jasmonic acid does not mediate root growth responses to wounding in arabidopsis thaliana

Jasmonic acid does not mediate root growth responses towounding in Arabidopsis thalianapce_2062 104..116

LILIAN SCHMIDT1, GRÉGOIRE M. HUMMEL1, MATTHIAS SCHÖTTNER2, ULRICH SCHURR1 & ACHIM WALTER1

1Institut Phytosphäre (ICG-3), Forschungszentrum Jülich GmbH, 52425 Jülich, Germany and 2Max Planck Institute forChemical Ecology, Department of Molecular Ecology, Hans-Knöll-Strasse 8, 07745 Jena, Germany

ABSTRACT

Jasmonic acid (JA) is a crucial plant defence signallingsubstance that has recently been shown to mediateherbivory-induced root growth reduction in the ecologicalmodel species Nicotiana attenuata. To clarify whetherJA-induced reduction of root growth might be a generalresponse increasing plant fitness under biotic stress, a suiteof experiments was performed with the model plant Ara-bidopsis thaliana. JA bursts were elicited in leaves ofA. thaliana in different ways. Root growth reductionwas neither induced by foliar application of herbivore oralsecretions nor by direct application of methyl jasmonate toleaves. Root growth reduction was observed when leaveswere infected with the pathogen Pseudomonas syringaepv. tomato, which persistently induces the JA signallingpathway. Yet, high resolution growth analyses of this effectin wild type and JA biosynthesis knock-out mutants showedthat it was elicited by the bacterial toxin coronatine thatsuggests ethylene- but not JA-induced root growth reduc-tion in A. thaliana. Overall, the results demonstrate thatthe reaction of root growth to herbivore-induced JA signal-ling differs among species, which is discussed in the contextof different ecological defence strategies among species.

Key-words: Pseudomonas syringae pv. tomato; coronatine;ethylene; image analysis; phytohormones; wounding.

INTRODUCTION

Plants have to cope with many abiotic and biotic stressesduring their development in order to grow and reproducesuccessfully. A number of plant defence hypotheses exist,each attempting to explain how plants follow their optimalstrategy within the triangle of resource availability, genera-tion of defence measures and sustained growth (for reviewssee Grime 1977; Stanton, Roy & Thiede 2000; Stamp 2003).Biotic stressors such as herbivores and pathogens damageplants in many ways and thereby induce different signallingpathways in the plant. The phytohormone jasmonic acid(JA) is one of the most important components of the plant

defence signalling system (Rojo, Solano & Sanchez-Serrano2003). In the model plant Arabidopsis thaliana, JA accumu-lates in the leaves after mechanical wounding (Laudertet al. 1996; Laudert & Weiler 1998; Park et al. 2002;Yan et al.2007; Glauser et al. 2008; Zhang & Turner 2008), afterpathogen attack and during feeding of herbivores (Stotzet al. 2002; Reymond et al. 2004; De Vos et al. 2005). Arabi-dopsis plants that are insensitive to JA, such as the coi1(coronatine insensitive) or the jar1-1 (jasmonate resistant)mutants, are more readily consumed by herbivores (Vijayanet al. 1998; Reymond et al. 2004; Bodenhausen & Reymond2007; Moreno et al. 2009) and are more susceptible topathogen attack (Thomma et al. 1998; Kunkel & Brooks2002), demonstrating the importance of an intact JA signal-ling pathway in A. thaliana as well.

The JA signalling pathway is most readily induced bythe bacterial toxin coronatine, which is structurally similarto jasmonates (Bender, Alarcon-Chaidez & Gross 1999;Glazebrook 2005). Infection of plants with Pseudomonassyringae pv. tomato (Pst) leads to coronatine-inducedactivation of the JA signalling pathway (Laudert & Weiler1998; Thilmony, Underwood & He 2006). The pattern ofJA accumulation upon infection with Pst differs betweenavirulent and virulent bacteria: JA accumulates morerapidly in A. thaliana upon infection with avirulent Pstcompared with an infection with virulent Pst (Spoel et al.2003; Grun et al. 2007).

In contrast to pathogens, herbivores can severelydamage leaves not only by introduction of toxins, but alsoby removing photosynthetically active tissue. Thus, theyimpact primary metabolism and may reduce the growth ofthe damaged leaf (Moore et al. 2003; Hummel et al. 2007;Schmidt, Schurr & Röse 2009). In Nicotiana attenuata,an increase of carbon allocation to the root (Schwachtjeet al. 2006) and a pronounced reduction of root growth(Hummel et al. 2007) in response to a single woundingor simulated herbivory event were discovered recently.Although this reaction pattern seems paradoxical at first,it might be the logical consequence of a defence strategyoptimized to retain important resources in the ‘safe’root for regrowth after herbivoral attack, while fosteringleaf growth to be stronger than root growth to maxi-mize carbon acquisition via photosynthesis. Such a ‘func-tional equilibrium’ for the balance between above- and

Correspondence: A. Walter. Fax: +49 2461 61 2492; e-mail: [email protected]

Plant, Cell and Environment (2010) 33, 104–116 doi: 10.1111/j.1365-3040.2009.02062.x

© 2009 Blackwell Publishing Ltd104

below-ground growth, which responds very sensitively tothe availability of resources offered above- and below-ground, has been described in the context of abioticresource capture (Poorter & Nagel 2000) and this isalso a conceivable strategy to cope with biotic stresssituations.

