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Plant Science 180 (2011) 686–693

Contents lists available at ScienceDirect

Plant Science

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ounding systemically activates a mitogen-activated protein kinase in foragend turf grasses

ames E. Dombrowskia,∗, Sarah R. Hindb, Ruth C. Martina, Johannes W. Stratmannb

USDA-ARS, National Forage Seed Production Research Center, 3450 SW Campus Way, Corvallis, OR 97331, United StatesUniversity of South Carolina, Department of Biological Sciences, Columbia, SC 29208, United States

r t i c l e i n f o

rticle history:eceived 30 September 2010eceived in revised form 15 January 2011ccepted 20 January 2011

a b s t r a c t

Forage and turf grasses are continually cut and grazed by livestock, however very little is known concern-ing the perception or molecular responses to wounding. Mechanical wounding rapidly activated a 46 kDaand a 44 kDa mitogen-activated protein kinase (MAPK) in six different grass species. In the model grassspecies Lolium temulentum, the 46 kDa MAPK was rapidly activated within 5 min of wounding both locally

vailable online 28 January 2011

eywords:APK

ystemic signalounding

and systemically in an adjacent unwounded tiller. This indicates that wounding generates a rapidly prop-agated long-distance signal that activates a MAPK in the distal portions of the plant. This 46 kDa MAPKactivity was not enhanced by the addition of the pathogen-associated signal salicylic acid (SA) to thewound site nor induced when exposed to methyl jasmonate (MJ), which is a potent inducer of the woundresponse in dicotyledonous plants. However, pretreatment with MJ increased the wound-induced activity

the a

rassolium temulentum

of the 44 kDa MAPK over

. Introduction

Plants are exposed to a wide array of environmental signals,hich they perceive through receptor and sensor proteins. Sinceost environmental signals are fairly specific, the number of sen-

ory proteins closely matches the number of signals. However, athe level of signal transduction, plant cells integrate input sig-als and employ common signaling components and moduleshat interact in a complex signaling network. Important converg-ng nodes for stress signals are mitogen-activated protein kinaseMAPK) modules, which consist of three functionally linked kinases.hese MAPK signaling cascades regulate various cellular activities,uch as gene expression, defense pathways, differentiation, growth,nd cellular survival. MAPKs are activated through phosphorylation

y MAPK kinases (MAPKKs or MEKs), which in turn are activatedy MAPKK kinases (MAPKKK or MEKKs or MP3Ks). The Arabidop-is MPK3 and MPK6, and their orthologs in other plants, such as

IPK and SIPK in tobacco, and MPK3 and MPK1/2 in tomato, are

Abbreviations: ABA, abscisic acid; ERK, extracellular signal regulated kinase; GTP,uanosine triphosphate; JA, jasmonic aci0d; Lt, Lolium temulentum; MAPK, mitogen-ctivated protein kinase; MJ, methyl jasmonate; NADPH, nicotinamide adenineinucleotide phosphate; PAGE, polyacrylamide gel electrophoresis; ROS, reactivexygen species; SA, salicylic acid; SDS, sodium dodecyl sulfate; UV, ultraviolet.∗ Corresponding author at: USDA-ARS, National Forage Seed Production Researchenter, Oregon State University, 3450 SW Campus Way, Corvallis, OR 97331-7102,nited States. Tel.: +1 54 17 384 147; fax: +1 54 17 384 160.

E-mail address: Jim.Dombrowski@ars.usda.gov (J.E. Dombrowski).

168-9452/$ – see front matter. © 2011 Published by Elsevier Ireland Ltd.oi:10.1016/j.plantsci.2011.01.010

ctivity in control plants.© 2011 Published by Elsevier Ireland Ltd.

the best characterized MAPKs and have been shown to be acti-vated by almost any disturbance of cellular homeostasis [1–7].The tomato MPK1 and MPK2 have been shown to be activatedby damage-related signals such as touch, mechanical wounding,herbivory, and systemin and systemin-like wound signaling pep-tides; by microbe-associated molecular patterns (MAMPs) suchas oligouronides, chitin, and chitosan, and a cell wall elicitor ofPhytophthora megasperma; by race-specific elicitors such as Avr4and Avr9 from the fungus Cladosporium fulvum and AvrPto fromPseudomonas syringae; and by ultraviolet radiation [8–16]. Thisraises the question as to how these MAPKs relay the input sig-nals to achieve a relatively specific output response. Recently, ithad been demonstrated that MAPKs have the potential to phos-phorylate a relatively high number of substrate proteins [15,17,18].This is thought to occur in a signal-specific manner, but the mecha-nisms that might lead to signal-specific substrate phosphorylationby MAPKs are not well understood in plants [6].

