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Environmental and Experimental Botany 100 (2014) 55– 63

Contents lists available at ScienceDirect

Environmental and Experimental Botany

jou rn al h om epa ge: www.elsev ier .com/ locate /envexpbot

olatile organic compound emissions from Alnus glutinosa undernteracting drought and herbivory stresses

ucian Copolovici a,b,∗, Astrid Kännastea, Triinu Remmela, Ülo Niinemetsa

Institute of Agricultural and Environmental Sciences, Estonian University of Life Sciences, Kreutzwaldi 1, Tartu 51014, EstoniaInstitute of Technical and Natural Sciences Research-Development of Aurel Vlaicu University, Elena Dragoi 2, Arad 310330, Romania

r t i c l e i n f o

rticle history:eceived 12 September 2013eceived in revised form7 December 2013ccepted 18 December 2013

eywords:iotic stress

a b s t r a c t

Plant volatile organic compounds (VOCs) elicited in response to herbivory can serve as cues for parasiticand predatory insects, but the modification of VOC elicitation responses under interacting abiotic stressesis poorly known. We studied foliage VOC emissions in the deciduous tree Alnus glutinosa induced by feed-ing by the larvae of green alder sawfly (Monsoma pulveratum) under well-watered and drought-stressedconditions. Drought strongly curbed photosynthesis rate and stomatal conductance, but there were noeffects of insect feeding on photosynthetic characteristics. Feeding induced emissions of volatile productsof lipoxygenase pathway and monoterpenes, and emissions of stress marker compounds (E)-�-ocimene

erbivory damagenteracting stresses

ethyl salicylatetress primingolatile organic compounds

and homoterpene DMNT. The emissions were more strongly elicited and reached a maximum value ear-lier in drought-stressed plants. In addition, methyl salicylate emissions were elicited in herbivory-feddrought-stressed plants. Herbivores were more strongly attracted to well-watered plants and consumedmore than a four-fold greater fraction of leaf area than they consumed from drought-treated plants.Overall, this study demonstrates an important priming effect of drought, suggesting that plants undercombined drought/herbivory stress are more resistant to herbivores.

© 2013 Elsevier B.V. All rights reserved.

. Introduction

Under natural conditions, plants are often exposed to a combi-ation of two or more simultaneous or sequential stress factorsHolopainen and Gershenzon, 2010; Mittler, 2006; Niinemets,010a,b). In the case of simultaneously occurring stresses, oneype of stress could weaken or enhance the effects of anotherimultaneous stress factor by direct physiological cumulative ornteractive effects (Ibrahim et al., 2008; Niinemets, 2010a). Sequen-ial or superimposed stress effects can be further complicated bytress priming (Frost et al., 2008; Heil and Kost, 2006; Kesslert al., 2006; Niinemets, 2010b) that can induce partial acclimationesponses to similar type of stress or result in metabolic changes

hat protect directly or indirectly against a different type of stress.or instance, abiotic stress-driven metabolic changes can affectiotic stress response, or priming in responses to one type of biotictress can affect responses to a different type of stress (Cardoza

∗ Corresponding author at: Institute of Agricultural and Environmental Sciences,stonian University of Life Sciences, Kreutzwaldi 1, Tartu 51014, Estonia.

E-mail address: lucian.copolovici@emu.ee (L. Copolovici).

098-8472/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.envexpbot.2013.12.011

et al., 2002; Dicke, 2009; Holopainen and Gershenzon, 2010; Penget al., 2011; Thaler et al., 2002).

Plant volatile emission patterns change quantitatively and qual-itatively in response to damage by herbivores (Arimura et al., 2005;Brilli et al., 2009; Dicke, 2009; Kant et al., 2009; Niinemets et al.,2013; Trowbridge and Stoy, 2013). Most plants have evolved effec-tive defense strategies in order to reduce insect attacks (reviewsin Dicke, 2009; Dicke and Baldwin, 2010; Dicke and Loreto, 2010;Holopainen and Gershenzon, 2010). Presence of multiple stressescan significantly affect the volatile emissions, but so far, the interac-tive effects are still poorly understood. For example, simultaneousfungal and lepidopteran herbivory treatment resulted in simi-lar emissions of herbivory-induced volatiles in Arachis hypogea(Cardoza et al., 2002) and ca. 50% reduced emissions in maize (Zeamays) (Rostás et al., 2006), but the herbivore performance wasnot affected (Rostás et al., 2006) or was even enhanced (Cardozaet al., 2002) in fungal-infected plants, demonstrating complexresponse to multiple biotic stresses. On the other hand, in Z. mays,herbivore-induced VOCs were reduced by 75% in the case of nutri-ent deficiency (Gouinguene and Turlings, 2002), indicating reduced

capacity for indirect defense.