Reduction in root growth of N. attenuata seedlings uponmechanical wounding and simulated herbivory wasascribed to JA and not to ethylene signalling, as shown bya suite of experiments involving JA signalling mutants anddifferent experimental treatments (Hummel et al. 2007,2009). The involvement of JA signalling in rapid alter-ations of root growth and carbon allocation patterns wasdemonstrated in other species, such as barley, as well(Henkes et al. 2008). Moreover, in A. thaliana, jasmonatesstrongly reduce root length (Staswick, Su & Howell 1992;Feys et al. 1994; Vellosillo et al. 2007; Yan et al. 2007) andleaf fresh weight (Zhang & Turner 2008) when applieddirectly to the growth medium. It was reported recentlythat repeated wounding of mature Arabidopsis leavesmediates a rapid increase in endogenous JA concentra-tions, resulting in reduced cell divisions and thus reducedsystemic leaf growth (Zhang & Turner 2008). However, itis not known whether wounding and biotic stress-inducedJA bursts mediate a reduction in root growth in Arabi-dopsis, a species that completes its life cycle very quickly,and which will rather avoid or tolerate herbivory, whereasspecies like N. attenuata and ‘typical crop plants’ induce asubstantial number of mechanisms to defend themselvesagainst herbivore attack (Baldwin 2001; Nunez-Farfan,Fornoni & Valverde 2007).

Thus, the aim of this study was to investigate whetherroot growth of the model species A. thaliana is affectedby wounding and pathogen attack via the JA signallingpathway. To test this we induced a JA burst via differenttreatments and then monitored root growth. First of all,methyl jasmonate (MeJA), which induces defenceresponses similar to caterpillar feeding (Moreno et al.2009), was directly applied to leaves and roots. To simulateherbivory, oral secretions of larvae of the generalist her-bivore Spodoptera littoralis were applied to mechanicallywounded leaves. In further experiments, root growth oftwo JA signalling mutants of A. thaliana was investigated:The coi1-1 mutant is insensitive to jasmonates and coro-natine (Feys et al. 1994) whereas the transgenic knock-outmutant aos is lacking the allene oxide synthase and is thusdefective in the rate-limiting step of JA biosynthesis(Léon, Rojo & Sanchez-Serrano 2001). In another set ofexperiments, Arabidopsis seedlings were mechanicallywounded and infected with a coronatine-producingavirulent Pst strain, and with a virulent Pst strain that isunable to synthesize coronatine. Finally, an experimentwith the ethylene reception blocker 1-methylcyclopropene(1-MCP) was performed to distinguish between ethyleneand JA responses of Pst infection. Ethylene generallyinhibits root elongation (e.g. Ellis & Turner 2002) and itsproduction increases following Pst infection (De Vos et al.2005).

MATERIALS AND METHODS

Plant material & cultivation systems

A. thaliana seedlings were grown in sterile agar in squarePetri dishes (120 ¥ 120 ¥ 17 mm). Five holes were meltedinto one side of the Petri dish with a glowing bolt understerile conditions. Subsequently the Petri dishes were com-pletely filled with sterile 1% plant agar (Duchefa, Haarlem,the Netherlands) in one-third Hoagland nutrient solution.When the medium had solidified, the Petri dish was closedand sealed with fabric tape (Micropore; 3M Health Care,Neuss, Germany).

The seeds of the aos knock-out mutant (N8149), the Col-6wild type (N8155) and the Col-0 wild type were surface-sterilized with 70% ethanol and 20% ‘glorix original’ (Uni-lever Belgium S.P.R.L., Brussels, Belgium) and placed intothe holes of the Petri dishes. The seeds on the agar werecovered with laboratory film (Parafilm, Pechiney PlasticPackaging, Menasha, WI, USA) to avoid water loss untilgermination and were stratified in the dark. After 3 d, thePetri dishes were transferred into a climate chamber with aconstant air temperature of 21 °C and 60% RH. Light wasprovided for 16 h with a phase of dawn and dusk of 15 min,each, and reached 150 mmol photons m-2 s-1 at seedlingheight. The Petri dishes were held vertically until germina-tion and were then set to an angle of 5° from the vertical toensure that the roots grew along the bottom of the Petri dish.Roots of the seedlings were inside the Petri dish,whereas theshoots grew outside. When the seedlings were 10–14 d old,they were used for experimental treatments.

For selection of homozygous coi1-1 mutants, the seedswere sterilized as described above and placed on Murashigeand Skoog medium (Sigma Aldrich Chemie GmbH, Stein-heim, Germany) supplemented with 3% sucrose and 30 mmMeJA.After 3 d of stratification, the plates were transferredinto the climate chamber. Approximately 5–7 d later, thehomozygous coi1-1 mutants were transferred to Petri dishesfilled with one-third Hoagland agar as described earlier.