While MAPKs in monocot plants are not as well characterizedas the Arabidopsis, tobacco, and tomato MAPKs, a better pictureis now emerging for rice MAPKs. The rice genome contains 17MAPKs (OsMPKs) versus the 20 and 21 MAPKs of Arabidopsis andpoplar, respectively [19,20]. Furthermore, 10 of the OsMPKs fallinto the D group of MAPKs that is characterized by a TDY phos-

phorylation motif. These D group rice MAPKs have been shown torespond to stress, including wounding and pathogens [21]. It wasestimated that about 50% of all rice MAPKs are transcriptionallyregulated by pathogen-related stress [22]. The D-group OsBWMK1(OsMPK17-1 or OsMPK12), was shown to be transcriptionally acti-

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J.E. Dombrowski et al. / Pla

ated by Magnaporthe grisea infection and mechanical wounding23,24].

In contrast A, B, and C-group MAPKs possess a TEY phosphoryla-ion motif, much like the mammalian extracellular signal-regulated

APKs (ERKs). The rice orthologs of AtMPK3 and AtMPK6 haveeen identified. Unfortunately, two different nomenclatures wereuggested for rice MAPKs to streamline previous confusing names19,20]. The AtMPK3 ortholog is called either OsMPK3 or OsMPK5here OsMPK3/5) and the AtMPK6 ortholog is called OsMPK6 orsMPK1 (here OsMPK6/1). While AtMPK3 orthologs are gener-lly activated at the transcriptional and posttranslational level,ranscripts of AtMPK6 and orthologs generally do not increase inesponse to signals that induce their activity. OsMPK3/5 (previouslyalled OsMAP1; OsMAPK2; OsMRSMK2; OsBIMK1; OsMAPK5)esponds at the transcriptional level to many stress signals includ-ng wounding [25], and positively regulates cold, drought and salttress, but negatively regulates resistance to bacterial and fun-al pathogens [26]. OsMPK6/1 functions as a negative regulator ofacterial resistance [27] and also responds at the transcriptional

evel to various stresses and signaling molecules such as JA, SA,thephon (ethlylene), ABA, H2O2, and chitosan; however, it didot respond to wounding, UV-C radiation, ozone, heat and drought28]. Although it was not determined if transcriptional activationorresponds to the induction of kinase activity, the study doesndicate that regulation of orthologous MAPKs in monocots andicots may have evolved differently. Like the dicot MPK3 and MPK6rthologs, OsMPK3/5 and OsMPK6/1 respond with an increase innzyme activity in response to wounding while protein levelsemain constant [29]. Taken together, this shows that woundingas the potential to activate different groups of rice MAPKs athe level of gene expression, but it remains to be determined ifncreased gene expression strictly correlates with increasing MAPKctivity.

Some stress signals are inflicted and perceived locally, butesponses to these signals occur systemically, often at the wholelant level. In addition to proteins such as kinases, receptors, phos-holipases, GTPases, ion channels, NADPH oxidase, calmodulin,alcium binding proteins and transcription factors utilized by theignaling pathways, plants use a variety of small molecules such asasmonic acid (JA) derivatives, salicylic acid (SA), reactive oxygenpecies (ROS), ABA and calcium to mediate responses to stress ortimuli [30–33]. The molecular characterization of wounding wasrst described and has been most extensively studied in tomatolants. The wound signaling peptide systemin had originally beenonsidered a mobile systemic wound signal produced in responseo wounding of tomato leaves [34]. However, extensive graftingxperiments demonstrated that the function of systemin in the sys-emic wound response is to induce the synthesis of jasmonic acidr a JA derivative which functions as the long-distance signal thatctivates the wound response in distant target leaves [35,36]. Inrabidopsis, it has been shown that the synthesis of JA-isoleucine,

he bioactive form of JA that interacts with the JA receptor complex,ncreases systemically in neighboring leaves within minutes after

ounding. Further evidence suggested that a physical systemic sig-al may cause the rapid systemic increase in JA-Ile levels [37]. Inddition to JA derivatives, hydrogen peroxide has also been showno function as an essential component of systemic wound signalingn Arabidopsis [38]. Other systemic wound signals include rapidlyropagated mechanical signals and electrical signals [14,39–41].