The typical fast response of the plants to herbivore attacks isthe emission of volatile products of lipoxygenase pathway – LOXproducts (also called green leaf volatiles) consisting of various

5 nd Exp

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6 L. Copolovici et al. / Environmental a

6 aldehydes and alcohols (Copolovici et al., 2011; Gosset et al.,009; Pinto et al., 2007; Toome et al., 2010). LOX product emis-ion is a ubiquitous stress response that has been observed inesponse to other biotic stresses such as fungal attacks (Steindelt al., 2005; Toome et al., 2010) and many abiotic stresses includ-ng heat and frost (Copolovici et al., 2012), flooding (Copolovici andiinemets, 2010), ozone (Beauchamp et al., 2005; Heiden et al.,003), high light (Loreto et al., 2006) and mechanical wound-

ng (Loreto et al., 2006). This initial rapid response is followedy emissions of induced volatile isoprenoids, in particular, mono-nd sesquiterpenes (Beauchamp et al., 2005; Copolovici et al.,012; Steindel et al., 2005; Toome et al., 2010) and in someases by emissions of benzenoid methyl salicylate (Beauchampt al., 2005; Cardoza et al., 2002; Zhao et al., 2010). These com-ounds can play a role in host detection of herbivores as wells in eliciting priming responses in plants (Choudhary et al.,008; Penuelas et al., 2007; Ton et al., 2007), thereby playing

major role in determining the integrated response of previ-usly imposed biotic or abiotic stress and subsequent herbivorytress.

Potential changes in host quality and both constitutive andnduced resistance induced by abiotic stresses could influenceerbivores’ development and health, and thereby determine theegree of herbivory damage (Lerdau et al., 1994). The share of theonstitutive vs. induced response is expected to depend on plantharacteristics (growth rate, defense strategy) and the specific typef stress. A key abiotic stress that strongly curtails plant carbonain and growth is drought, but the influence of water deficiencyn the strategies of plants to cope with different biotic attacks haseceived limited attention, and the studies have mainly focused onon-volatile primary or secondary metabolites (e.g., Gutbrodt et al.,012; Khan et al., 2011) or plants’ growth characteristics (Eranent al., 2009; Halpern et al., 2010). For deciduous trees in general,here are very few studies investigating the impact of multipletresses on plant performance and, especially limited are stud-es combining abiotic and biotic stresses (Dicke and Loreto, 2010;olopainen and Gershenzon, 2010; Niinemets, 2010a; Niinemetsnd Monson, 2013). This is an important omission because in treesith longer-living foliage and slower leaf turnover (Wright et al.,

004), the constitutive stress response is expected to be of greaterignificance than in herbaceous species with faster leaf turnovernd more dynamic stress responses.

Here we investigated the kinetics of VOC emission in blacklder (Alnus glutinosa) leaves in response to combined droughtnd herbivory by green alder sawfly (Monsoma pulveratum) larvae,nd studied the degree of herbivory damage and plant attractive-ess to herbivores in drought-stressed and well-watered plants. A.lutinosa is a relatively short-living (ca. 40 yrs) wide-spread fast-rowing tree species colonizing habitats along stream banks andet forests. It has a very low drought tolerance (Niinemets andalladares, 2006). Herbivores and pathogens have a strong impactn Alnus spp. altering their survival, growth and even reproduction,hereby influencing the composition of early successional alderominated ecosystems. So far, only a few studies have investi-ated stress induced by herbivore attack (e.g., Blande et al., 2010;olch and Tscharntke, 2000; Giertych et al., 2006; Tscharntke et al.,001) or by drought (Arbellay et al., 2010; Francis et al., 2005;acke and Sauter, 1996; Sundstrom and Hussdanell, 1995) in Alnus

pp., but the combined stress has not been studied in alder to ournowledge.