Throughout the cultivation and the experimental treat-ments, the roots of the seedlings were not covered and thuswere exposed to light. To avoid possible effects of the treat-ments on root growth being masked by light effects, allplants were exposed to the same conditions.

The agar temperature at the root tip and the air tempera-ture at the height of the seedlings were monitored on arepresentative Petri dish using a thermologger (K204,Conrad Electronic SE, Hirschau, Germany).

Pathogens

The coronatine-producing, avirulent bacteria Pseudomonassyringae pv. tomato DC3000 avrRpt2 (Pst DC3000 avrRpt2)were grown in an incubator set to 28 °C on King’s B agar(King, Ward & Raney 1954) with kanamycin for selection.For experiments, the bacteria were transferred to liquidlysogeny broth medium (Fluka Feinchemikalien GmbH,Neu-Ulm, Germany) with kanamycin and were allowedto grow overnight at 28 °C. The bacteria were purified by

Root growth wound-response in Arabidopsis thaliana 105

© 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 104–116

centrifugation with washing steps in MgSO4 and finallyadjusted to a concentration of 1 ¥ 107 cfu mL-1.

The virulent strain Pseudomonas syringae pv. tomatoNCPPB 1008 (Pst DC3000 NCPPB 1008), which is deficientin coronatine biosynthesis, was grown on LB agar plateswithout antibiotics. The same purification procedure asdescribed above was used for experiments.

Herbivores

Spodoptera littoralis (Lepidoptera) eggs were reared on adiet consisting of shredded beans and vitamins. Two daysbefore starting the collection of oral secretions, the third-instar larvae were placed in a plastic container and wereallowed to feed on A. thaliana Col-0 leaves. In order toinduce regurgitation of oral secretions, a caterpillar washeld with lightweight forceps in the head region. Collectionof oral secretions used two 50 mL pipettes inserted into avial through a septum. One of the pipettes was held onto themouth part of the caterpillar whereas the other pipette wasconnected to a low vacuum.The oral secretions were storedat -80 °C immediately after collection.

Experimental treatments

An outline of the experimental design, specifying genotypesand replicate numbers of each individual experiment, isgiven in Table 1.

For MeJA treatment on roots, the Petri dish was openedand 1 mL of pure MeJA was applied to the agar at 5 mmdistance to the root tip. In this experiment, control plantswere treated with 1 mL of sterile deionized water (MilliporeGmbH, Schwalbach, Germany) in the same manner as theMeJA-treated plants.

For the two MeJA treatments on leaves, 400 or 4000 ngMeJA were dissolved in 1 mL of lanolin, and applied asdescribed by Hummel et al. (2007). Control plants weretreated with 1 mL of pure lanolin.

For wounding and bacteria treatments, two leaves of theplants were wounded twice using sterile tweezers punchingsmall holes into the leaves without damaging the majorveins. For experiments with Pseudomonas syringae strains,2 mL of the bacterial suspension were applied to each

wounded leaf with sterile pipette tips. To facilitate theinfection of the leaf, the bacteria suspension was ejectedand retrieved several times with the pipette tip to ensuremaximal coverage of the leaf surface with the pathogens.

To analyze long-term effects of simulated caterpillar her-bivory, two leaves per seedling were wounded and immedi-ately treated with 1 mL of Spodoptera littoralis regurgitant(diluted 1:5 with 50 mm phosphate buffer, Hummel et al.(2007). Control plants were treated with 1 mL bufferedwater after wounding.

For experiments with 1-MCP (obtained as SmartFreshfrom AgroFresh Inc., Spring House, PA, USA), a strongethylene receptor blocker, the Petri dishes were placed in atightly closed glass chamber as described by Hummel et al.(2009). Plants were allowed to adapt to the conditions in theglass chamber for 5 d. 1-MCP was applied to the chamber ata concentration of 45 mL l-1 by dissolving the SmartFreshpowder in 100 mL of water according to Hummel et al.(2009). The glass chamber was opened only for a fewminutes to mark growing root tips as described in ‘basicroot growth analysis’. After each opening of the glasschamber, the 1-MCP pretreatments were renewed. Experi-mental treatments were performed 24 h after the first1-MCP application to ensure that all ethylene receptorswere blocked by 1-MCP before treatment.

Basic root growth analysis

For basic root growth analysis, the position of the root tipwas marked every 24 h with a pen on the bottom of thedishes, starting 2 d before the experimental treatment. Theincrease in length of the primary root of each seedling wasmonitored with a ruler. Only seedlings with initial primaryroot length of more than 20 mm (on treatment day, day 0)were included in experiments with caterpillar regurgitant.For experiments with 1-MCP, seedlings were selected withroot lengths of more than 29 mm on the day of 1-MCPtreatment. The data were normalized by dividing the veloc-ity of the root tip (vTip) on the days following the treatmentby vTip for the day before treatment (day 0). The vTip valuesof the individuals per Petri dish were averaged and thenumber of Petri dishes per treatment was taken as thereplicate number.