In monocots, systemic responses have rarely been studied.nlike most cereal grasses, forage and turf grasses are continu-

lly being cut and grazed by animals. Currently very little is knownoncerning the perception or molecular responses to wounding inhese forage and turf grasses and how these responses affect persis-ence and the long-term nutritional quality of the grass. We reportere that mechanical wounding in forage and turf grass species

ence 180 (2011) 686–693 687

rapidly activated a 46 kDa and a 44 kDa MAPKs and generated arapidly propagated long-distance signal that activated the 46 kDaMAPK in the distal unwounded portions of the plant.

2. Materials and methods

2.1. Plant materials

Festuca arundinacea Schreb. (Tall fescue) cv. Kentucky 31non-infected and infected with the endophyte Neotyphodiumcoenophialum; Dactylis glomerata L. (Orchardgrass) cv. Hallmark,Lolium multiflorum Lam. (Annual ryegrass) cv. Surrey: Loliumperenne L. (Perennial ryegrass) cv. Manhattan and Derby Supreme;Lolium temulentum L. (Darnel ryegrass) cv. Ceres and Brachypodiumdistachyon cv. Bd21 seeds were planted in 4 inch square pots or in3X3X4 inch pots containing SB40 Sunshine Growing Mix (Sun GroHorticulture, Canada). Plants were fertilized weekly using Techni-gro 20-18-20 all-purpose fertilizer (Sun Gro Horticulture, Canada).Plants were grown in a Conviron E15 (Conviron, Winnipeg, Canada)growth chamber under 8 h and 12 h photo-periods at 21 ◦C day and17 ◦C night.

2.2. Plant treatments

2.2.1. WoundingPlants were removed from the growth chamber and brought

into the lab after a minimum of 3 h of light, and allowed to restundisturbed on the bench for a minimum of 2 h prior to treatment.Three to four-week-old plants were wounded perpendicular to thetissue using a hemostat. The region of the plant or specific tillerthat was wounded for each experiment is illustrated in each fig-ure in the experimental results. For plants subjected to multiplewounds, it took less than 5 s to inflict the 3 consecutive woundsto the plant or tiller. The entire aerial portion of a single plant ortiller was collected for each time point, except for the experimentsdescribed in Fig. 3. In experiments described in Fig. 3, the tissuesamples collected for each time point and region were derived from3 different plants. The wounded tissue collected in Region 2 shownin Fig. 3 also included approximately 5–8 mm2 of unwounded tis-sue on both sides of the 1 cm2 wound site. All tissues were collectedat the times indicated after wounding or treatment, placed in foilpackets, quick frozen in liquid nitrogen and stored at −80 ◦C untilprocessed.

2.2.2. Catalase and salicylic acidTen or 15 �L of sample was pipetted onto the junction between

the leaf blade-collar and the pseudostem of three-week-old L.temulentum plants. Samples tested: 10 �g/�L BSA FRAC V (Sigma,A3294-100G); 10 �g/�L catalase from bovine liver (Sigma C1345-1G); 1 mM and 100 �M salicylic acid (Sigma, 24,758-8) in 20 mMsodium phosphate pH 6.8; 20 mM sodium phosphate pH 6.8 anddeionized water. The tiller was then wounded once at the applica-tion site.

2.2.3. Methyl jasmonateThree-week-old L. temulentum plants were placed in a 10 in.

tall × 9 in. diameter plexiglass cylinder fitted with a lid. Plants wereeither exposed to MJ vapor produced by pipetting 10 �L of a 1:1ethanol/MJ (v/v) (Sigma, Cat # 392707) mixture onto a cotton

a 2:1 MJ/ethanol mixture directly onto the plant and incubatingthe plants in a closed chamber for the time indicated. In someexperiments 10 min after application of the MJ the tiller was thenwounded once at the application site and incubated in the closedplexiglass cylinder for the times indicated.

