okadaic acid, a protein phosphatase

inhibitor, blocks calcium changes, gene expression, and cell

death induced by gibberellin in wheat aleurone cells. Plant Cell,

8, 259–269.

Laudert, D., Pfannschmidt, U., Hollander-Czytko, H. and Weiler,

E.W. (1996) Cloning, molecular and functional characterization

of Arabidopsis thaliana allene oxide synthase (CYP 74), the first

enzyme of the octadecanoid pathway to jasmonates. Plant Mol.

Biol. 31, 323–335.

Leung, J., Bouvier-Durand, M., Morris, P., Guerrier, D., Chefdor, F.

and Giraudat, J. (1994) Arabidopsis ABA response gene ABI1:

features of a calcium-modulated protein phosphatase. Science,

264, 1448–1452.

Liscovitch, M. and Cantley, L.C. (1994) Lipid second messengers.

Cell, 77, 329–334.

Logemann, J., Schell, J. and Willmitzer, L. (1987) Improved method

for the isolation of RNA from plant tissues. Anal. Biochem. 163,

16–20.

Ma, H. (1994) GTP-binding proteins in plants: new members of an

old family. Plant Mol. Biol. 26, 1611–1636.

McConn, M. and Browse, J. (1996) The critical requirement for

linolenic acid is pollen development, not photosynthesis, in an

arabidopsis mutant. Plant Cell, 8, 403–416.

MacKintosh, C. and Cohen, P. (1989) Identification of high levels

of type 1 and type 2A protein phosphatases in higher plants.

Biochem. J. 262, 335–339.

MacKintosh, C. and Klumpp, S. (1990) Tautomycin from the

bacterium Streptomyces verticillatus. Another potent and

specific inhibitor of protein phosphatases 1 and 2A. FEBS Lett.

277, 137–140.

MacKintosh, C. and MacKintosh, R.W. (1994) Inhibitors of protein

kinases and phosphatases. Trends Biochem. Sci. 19, 444–448.

MacKintosh, C., Coggings, J. and Cohen, P. (1991) Plant protein

phosphatases: subcellular distribution, detection of protein

phosphatase 2C and identification of protein phosphatase 2A

as the major quinate dehydrogenase phosphatase. Biochem. J.

273, 733–738.

MacKintosh, C., Lyon, G.D. and MacKintosh, R.W. (1994) Protein

phosphatase inhibitors activate anti-fungal defence responses

of soybean cotyledons and cell cultures. Plant J. 5, 137–147.

Mason, H.S. and Mullet, J.E. (1990) Expression of two soybean


vegetative storage protein genes during development and in

response to water deficit, wounding and jasmonic acid. Plant

Cell, 2, 569–579.

Meyer, K., Leube, M.P. and Grill, E. (1994) A protein phosphatase

2C involved in ABA signal transduction in Arabidopsis thaliana.

Science, 264, 1452–1455.

O’Donnell, P.J., Calvert, C., Atzorn, R., Wasternack, C., Leyser,

H.M.O. and Bowles, D.J. (1996) Ethylene as a signal mediating

the wound response of tomato plants. Science, 274, 1914–1917.

Pelacho, A.M. and Mingo-Castel, A.M. (1991) Jasmonic acid

induces tuberisation of potato stolons cultured in vitro. Plant

Physiol. 97, 1253–1255.

Pena-Cortes, H., Albrecht, T., Prat, S., Weiler, E.W. and Willmitzer,

L. (1993) Aspirin prevents wound-induced gene expression in

tomato leaves by blocking jasmonic acid biosynthesis. Planta,

191, 123–128.

Pena-Cortes, H., Fisahn, J. and Willmitzer, L. (1995) Signals

involved in wound-induced proteinase inhibitor II gene

expression in tomato and potato plants. Proc. Natl Acad. Sci.

USA, 92, 4106–4113.

Pena-Cortes, H., Liu, X., Sanchez-Serrano, J.J., Schmid, R. and

Willmitzer, L. (1992) Factors affecting gene expression of patatin

and proteinase-inhibitor-II gene families in detached potato

leaves: implications for their co-expression in developing tubers.

Planta, 186, 495–502.

Raz, V. and Fluhr, R. (1993) Ethylene signal is transduced via

protein phosphorylation events in plants. Plant Cell, 5, 523–530.

Reinbothe, S., Mollenhauer, B. and Reinbothe, C. (1994) JIPs and

RIPs: the regulation of plant gene expression by jasmonates in

response to environmental cues and pathogens. Plant Cell, 6,

1197–1209.

Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular

Cloning. A Laboratory Manual, 2nd Edn. NY: Cold Spring Harbor

Laboratory Press.

Sembdner, G. and Parthier, B. (1993) The biochemistry and the

physiological and molecular actions of jasmonates. Annu. Rev.

Plant Physiol. Plant Mol. Biol. 44, 569–589.

Seo, S., Okamoto, M., Seto, H., Ishizuka, K., Sano, H. and Ohashi,

Y. (1995) Tobacco MAP kinase: a possible mediator in wound

signal transduction pathways. Science 270, 1988–1992.

Smith, R.D. and Walker, J.C. (1996) Plant protein phosphatases.

Annu. Rev. Plant Physiol. Plant Mol. Biol. 47, 101–125.

Smith, R.D., Wilson, J.E., Walker, J.C. and Baskin, T.I. (1994)

Protein phosphatase inhibitors block root hair growth and alter

cortical cell shape of Arabidopsis roots. Planta, 194, 516–524.

Tamaoki, T., Nomoto, H., Takahashi, I., Kato, Y., Morimoto, M. and

Tomita, F. (1986) Staurosporin, a potent inhibitor of

phospholipid/Ca21 dependent protein kinase. Biochem. Biophys.

Res. Commun. 135, 397–402.

Thornburg, R.W. and Li, X. (1991) Wounding Nicotiana tabacum

leaves causes a decline in endogenous indole-3-acetic acid.

Plant Physiol. 96, 802–805.

Titarenko, E., Rojo, E., Leon, J. and Sanchez-Serrano, J.J. (1997)

JA-dependent and independent signalling pathways control

wound-induced gene activation in Arabidopsis thaliana. Plant

Physiol. 115, 817–826.

reversible protein phosphorylation regulates jasmonic acid-dependent and -independent wound signal...

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