We tested the hypotheses that priming by drought stress resultsn altered kinetics of VOC emissions, with drought-stressed plants

esponding earlier to herbivory stress than well-watered plantsnd we also expected the drought-stressed plants to be less attrac-ive and less palatable to the herbivores due to priming of defenseesponses by water stress.

erimental Botany 100 (2014) 55– 63

2. Materials and methods

2.1. Plant material

A. glutinosa seedlings of local (Estonian) origin were grownin 5 L clay pots filled with a 1:1 mixture of commercial pottingsoil (Biolan Oy, Finland) and quartz sand under light intensity of200 �mol m−2 s−1 (HPI-T Plus 400 W metal halide lamps, Philips)with day/night temperatures of 24/18 ◦C for a 12 h light period. Theplants were watered daily and fertilized once per month with aslow release NPK (3-1-2 ratio) fertilizer containing microelements.In all experiments, we used similar-sized 2-year-old seedlings with20–25 leaves. The experiment was conducted with plants hav-ing fully mature leaves, and no leaf area expansion was observedthroughout the experiment in both the control and insect-treatedplants.

2.2. Insects

Larvae of green alder sawfly M. pulveratum (Hymenoptera: Ten-thredinidae) at final instar were used as herbivores. M. pulveratumis a common Palearctic species (recently also found in North Amer-ica) whose folivorous larvae feed on Alnus species, occasionallycausing total defoliation of the trees. The larvae were collected fromthe vicinity of Tartu, Estonia (58◦22′ N 26◦43′ E) a few days prior tothe experiment and kept in plastic containers with access to freshAlnus leaves. Larvae of similar size, approximately 1.2 cm in length,were selected for the experiment.

2.3. Experimental setup

Whole plants were placed in a dynamic headspace samplingcuvette system consisting of eight 3 L glass chambers similar to thesystem described in detail in Toome et al. (2010) and Copoloviciet al. (2011). In this system, roots with the potting medium stayedoutside the chambers, and above-ground plant parts were her-metically sealed in individual cuvettes. The air flow through eachchamber was 0.25 L min−1 and a fan (Sunon Group, Beijing, China)was installed in the chamber resulting in high turbulence. Duringthe experiment, light was provided by Philips HPI-T Plus 400 Wmetal halide lamps for 12 h photoperiod at a level of light inten-sity of 200 �mol m−2 s−1. After hermetic installation of the plants(day −1), all plants were watered to field capacity. Three plantswere randomly chosen for drought treatment (no water providedfor 6 days), while the remaining five were watered daily. After 48 h(day 1), the larvae of M. pulveratum were placed on three drought-stressed and on three well-watered plants to initiate the herbivorystress, while two well-watered plants were left untreated as con-trols. Five larvae were placed on each herbivory-treated plant. Afterthe introduction of the larvae, the plants were stabilized in thesystem with dynamic air flow for two more hours before VOC samp-ling. The larvae were left to feed on the leaves for 7 days. After 5 daysof feeding, the plants under drought stress were watered to fieldcapacity and their recovery was followed for another 2 days withthe larvae still feeding. A similar experiment was performed usingthe same set-up with 4 plants as controls and 4 plants drought-stressed without the herbivory treatment. Both experiments werereplicated twice.

2.4. Leaf structural measurements

After the last collection of VOCs, all leaves were harvested

and scanned at 300 dpi. Projected leaf area was determined fromthe scanned images by a custom made software. Both the totalleaf area after the experiment and before the feeding treatment(non-damaged area, i.e., the holes and fed margins added) were

nd Experimental Botany 100 (2014) 55– 63 57

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Fig. 1. Illustration of the custom-made Y-tube olfactometer (Teflon tubing, internaldiameter of 0.65 cm; length of arms 6 cm) to assess the responses of herbivoresto plant volatiles. For the olfactometric bioassay, the Y-tube was positioned in thecenter of a box (10 cm × 10 cm × 5 cm), and two jars, one enclosing a well-wateredplant and the other a drought-stressed plant, were connected to each of the Y-tube

L. Copolovici et al. / Environmental a

stimated. M. pulveratum feeding mainly consisted of holes anderforations with a minor contribution of feeding at the margins.oreover, the leaf area lost due to the herbivore damage was

stimated daily by counting the new holes. Leaf fresh mass wasetermined immediately after the area estimation, and leaf dryass after drying the leaves for 48 h at 70 ◦C.