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6 nt Science 180 (2011) 686–693

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Fig. 1. Wound activation of a p46 and p44 MAPK in different grass species. Dif-ferent grass species were wounded 3 times across all tillers using a hemostat asillustrated above. The entire aerial portion of the plant (one plant per time point)was collected over a 2-h period. To determine MAPK activity, immunoblots of pro-tein extracts were performed using anti-phospho-MAPK (Erk1/2) antibody. TF E−:tall fescue without endophyte; TF E+: tall fescue with endophyte; OG: orchard-grass; PRG: perennial ryegrass; ARG: annual ryegrass; Lt: Lolium temulentum; Bd:

88 J.E. Dombrowski et al. / Pla

.3. Sample preparation

Frozen tissues were transferred to a mortar and homogenized in00–1000 �L extraction buffer (50 mM HEPES·KOH, pH 7.6, 2 mMTT, 1 mM EDTA, 1 mM EGTA, 20 mM glycerophosphate, 20% glyc-rol, 4 mM Na3V04, 2 mM NaF and 1 complete proteinase inhibitorocktail tablet [Boehringer Mannheim, IN] per 50 mL) dependingn the amount of tissue collected to be extracted. Some samplesith a large amount of tissue were ground and then only a por-

ion of the resulting powder was extracted. Homogenates wereentrifuged at 14,000 rpm for 20 min at 4 ◦C in a microcentrifuge.rotein concentrations in the supernatants were determined withhe Pierce (Rockford, IL) BCA protein assay. Protein extracts (50 �gf extract/time point) were fractionated on 10% SDS–PAGE gels0.75 mm thickness). Duplicate gels were run for some of the sam-le preps. These gels were stained with Coomassie Blue to confirmhe equal loading of protein per lane on the SDS–PAGE gels and toerify the accuracy of the BCA protein assay.

.4. MAPK activation assays

.4.1. Immunoblot analysisProtein extracts were fractionated (50 �g per lane) on 10%

DS–PAGE gels. The separated proteins were electro-transferrednto Immobilon-P (Millipore) membranes. The consistency of pro-ein transfer onto the membranes was confirmed by Ponceau Staining. Membranes were blocked for 1 h at RT with 5% (w/v)SA, 1× Tris buffered saline (TBS), 0.1% Tween-20 with gentlehaking. The primary antibody [Phospho-p44/42 MAPK (Erk1/2)Thr202/Tyr204) (D13.14.4E) XPTM Rabbit mAb (cat# 4370) fromell Signaling Technology, Beverly MA] was diluted (1:2000) in% (w/v) BSA (fraction V), 1× TBS, 0.1% Tween-20. Membranesere gently shaken with the diluted antibody at 4 ◦C overnight.

he blots were then washed 3 times for 12 min/wash with 1× TBS,.1% Tween-20 at RT with gentle shaking. The blots were then incu-ated with the secondary antibody (Sigma Goat anti Rabbit Alkalinehosphatase conjugate #A3937) diluted (1:2000) in 1% (w/v) BSAFraction V), 1× TBS, 0.05% Tween-20 at RT in the dark for 2 h withentle shaking. The blots were washed 3 times for 12 min/washith 1× TBS, 0.1% Tween-20 at RT with gentle shaking, and theneveloped using a standard nBT/BCIP substrate mixture.

.4.2. In-gel kinase assaysThe In-gel kinase assays were performed as described by Strat-

ann and Ryan [14]. The resulting gels were dried betweenellophane sheets and the radioactive signals were analyzed byutoradiography.

. Results

.1. Wound activation of a p46 MAPK in different grass species

To determine if a MAPK is associated with wound signaling inorage and turf grass grasses, various grass species were woundedith a hemostat and the activation of MAPKs was investigated.

hosphorylation of the tyrosine and serine/threonine residues inhe activation motif TE/DY of many MAPKs is an indicator of itsctivation. To determine the state of phosphorylation/activation ofhe MAPK, we used an anti-phospho-ERK (phospho-p44/42 MAPK)ntibody which has been used in many studies to test MAPK activityn plants [42–44]. This antibody, specifically detects MAPKs phos-

horylated on either both the threonine and tyrosine residues orn the threonine alone in the TEY motif. Plant MAPKs of the A, B,nd C group all contain the TEY phosphorylation motif, but onlyrabidopsis MPK3 and 6 and their orthologs in other species can beonsistently detected by this antibody [43,44].