Table 1. Experimental designExperiment Genotypes Replicates

MeJA on roots Col-0 3–4MeJA on leaves Col-0 3–6Control Col-0, Col-6, coi1-1, aos 4Wounding Col-0, Col-6, coi1-1, aos 5–8Wounding + OS Col-0 5–6Pst DC3000 avrRpt2 Col-0, Col-6, coi1-1, aos 4–5Pst DC3000 NCPPB 1008 Col-0, Col-6, coi1-1, aos 3–4Pst DC3000 avrRpt2 and 1-MCP Col-0 3–4

Overview of the A. thaliana lines used in the individual experiments.1-MCP, 1-methylcyclopropene; MeJA, methyl jasmonate.

106 L. Schmidt et al.


High-resolution root growth analysis

An image of the primary root growth zone was taken every30 s with a charge-coupled device (CCD) camera (SonyXC-55 and XC-75, Sony, Köln, Germany) at a resolutionof 740 ¥ 480 pixels that corresponds to a total area of2.7 ¥ 1.8 mm. During the dark phase, infrared illumination(l = 940 nm) enabled image acquisition. The cameras wereequipped with low-pass infrared filters (RG, Schott, Mainz,Germany) to block visible irradiation. The root tips werefollowed via a tracking algorithm that controlled a set ofx–y moving stages that repositioned the Petri dish, and thusthe root tip, when the tip approached the border of theimage field (for more details see Hummel et al. 2007). Eachreplicate was measured for at least 24 h. The velocity ofthe root tip (vTip) was calculated using image processingalgorithms described elsewhere (Walter et al. 2002). ThevTip data were normalized by dividing them by the value atthe time of treatment to facilitate the comparison of theexperimental treatments.

Determination of jasmonates in shoots

For analysis of the concentrations of JA in A. thaliana,entire shoots of five to seven seedlings per replicate werepooled. The samples were harvested and immediatelyfrozen in liquid nitrogen. Prior to the extraction of phyto-hormones, the frozen samples were homogenized with twosteel balls in a Geno/Grinder 2000 (OPS Diagnostics, LLC;Bridgewater, NJ, USA) with 250 stokes min-1 for 30 s. Eachsample was extracted with one millilitre of ethyl acetatespiked with 40 ng D2-dihydro-JA and 8 ng JA-13C6-Ile byshaking for 10 min. After centrifugation at 16 100 g for20 min at 4 °C, the extraction was repeated with 500 mL ofethyl acetate. The supernatants were combined and evapo-rated to dryness in a vacuum concentrator at 30 °C. The dryresidue was dissolved in 200 mL methanol and centrifugedat 16 100 g for 10 min. An aliquot was transferred to HPLCvials and measured on a 1200 L liquid chromatography–triple quadrupole mass spectrometry system (Varian,Palo Alto, CA, USA). Ten microlitres was injected ontoa ProntoSIL C18-ace-EPS column (50 ¥ 2 mm, 5 mmdiameter, Bischoff, Germany) attached to a pre-column(C18, 4 ¥ 2 mm, Phenomenex, Torrance, CA, USA). Amobile phase composed of 0.05% formic acid and methanolwas used in a gradient mode for the separation. The mobilephase comprised of solvent A (0.05% formic acid) andsolvent B (methanol) used in a gradient mode (time/concentration for (min/%B): 0/15; 1.5′/15; 4.5′/98; 12′/98;13′/15; 15′/15 with a variable flow rate time/flow (mL/min):0′/0.4; 1′/0.4; 1,5′/0.2; 10′/0.2; 10.5′/0.4; 15′/0.4). Compoundswere detected as negative ions in an mutiple reaction moni-toring mode. Molecular ions M-H(-) at m/z 209 and 322generated from endogenous JA and JA-Ile and from theirinternal standards 213 and 328 were fragmented under 12and 19 V CE for JA and JA-Ile, respectively. The productIon of JA and its internal standard is m/z 59, JA-Ile and theinternal standard forms the product ions m/z 130 and m/z

136, respectively. The ratio of ion intensities of the responseof the product ions was used to quantify JA and JA-Ile.

Statistical analyses

Statistical analyses were performed with SigmaStat, version2.03 (San Jose, CA, USA). To test for significant effectsof caterpillar regurgitant on root growth, the treatments‘wounding’ and ‘wounding + oral secretions’ (OS) weretested with a t-test for each day following treatment. Con-centrations of JA and JA-Ile were compared between con-trols and wounded plants for each time point followingtreatment with t-tests or Mann–Whitney rank sum tests.

Total increases in root length of untreated and bacteria-exposed plants following 1-MCP application were per-formed with one-way analysis of variance (anova) followedby Fisher least significant difference (LSD) post hoc test.