.5. Photosynthesis measurements

The photosynthetic characteristics of the plants during lar-al feeding were monitored throughout the experiment as inopolovici et al. (2011). Shortly, plants were placed in a ther-ostable glass chamber with controlled conditions: light intensity

f 200 �mol m−2 s−1, temperature of 28 ◦C, and ambient CO2 con-entration of 380–400 �mol mol−1. CO2 and H2O concentrationst the chamber in- and outlets were measured with an infra-redual-channel gas analyzer operated in differential mode (CIRAS II,P-systems, Amesbury, MA, USA). The rates of net assimilation (A),ranspiration (E), and stomatal conductance to water vapor (gs)ere determined from the chamber in- and outgoing gas concen-

rations according to von Caemmerer and Farquhar (1981).

.6. VOC sampling and GC–MS analysis

VOC sampling was performed on adsorbent cartridges via theutlets of each cuvette every day with a flow rate of 200 ml min−1

or 20 min by using a constant flow air sample pump with fourorts (1003-SKC, SKC Inc., Houston, TX, USA). In addition, a sam-le was taken from the air inlet prior to the cuvettes to estimatehe background VOC concentrations. Multibed adsorbent cartridgesere filled with different grades of Carbopack and were opti-ized for trapping of all plant volatiles between C5 and C15

Copolovici et al., 2009; Toome et al., 2010). Adsorbent cartridgesere analyzed for lipoxygenase pathway products (LOX), mono-,omo- and sesquiterpene concentrations using a Shimadzu TD20utomated cartridge desorber and Shimadzu 2010Plus GC-MSnstrument (Shimadzu Corporation, Kyoto, Japan) according tohe GC–MS method detailed in our previous studies (Copolovicit al., 2009; Toome et al., 2010). The identifications and quantifica-ions of different compounds were done using authentic standardsSigma–Aldrich, Taufkirchen, Germany). The background (blank)OC concentrations were subtracted from the emission samplesith the seedlings. Separate samples were collected from the lar-

ae without the plants. The emissions of volatile compounds fromhe larvae were below the detection limit of our system.

.7. Olfactometer bioassays

Responses of herbivores to plant-derived volatiles weressessed using a home-made Teflon Y-tube olfactometer (internaliameter of 0.65 cm; length of arms 6 cm). The olfactometer assayas carried out in the same environmental conditions as the her-

ivory treatments. The Y-tube was placed in the center of a box10 cm × 10 cm × 5 cm). Two jars enclosing plants similar to thosesed for the experiments, one with a well-watered plant and thether with a drought-stressed plant, were connected to each of thends of the Y-tube olfactometer. The air was circulated through theystem using a small pump as indicated in Fig. 1. Before the firstarva was released, the whole setup was left to stabilize for 2 h. Thearvae of M. pulveratum were kept without food for 1 day before

he experiment started. In each time, 30 larvae were released inhe box and the number of larvae choosing either one or the otherlant were counted after 10 h. The test was replicated five timesith independent plants.

olfactometer ends.

2.8. Statistical analysis and data handling

The experiments were replicated twice with independent plantsand larvae (in total 6 plants per treatment and 4 controls). As theeffect of the replicate experiment (block effect) was not statisticallysignificant, the replicate experiments were pooled. The treatmentmeans were statistically compared by Tukey’s test and StudentANOVA post hoc test using ORIGIN 8 (OriginLab corporation, MA,USA) and GraphPad PRISM 5 (GraphPad Software Inc., La Jolla, CA,USA).

The plants’ emissions were evaluated by principal componentanalysis (PCA). After mean-centering, logarithmical data trans-formation was used. From PCA analysis, resulting loading andscore plots were derived. The differences between the bouquetswere tested by Monte Carlo permutation test using redundancydata analysis (RDA). The multivariate analyses were conductedby Canoco 4.5 software (ter Braak and Smilauer, Biometris PlantResearch International, The Netherlands). All statistical tests wereconsidered significant at P < 0.05. Means in olfactometer assay werecompared by a Kruskal–Wallis non-parametric ANOVA.