Brachypodium distachyon. All experiments were repeated at least 3 times using sam-ples from independent experiments for each grass species. Representative blots arepresented.

The immunoblot analysis in Fig. 1 shows the pattern of phos-phorylation of MAPKs in response to wounding in different grassspecies over a 2-h period. Tall fescue (F. arundinacea Schreb) andorchardgrass (D. glomerata L.) are widely used as forage grasses,whereas annual (L. multiflorum Lam.) and perennial (L. perenne L.)ryegrasses are commonly used for both turf and forage applica-tions. L. temulentum [45,46], commonly known as darnel ryegrass,and B. distachyon [47] are currently being utilized as model grassspecies. In all grasses tested, the activation of a MAPK, with anapparent molecular weight of 46 kDa, was generally observed 5 minafter wounding and began to wane around 15 min post-wounding.It should be noted that in all species tested, we would occasion-ally observe activation of the 46 MAPK 2 min after wounding. Inperennial ryegrass and L. temulentum, activation was observed asearly as 2 min after wounding, and in perennial ryegrass, it lastedas long as 45 min post-wounding. In addition to the p46 MAPK,another MAPK with an apparent molecular weight of 44 kDa (p44

MAPK), was found to be activated within 30 min after woundingand was found to persist as long as 60 min post-wounding in mostof the grass species tested. Since grass-endophyte symbiosis hasbeen shown to enhance the host plant’s tolerance to multiple biotic

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J.E. Dombrowski et al. / Plant Science 180 (2011) 686–693 689

Fig. 2. MAPK response to various wound stimuli and touch in the model grassspecies L. temulentum. (A) Three to four-week-old plants were wounded one timewith a hemostat either at a single location or at 3 different sites across all tillersas indicated in the illustration. (B) Plant tillers were either wounded one time at3 different locations on the plant using a hemostat or touched by gently press-ing between two fingers without crushing at the location indicated above. Theentire aerial-portion of the plant/tiller (one plant/tiller per time point) was collectedover a 2-h period. To determine MAPK activity, immunoblots of protein extractswrR

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Fig. 3. Activation of a p46 and p44 MAPK in tissues adjacent to the wound sitein L. temulentum. Three-week-old plants at the single tiller stage were wounded3 times in Region 2 at intervals of 1 s per wound resulting in a 1 cm2 wound site(see illustration). Tissue samples were collected over a 2-h period for the 3 dis-tinct regions shown (see illustration). Protein extracts for each time point andregion were derived from the combined tissue collected from 3 different plants. Todetermine MAPK activity, immunoblots of protein extracts were performed using

ere performed using anti-phospho-MAPK (Erk1/2) antibody. All experiments wereepeated a minimum of 3 times using samples from independent experiments.epresentative blots are presented.

nd abiotic stresses [48,49] we wanted to see if the presence of thendophyte would affect the wound activation of the MAPK. How-ver, when comparing the wound activation of the p46 MAPK inndophyte infected verses non-infected tall fescue plants, we foundo significant difference in the pattern of wound activation of thewo MAPKs.

.2. Sensitivity of MAPK activation in L. temulentum

Grass species are generally polymorphic, obligate out-crossershat are self-incompatible, which results in considerable geneticiversity between individual plants of the same variety. This het-rogeneity between individual plants can result in inconsistent orndesirable variation between plants when investigating a partic-lar response to a treatment or stress. In contrast, L. temulentumLt) and B. distachyon are diploid self-fertile grass species that havenbred lines available [43–45]. Since both of these species exhib-ted wound activation of the MAPK (Fig. 1), we chose Lt for furthernalysis since it is member of the Lolium genus and it possessesorphological traits more similar to common forage and turf grass

pecies. To further investigate the wound activation of the p46APK, we determined the sensitivity of Lt plants to wounding. As

hown in Fig. 2A, multiple wounds (one wound at 3 different siteser plant) generated a much stronger activation of both the p44 and46 MAPKs when compared to a single wound. In addition, whenssaying the entire tiller for MAPK activation, the intensity of acti-ation was similar when the 3 wounds were dispersed across thelant/tiller as shown in Fig. 2 or concentrated in the middle of the

iller as shown in Fig. 3. In addition, gently touching plants betweenhe fingertips at locations similar to those that were wounded usinghemostat failed to activate the MAPKs above background levels

Fig. 2B).

anti-phospho-MAPK (Erk1/2) antibody. The experiment was repeated at least 3times using samples from independent experiments. Representative blots are pre-sented.