RESULTS

MeJA application to roots and leaves

Direct application of MeJA to the agar next to the rootgrowth zone immediately reduced root growth (Fig. 1a),clearly demonstrating the inhibiting growth effect of JA.Yet, neither foliar application of 400 nor of 4000 ng MeJAled to decreased root growth (Fig. 1b). This result showsthat the perception of JA in leaves and roots can differenormously and that root growth is not necessarily reducedwhen JA is sensed by leaves.

Root growth patterns of untreated plants

As the initial experiment showed that root growthdecreased during the day and increased at night, 24 h (diel)growth dynamics of all investigated plant lines were ana-lyzed carefully. The A. thaliana wild types Col-0 and Col-6,as well as the JA signalling mutants coi1-1 and aos, showedthe same basic diel pattern of root growth (Fig. 2a). Duringthe day, the velocity of the root tip (vTip) decreased slightly(daily average 0.25 mm h-1). During the night, root growthincreased, showing maximal values of up to 0.4 mm h-1

around the night–day transition (Fig. 2a).As the diel fluctuation of root growth is often strongly

correlated to temperature (Hummel et al. 2007; Walter &Hummel 2008), temperature was measured within the agarduring the diel cycle (Fig. 2b). Temperature varied morestrongly within the agar than in the air and was 1–1.5 °Chigher during the day than during the night (Fig. 2b). Yet,the observed diel variation of root growth was not corre-lated to diel temperature variations. This temporal growthvariation served as the reference frame for all subsequentexperiments, with the consequence that all treatments hadto be applied at the same time of the day.

Wounding

Wounding two leaves immediately reduced root growthby 26 � 4% in all Arabidopsis seedlings (Fig. 3a–d). This



decrease was transient; root growth recovered to pretreat-ment levels at the beginning of the next day. No differencesin root growth following a single wounding treatment oftwo leaves were found between the different Arabidopsislines (Fig. 3).

The concentrations of JA and JA-Ile increased signifi-cantly within 30 min following the mechanical woundingtreatment and then decreased slowly (Fig. 4).

Foliar wounding and application ofcaterpillar regurgitant

When leaves were wounded and treated with oral secre-tions of Spodoptera littoralis, the velocity of the root tip(vTip) was clearly reduced. However, no differencebetween mere mechanical wounding and the wounding and

application of oral secretions was found concerning rootgrowth (Fig. 5).

Wounding and application of differentPseudomonas syringae pv. tomatoDC3000 strains

Immediately after wounding and application of Pst DC3000avrRpt2, root growth decreased strongly in the aos mutantand the wild types (Fig. 6a). Compared with the meremechanical wounding treatment (Fig. 3), root growth wasdecreased more severely here and the reduction lasted forat least 2 d. Throughout the 2 d analysed, the decrease inroot growth was more pronounced in the aos mutant than inthe wild types (Fig. 6a). In the coi1-1 mutant, root growthdecreased only slightly during the first hours following thetreatment and rapidly recovered to the pre-treatment level

Figure 1. Normalized values of root tipvelocities (vTip) of A. thaliana wild types(a) after application of either 1 mL wateror 1 mL methyl jasmonate to the agarnear the root tip and (b) after applicationto a leaf of 0, 400 or 4000 ng methyljasmonate dissolved in 1 mL lanolin. Thenight period is highlighted in grey(mean � standard error, n = 3–6). MeJA,methyl jasmonate.Time after treatment (h)

–4 0 4 8 12 16 20 24

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(Fig. 6a). Immediately after joint mechanical wounding andapplication of the virulent coronatine-deficient Pst DC3000strain NCPPB 1008, root growth decreased transiently,recovering soon to values of untreated plants in all Arabi-dopsis lines (Fig. 6b).

Treatment with 1-MCP, which blocks ethylene perceptionin plants, led to significantly increased root growth ofArabidopsis seedlings compared with plants that were notexposed to 1-MCP (P � 0.002; Fig. 7a,b). Seedlings thatwere wounded and treated with the coronatine-producingPst DC3000 avrRpt2 grew significantly slower for 3 d com-pared with untreated control plants (P = 0.028; Fig. 7b).Four days after 1-MCP treatment, there was no significant

difference between the root length of untreated controlseedlings and seedlings that were wounded and treated withbacteria (P > 0.05; Fig. 7b).

DISCUSSION

Impaired JA signalling does not affect rootgrowth of A. thaliana

As root growth is regulated by different plant hormones, itwas essential to test whether root growth of wild type plantsand of the two JA signalling mutants applied in this study iscomparable. Indeed, there was no difference between lines

Figure 2. Time courses of (a)normalized values of root tip velocities(vTip) of two wild types (Col-0 and Col-6)and two jasmonic acid signalling mutants(coi1-1 and aos) of A. thaliana and of(b) corresponding temperature of the airand the agar of a representative Petridish. The night period is highlighted ingrey. For (a), (mean � standard error,n = 4).