3.3. Systemic wound activation of p46 MAPK

To determine if the p46 and p44 MAPKs are activated in adja-cent unwounded tissues on the same tiller, Lt plants were wounded3 times in an approximate 1 cm2 area in the middle of the tiller(Region 2). The activation of MAPKs at the wound site and the adja-cent tissues (Regions 1 and 3) were investigated. As shown in Fig. 3,there is a strong increase in p46 MAPK activity in Region 2 (thewound site) from 5 min to 15 min after wounding, and the increasedactivity persisted for at least 60 min post-wounding. The p46 MAPKwas also activated in the adjacent unwounded portions of the tillerin the upper leaf tissues (Region 1) and in the pseudostem tissues(Region 3) within 5 min after wounding. The activity of the p46MAPK in Region 1 began to wane after 15 min and returned to back-ground levels by 30 min, while the p46 MAPK in Region 3 showeda similar activation pattern to the one observed in Region 2. In all3 regions the p44 MAPK was activated by 30 min after woundingand remained active until 60 min.

Upon detecting the activation of the MAPKs in the unwoundedsystemic tissues bordering the wound site (Regions 1 and 3, Fig. 3),we wanted to determine if a wound-generated signal could betransmitted through the root crown to an adjacent unwoundedtiller. Four-week-old Lt plants at the two-tiller stage were wounded3 times (1 wound per second) in the middle of the largest tiller asillustrated in Fig. 4. The wounded tiller displayed a typical responseof the p46 MAPK for Lt as shown in Fig. 4A. In this particular set ofexperiments, activation of the p44 MAPK could not be detected.Surprisingly, in the unwounded systemic tiller, the p46 MAPK wasactivated within 5 min of wounding and increased activity was stilldetectable 30 min post wounding.

To confirm that the phosphorylated MAPKs have enzyme activ-

ity and can actively phosphorylate an artificial substrate protein(myelin basic protein), an in-gel kinase assay was performed. Theidentical tissue samples used for the immunoblot analysis withanti-phospho-ERK antibodies (Fig. 4A) were also used for the in-

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690 J.E. Dombrowski et al. / Plant Science 180 (2011) 686–693

Fig. 4. Systemic MAPK activation in unwounded tiller adjacent to the woundedtiller in L. temulentum. Four-week-old plants at the two-tiller stage, were wounded3 times (1 wound per second) in the middle of the largest tiller in an 1 cm2 area.Extreme care was taken not to disturb the smaller second tiller, as illustrated.The wounded and unwounded systemic tillers were collected over a 2-h period.(A) To determine MAPK activity, immunoblots of protein extracts were performedusing anti-phospho-MAPK (Erk1/2) antibody. (B) The identical protein samplesused for the immunoblots were also analyzed by an in-gel kinase assay using MBP(ii

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Fig. 5. Catalase reduces wound-induced p46 MAPK activation in L. temulentum.Three-week-old plants at the single tiller stage were wounded once across the tillerusing a hemostat with no prior application or after pipetting 10 �L BSA (10 �g/�LBSA FRAC V) or catalase (10 �g/�L) onto the plant at the area to be wounded (seeillustration). Samples were collected over a 2-h period. To determine MAPK activ-

while the pretreatment of plants with MJ 10 min prior to wound-ing did not enhance the activation of the p46 MAPK, it enhancedthe wound-induced activation of the p44 MAPK and activatedthe p44 MAPK 15 min earlier as compared to wounded controls(Fig. 7B).

Fig. 6. Salicylic acid did not alter wound-induced MAPK activity in L. temulentum.Fifteen microliters of 100 �M salicylic acid solution in 20 mM sodium phosphate pH6.8 or 20 mM sodium phosphate pH 6.8 was pipetted onto three-week-old plants

myelin basic protein) as an artificial MAPK substrate. The experiment for both themmunoblot and the in-gel kinase assays were repeated twice using samples fromndependent experiments. Representative blots are presented.

el kinase assay. As shown in Fig. 4B, the time course of p46 MAPKctivity in the wounded and systemic tillers were very similar tohe time courses obtained in the immunoblot experiments (Fig. 4A).hese results indicate the corresponding phosphorylated MAPKsdentified by immunoblot analysis are active and able to phospho-ylate a potential substrate.