Time of day (h)

9:00:00 17:00:00 1:00:00 9:00:00 17:00:00 1:00:00

9:00:00 17:00:00 1:00:00 9:00:00 17:00:00 1:00:00

vT

ip (

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Time of day (h)

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)

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in control conditions (Fig. 2a) suggesting that JA does notplay a major role in controlling diurnal or nocturnal rootgrowth dynamics in Arabidopsis under ‘normal’, unstressedcircumstances. The application of the ethylene blocker1-MCP increased root growth (Fig. 7), indicating that alsoin conditions without experimental treatments, ethyleneaccumulates inside the Petri dish and thereby preventsutilization of the full root growth potential (Eliasson &Bollmark 1988).

A repetitive variation of root growth within 24 h wasobserved in all Arabidopsis lines and under all treatments,with maximal values of vTip around dawn and minimalvalues at the end of the day (Fig. 2a). This result contradictsother observations from our group (Walter et al. 2002;Walter & Schurr 2005; Walter & Hummel 2008) and can-not be explained by temperature variation (Fig. 2b). Theunusual fluctuation might originate from progressive rootgrowth inhibition induced by the high prevailing light inten-sities in the climate chamber during the day, which is abro-gated each night. In contrast to the studies on N. attenuatawhere illumination of the Petri dishes from the side wasapproximately 3 mmol photosynthetically active radiation(PAR) m-2 s-1, the equivalent light intensity reflected was afactor of 20 higher (50–60 mmol PAR m-2 s-1). As all plantsof this study were exposed to the same light conditionsduring pre-cultivation and experiments, and as diel growthvariations were comparable in all populations, we can

exclude the possibility that treatment effects on root growthwere caused by the exposure of the roots to light.

MeJA applied to leaves does not reduce rootgrowth in Arabidopsis

Foliar application did not affect root growth (Fig. 1b). Incontrast, direct application of MeJA to the growth mediumreduced root growth (Fig. 1a). Root growth reduction wascomparable with that in experiments described in theliterature (Feys et al. 1994; Vellosillo et al. 2007; Yan et al.2007; Zhang & Turner 2008). Foliar application was per-formed at different concentrations that were reported tolead to clear effects on gene expression and concentrationof phenolics of the leaves (Moreno et al. 2009). There arethree potential reasons why root growth might not havebeen decreased here, although leaves were provided withJA: (1) the jasmonate signal was not transported fromleaves to root growth zones; (2) the JA signal arrived at theroot growth zone but was not sensed there; (3) the signalwas sensed but did not lead to a growth reduction and wasinterpreted differently than in N. attenuata. Possibility (1) isunlikely as it is shown that, in Arabidopsis, the JA signalis systemically distributed along the phloem (Trumanet al. 2007) and reduces cell division of leaves systemically(Zhang & Turner 2008). Possibility (2) can be excludedbecause of the experiment with direct application of MeJA

Figure 3. Velocities of the root tip (vTip),normalized to the time of treatment, ofmechanically wounded A. thaliana wildtypes (a) Col-0 and (b) Col-6 and (c) theJA-insensitive mutant coi1-1 and (d)allene oxide synthase knock-out mutantaos. The night period is highlighted ingrey (mean � standard error, n = 5–8).

(b) Col-6

(d) aos(c) coi1-1

Time after treatment (h)

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to roots, which is also supported by literature (Spoel et al.2003; Badri et al. 2008). Hence, the following parts of thediscussion have to elaborate on possibility (3).

Wounding and simulated herbivory transientlyreduce root growth in A. thaliana

Mechanical wounding of the leaves induces strong burstsof plant-internal jasmonates (Fig. 4) in the same timingand range as in N. attenuata (Hummel et al. 2009) and asreported earlier for wounded Arabidopsis plants (Laudertet al. 1996; Park et al. 2002; Reymond et al. 2004; Yan et al.2007). Moreover mechanical wounding led to a transientroot growth reduction of 26 � 4% compared with the pre-treatment value in all investigated plant lines within 2 h(Fig. 3). Root growth recovered throughout the treatmentday but remained some percent lower compared with

control plants. The initial transient root growth reductionis clearly not mediated by JA, as JA signalling mutantsshowed this response in the same way as wild type plants(Fig. 3). This is different to reports on JA-mediated reduc-tions of leaf growth following repeated mechanical wound-ing (Zhang & Turner 2008). A combination of decreasedturgor, and decreased carbohydrate availability to the root,because of the damaged photosynthetic tissue, accountsfor this pronounced short-term effect, and for a minusculeroot growth reduction in the following days (Nagel, Schurr& Walter 2006; Hummel et al. 2007). Application of oralsecretions of the larvae of Spodoptera littoralis did notsignificantly increase the slight effect of wounding on rootgrowth (Fig. 5).

It is conceivable that the wounding effect was too small toevoke pronounced reactions in the root, even though itresulted in increased concentrations of jasmonates in the

Figure 4. Time course of jasmonic acid(JA) (a) and JA-Ile (b) concentrationsfollowing mechanical wounding of A.thaliana Col-0 seedlings (mean �

standard error, n = 4–6).

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shoot (Fig. 4). It is known that abundances of the AOSprotein (Laudert & Weiler 1998) and accumulation of polarjasmonates (Glauser et al. 2008) are lower in Arabidopsisroots compared with leaves after mechanical wounding ofleaves. It was possible to measure concentrations of JA andits conjugated form JA-Ile in the shoot only, and thus we can

not predict the real JA concentrations at the root tip. Fur-thermore, the repeated mechanical wounding imposed byZhang & Turner (2008) was more severe than our singlewounding treatment, which may explain why they observedstunted leaf growth whereas there were no effects on rootgrowth of Arabidopsis in our study. Yet, a more severe

Figure 5. Root growth of A. thalianaCol-0 lines in untreated control plants(con), following mechanical wounding(wounding) and mechanical woundingand application of oral secretions ofSpodoptera littoralis (wounding + OS)(mean � standard error, n = 5–6).Time after treatment (days)

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Figure 6. Normalized values of root tip velocities (vTip) of different A. thaliana lines (a) after concomitant mechanical wounding andapplication of Pseudomonas syringae pv. tomato DC30000 avrRpt2 and (b) after concomitant mechanical wounding and application of thecoronatine-deficient strain P. syringae pv. tomato DC30000 NCPPB 1008. The bacteria were applied at concentrations of 1 ¥ 107 cfu mL-1.The night period is highlighted in grey (mean � standard error, n = 3–5).



destruction of leaf material would have affected primarymetabolism in such a serious way that a distinctionbetween defence signalling and carbon starvation wouldhave been difficult. The severity of the wound treatmentswas comparable with that in experiments with N. attenuata,where a single wounding event induced a clear reduction ofvTip to 50% of the control value, lasting for 16 h. Hence

Arabidopsis root growth is clearly less susceptible to leafwounding and herbivory compared with N. attenuata. If theobserved reaction is not only caused by hydraulic effectsand reduced plant photosynthesis but by JA signalling, thenthe JA signal perceived by the root leads to a very subtlegrowth reduction.

Pseudomonas syringae pv. tomato infectionreduces root growth via coronatine-mediatedethylene signalling

Pseudomonas syringae pv. tomato was found to induce avery strong and persistent JA formation in leaves, as, in theavirulent strain, coronatine is continually produced, leadingto a continuous induction of the JA signalling pathway(Laudert & Weiler 1998; Thilmony et al. 2006).

Upon wounding and treatment with the avirulent,coronatine-producing bacteria strain Pst DC3000 avrRpt2,root growth decreased immediately in all Arabidopsis lines,and was only able to recover during the night in thecoronatine-insensitive mutant coi1-1 (Fig. 6a). Interestingly,the aos mutant behaves like the wild types suggesting thatnot the JA signalling pathway per se induces the observedreduction of root growth by 50% but that a JA-independentpathway is involved as well. This points to the bacterialtoxin coronatine as reason for root growth reduction, whichwas clearly proven by the following experiment with thevirulent bacteria strain Pst DC3000 NCPPB 1008. In thisexperiment, Pst DC3000 NCPPB 1008, which are unable toproduce coronatine, led to very similar root growth reac-tions in wild type and JA signalling mutant plants (Fig. 6b).It was reported earlier that coronatine reduces the rootlength of Arabidopsis wild type plants similarly to MeJA,whereas root growth of the coi1-1 mutants is not affected(Feys et al. 1994). Coronatine acts via a COI1 dependentpathway (He et al. 2004), which makes the Arabidopsiscoi1-1 mutant insensitive to coronatine-producing Pseudo-monas syringae strains (Feys et al. 1994).

Up-regulation of the JA signalling pathway by a con-tinuous supply of coronatine has probably acted synergis-tically with ethylene to mediate root growth reductionupon infection with Pst. It is known that coronatine andanother Pst virulence factor, the type III effector avrRpt2,mediate increased ethylene biosynthesis via auxin (Chenet al. 2007). As ethylene is known to reduce the root cellelongation by locally up-regulating auxin biosynthesis andtransport (Ruzicka et al. 2007; Swarup et al. 2007), we con-clude that ethylene was the major factor for root growthreduction upon infection of Arabidopsis with Pst. Thishypothesis is supported by the experiment with 1-MCP,which blocks ethylene perception. Upon the applicationof 1-MCP, the root growth increase was similar in bothuntreated control plants (as reported for N. attenuata,Hummel et al. 2009) and in plants that were inoculatedwith Pst (Fig. 7), confirming that ethylene is involved inmediating the reduction in root growth upon infectionwith the bacteria.

con bact con 1-MCP bact 1-MCP

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Figure 7. Root growth of untreated Arabidopsis seedlings(con), seedlings that were wounded mechanically and treatedwith Pseudomonas syringae pv. tomato DC3000 avrRpt2 at1 ¥ 107 cfu mL-1 for 3 d (bact), seedlings treated with1-methylcyclopropene (con + 1-MCP) and seedlings exposed to1-MCP for 4 d and concomitantly wounded and treated withbacteria for 3 d (bact + 1-MCP). (a) Time course of root lengthincrease; (b) total increase in root length after 4 d. Differentletters indicate significant differences at P < 0.05 level(mean � standard error, n = 3–4).