.4. Catalase reduces MAPK activation

It has been shown that an oxidative burst occurs after woundingn many plant species [38,50,51]. To determine if hydrogen per-xide plays a role in the activation of the p46 MAPK, catalase, annzyme that rapidly breaks down hydrogen peroxide to water andxygen, was applied to the plant. The tiller was then wounded athe same location where the catalase droplet was applied (see illus-ration Fig. 5). Fig. 5 shows that the activation of the p46 MAPK waseduced but not totally suppressed, when catalase was present athe time of wounding as compared to the application of BSA oround controls. The presence of catalase did not appear to signifi-

antly affect the p44 MAPK.

.5. MJ and SA do not activate the p46 MAPK

In plants, many signaling components have been shown to bectivated, or induced by various small signaling molecules. Fig. 6Ahows that the presence of the pathogen-associated signal SAt the time of wounding had no effect on the activation of the46 MAPK. When plants were exposed to methyl jasmonate (MJ),

ity, immunoblots of protein extracts were performed using anti-phospho-MAPK(Erk1/2) antibody. All experiments were repeated a minimum of 3 times usingsamples from independent experiments. Representative blots are presented.

the p46 and p44 MAPKs (Fig. 7A) were not activated. However,

prior to wounding the plants by closing a hemostat once across the leaves at the samelocation (see illustration). The entire aerial portion of the plant was collected for eachtime point at the times indicated for each treatment. To determine MAPK activ-ity, immunoblots of protein extracts were performed using anti-phospho-MAPK(Erk1/2) antibody. The experiment was repeated 3 times using samples from inde-pendent experiments. Representative blots are presented.

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Fig. 7. Methyl jasmonate potentiates p44 MAPK activity in L. temulentum. (A) Three-week-old plants at the one tiller stage were exposed to 3 �L of a 2:1 ethanol/methyljasmonate (MJ) mixture in an airtight plexiglass cylinder. Samples were collected atthe times indicated after start of exposure. ‘5W’ (last lane) denotes a positive controlof wound-activated p46 MAPK in L. temulentum—5 min after wounding with a hemo-stat. (B) In an airtight plexiglass cylinder, 3 �L of a 2:1 ethanol/methyl jasmonate(MJ) mixture or a 66% ethanol–water mixture were pipetted directly onto three-week-old plants. After 10 min incubation, the plants were wounded by a hemostatonce across the leaves at the same location as the pretreatment. The entire aerialportion of the plant was collected for each time point at the times indicated foreach treatment after wounding. To determine MAPK activity, immunoblots of pro-teR

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Acknowledgments

ein extracts were performed using anti-phospho-MAPK (Erk1/2) antibody. Thesexperiments were repeated 3 times using samples from independent experiments.epresentative blots are presented.

. Discussion

Wounding by mechanical cutting or by grazing animals is a typef stress that forage and turf grasses are constantly subjected to.urrently very little is known on how these grasses perceive andeact to wounding on the molecular level. Our results identified aound-activated p46 and p44 MAPK in six different grass species.

n the model grass species L. temulentum, we have shown that theAPK is activated locally and systemically within 5 min of wound-

ng. While systemic wound activation of MAPKs has been shown inicots, such as tomato, tobacco and Arabidopsis, to our knowledge,his is the first report of a systemically wound-activated MAPK in

onocots. These results indicate that a MAPK signaling cascade isctivated as a response to wounding in forage grasses. It will benteresting to determine if this wound-activated Lt p46 MAPK is anrtholog to the rice OsMPK3/5 or OsMPK6/1 and their correspond-ng orthologs in tomato, tobacco and Arabidopsis or if it is a memberf a different functional group.