ECOLOGICAL ASPECTS OF ROOT GROWTHREACTIONS AFTER HERBIVORY

Plants live in different habitats and are faced with differentstrengths and modes of herbivory and pathogen attack.Hence, they evolved specific defence responses to minimizethe negative consequences. As many biochemical signallingpathways like JA, salicylic acid or ethylene pathways arehighly conserved in plants, the complex sets of signals mustbe integrated by the different plant organs and organizedinto specific reactions concerning growth, defence, storageand reproduction according to their ecological context.Thus, wounding signals must be translated into specificreactions in order to reduce the negative impact of her-bivory on plant fitness. In this study we demonstrated thatwound-induced JA does not affect root growth in Arabi-dopsis, although JA is well known to inhibit root growthstrongly. In contrast to that result, Hummel et al. (2007,2009) demonstrated that wound-induced JA reduces rootgrowth in N. attenuata markedly. Hence, the same woundsignal triggers different root growth reactions in the twospecies. This difference in growth response might beexplained by the ecologies of the habitats in which theplants evolved. Arabidopsis is found in temperate climatesand has to cope with shorter growth periods than Nicotiana,which is a subtropical plant. Arabidopsis therefore has tofinish its life cycle more quickly than N. attenuata, and doesnot suffer from high herbivore pressure. For instance nospecialist herbivore is known to attack Arabidopsis. Thus,Arabidopsis is focused on a rapid turnover and adjusts itsgrowth response only marginally upon wounding, by sup-pressing the rate of leaf cell division. The resulting reduc-tion of cell numbers requires fewer nutrients, which savesresources for the completion of the life cycle of the plant(Zhang & Turner 2008). In contrast, N. attenuata has longergrowth periods and mostly grows in monocultures. Hence,herbivore pressure and intraspecific competition is farhigher compared to Arabidopsis (Baldwin 2001). Oftenwhen N. attenuata is attacked by its specialist herbivoreManduca sexta, the shoot is entirely defoliated. In thiscase, tolerance is suggested to be the best strategy for aplant to cope with its species-specific, adapted herbivores(Schwachtje et al. 2006). Thus, when attacked by its special-ist herbivore M. sexta, N. attenuata: (1) increases its defencemechanisms; but (2) also allocates recently fixed carbohy-drates into its roots, which can be used for regrowthwhenever the aboveground threat has passed by. Such atolerance reaction only makes sense when, simultaneously,root growth is reduced and carbon is stored in roots insteadof being used for root growth. At the same time, the bio-synthesis of nicotine takes place in the roots of Nicotiana,incorporating up to 8% of the plant’s nitrogen pool(Baldwin 2001), which will not be available for growth pro-cesses. Because in Arabidopsis, the main defensive sub-stances, the glucosinolates, are synthesized in the leaves andnot the roots, the resources of the roots would not be usedfor defence against herbivores, and thus help root growth tocontinue following herbivory.

CONCLUSIONS

Overall, this study demonstrates that the relationshipbetween plant defence patterns, resource capture and veg-etative growth is regulated in a complex manner with dif-ferent species following different strategies that optimizetheir fitness within their specific ecological habitat. More-over, the results reveal limits for the use of model organ-isms. Although A. thaliana and N. attenuata use the JAsignalling pathway and although JA leads to a reduction ofroot growth in both species when applied directly to roots,the two species follow different rules for the response oftheir belowground growth when biotic stresses occuraboveground. It seems likely that these rules are stronglydependent on the ecological context in which a species hasevolved.

ACKNOWLEDGMENTS

We thank John Turner (University of East Anglia, UK)for donation of A. thaliana coi1-1 seeds, and the NASC(Nottingham, UK) for A. thaliana aos and Col-6 seeds.

Pseudomonas syringae pv. tomato DC3000 avrRpt2 werea kind gift of Corné Pieterse (Utrecht University, the Neth-erlands) and Pseudomonas syringae pv. tomato DC3000NCPPB 1008 were kindly provided by Diane Cuppels andTeresa Ainsworth (Agriculture and Agri-Food Canada,London ON, Canada).

We thank Roland Reist (Syngenta Crop Protection,Stein, Switzerland) for the Spodoptera littoralis eggs andJon F. Fobes (AgroFresh Inc., Spring House, USA) for theSmartFresh powder.

Vicky Temperton, Michael Thorpe and several other col-leagues are thanked for critical discussion and helpful com-ments. This work was supported by funding from theForschungszentrum Jülich. L.S. acknowledges the supportfor her PhD thesis at the Heinrich-Heine-UniversitätDüsseldorf.

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Received 12 May 2009; received in revised form 21 August 2009;accepted for publication 9 October 2009



jasmonic acid does not mediate root growth responses to wounding in arabidopsis thaliana

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