The wound activation of the MAPKs in the forage and turf grasspecies may not be related to a defense response, since plant-enerated chemical barriers to herbivory have not been observedn many grasses, such as is displayed in many dicot species. Inrder to defend against herbivores or sucking insects and to deterrazing animals, some of these grasses have developed a mutual-stic symbiosis with endophytes. This symbiosis has been showno increase the plant’s tolerance to abiotic stresses and providerotection against herbivory through production of toxic alkaloids

52]. The response to wounding in forage grasses may be to ini-iate rapid growth to replenish lost tissues. We have observed aapid growth spurt in forage and turf grasses after cutting in ourreenhouses (over 1 cm/day). In rice, it was recently shown that

ence 180 (2011) 686–693 691

mechanical wounding increased the expression of a cell wall asso-ciated enzyme, and a 16% increase in leaf elongation was observedrelative to that of control seedlings [53]. It is tempting to speculatethat this growth after wounding is mediated by the p46 and p44MAPKs.

Wound signals are generated and perceived locally, butresponses to these signals can occur systemically in the distalportions of the plant. The architecture of grasses is probably notconducive for rapid vasculature-dependent signals. In our studies,the activation of the MAPK demonstrated the existence of a sys-temic signal and provided us a means to monitor the speed at whichthe wound-generated signal was transmitted. This signal needs tobe transmitted down a tiller into the root crown and then back upinto the adjacent tillers. The wound site was located 5–7 cm abovethe root crown and the adjacent systemic tiller that received the sig-nal had a total length of 20–25 cm from the root crown to the tip ofthe longest leaf. Using MAPK activity as a reporter, we determinedthat the wound generated signal took less than 5 min to traversethis distance, which included the time it takes to perceive the sig-nal and activate a signaling pathway resulting in MAPK activation.The speed of transmission (roughly 0.3 mm/s) of this systemic sig-nal would be consistent with JA-induced signaling [37,54], ROSpropagated [38], mechanical and electrical based signaling mech-anisms [14,39,41,55], but not a signal that would require transportof chemicals through the phloem [56].

The speed of the ROS generated signal in Arabidopsis (estimatedto be 1.4 mm/s) [38] coupled with the evidence of an oxidative burstafter wounding in ryegrass [51] led us to investigate if ROS wasinvolved in the wound activation of the Lt MAPKs. The addition ofcatalase, which is an inhibitor of ROS accumulation, to the woundsite reduced the activation of the p46 MAPK, however it did notfully suppress it. One potential explanation for this is that thereis more than one component to the signal being generated. Or thegeneration of the signal and its subsequent movement is faster thanthe ability of catalase to fully suppress it at the wound site. Anotherpossibility is that the solution containing the catalase was unable todiffuse rapidly enough to fully suppress the signal from activatingthe MAPK. These results suggest that ROS species may play a role inthe wound activation of the Lt MAPK and further research is neededto determine its actual role in wound signaling in forage and turfgrasses.

Other potential signaling molecules such as the pathogen-associated signal SA and methyl jasmonate (MJ), a potent inducerof the wound response in dicotyledonous plants, were investigatedto determine their effect on the activation of the Lt MAPK. We foundthat SA and MJ did not enhance or suppress the activation of the Ltp46 MAPK. However pretreatment with MJ prior to wounding didaccelerate and potentiate the wound activation of the p44 MAPK.This result suggests that MJ specifically regulates p44 MAPK activitythrough an unknown mechanism.

We have shown that mechanical wounding in forage and turfgrasses generates a long distance signal that systemically activateda MAPK in an adjacent tiller. This is to our knowledge the first reportof a wound signal being transmitted from one tiller to an adjacenttiller through the root crown in grasses. Future research will befocused on identifying what this particular wound signal is, howit is propagated and how it is able to transmit through the rootcrown architecture into the adjacent tiller, and to determine thedownstream responses to this wound signal.

Special thanks are extended to Vicky Hollenbeck (USDA-ARS,Corvallis, OR) for her assistance in performing the tissue extrac-tions and immunoblot analysis of the MAPKs, and to Dr. Lloyd T.

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vans (CSIRO, Canberra Australia) for his generous gift of the L.emulentum cv. Ceres seed. A portion of the research was supportedy National Science Foundation grant IOS-0745545 (to J.W.S.).xperimental methods performed in this research complied withurrent laws and regulations of the U.S.A. The use of trade, firm,r corporation names in this publication is for the information andonvenience of the reader. Such use does not constitute an offi-ial endorsement or approval by the United States Department ofgriculture or the Agricultural Research Service of any product orervice to the exclusion of others that may be suitable.

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