3. Results

3.1. Herbivore preference and degree of feeding damage inwell-watered and drought-stressed plants

When the larvae of M. pulveratum could choose between well-watered and drought-stressed A. glutinosa plants in the Y-tubeolfactometer test, ca. 60% of them choose the well-watered plants,and 40% the drought-stressed plants (Fig. 2a). The leaf area eaten

by the insects was significantly higher (P < 0.01) for well-wateredplants than for drought-stressed plants (Fig. 2b).

58 L. Copolovici et al. / Environmental and Experimental Botany 100 (2014) 55– 63

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Fig. 2. Average (±SE) behavioral responses of alder sawfly (Monsoma pulveratum) larvae in a Y-type olfactometer to odors of well-watered and drought-stressed Alnusglutinosa plants (a) and average leaf area eaten (±SE) by five larvae of M. pulveratum at the end of the experiment in the well-watered and drought-stressed treatments( 5), ans

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.2. Herbivory effects on foliage structure and photosyntheticharacteristics

Average values (±SE) of leaf dry to fresh mass ratio of.265 ± 0.023 g g−1 and leaf dry mass per unit area of 65 ± 6 g m−2

ere observed in A. glutinosa seedlings. There were no sta-istical differences among the treatments in both traits (P >.1).

In control (well-watered, non-herbivory treated) plants, the netssimilation rate, A, was stable at 10–11 �mol m−2 s−1 throughouthe experiment (Fig. 3a). In M. pulveratum infested well-wateredlants, A was between 9 and 10 �mol m−2 s−1 during the exper-

ment, and did not differ from the control treatment (P > 0.05;ig. 3a). In the case of infested drought-stressed plants, A decreasedo a level of 1.55 ± 0.37 �mol m−2 s−1 after 7 days of drought andecovered to a level of 10.13 ± 0.48 �mol m−2 s−1 after re-wateringFig. 3a). The trend was the same for the drought-stressed plantsithout insects.

Stomatal conductance was stable at 85 mmol m−2 s−1 through-ut the study in both control and well-watered herbivore-infestedlants (Fig. 3b). In the drought-stressed plants, the stomatal con-uctance to water vapor decreased drastically after 7 days of water

estriction to a very low level of 17.8 ± 2.1 mmol m−2 s−1 in plantsith the herbivore feeding and to a level 13 ± 3 mmol m−2 s−1 inlants without herbivore feeding. After re-watering, stomatal con-uctance recovered to the initial level.

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ig. 3. Changes in net assimilation rate (a) and stomatal conductance to water vapor (b) irought without insects (©), and with Monsoma pulveratum larvae, either well-wateredulveratum were placed on each plant. The measurements were conducted at a quantuoncentration of 380 ± 20 �mol mol−1. The data are expressed per unit projected leaf are

d in (b) by a standard ANOVA (n = 6) and different letters indicate means that are

3.3. LOX product emissions

Volatile lipoxygenase pathway products (LOX compounds) werethe first volatiles induced by stresses. Even before the larvae wereadded to the plants (day 0), the emission of LOX had alreadysignificantly increased in drought-stressed plants compared withwell-watered plants (P < 0.01). After the larvae started to eat theleaves (day 1), the emission of LOX increased significantly in well-watered plants relative to controls (P < 0.05), but even more in thedrought-stressed plants (P < 0.001; Fig. 4). In the following days,the emissions of LOX compounds in well-watered plants stayedalmost constant at the level of 57 ± 8 pmol m−2 s−1 until day 8.In drought-stressed plants, LOX emission rate was on average182 ± 32 pmol m−2 s−1 between days 1 and 6 for plants with insects,and 135 ± 32 pmol m−2 s−1 for plants without insects. Lox emis-sions decreased significantly (P < 0.01) on day 7 when the plantswere re-watered (Fig. 4). At the end of the experiment on day8, LOX emission rate of 62 ± 8 pmol m−2 s−1 in infested drought-stressed plants was not different from LOX emissions in infestedwell-watered plants, but the emissions were significantly different(P < 0.01) in non-infested drought-stressed plants (Fig. 4).

3.4. Monoterpene emissions

The monoterpenes detected were �-pinene, �-pinene, cam-phene, limonene, 3-carene, p-cymene, �-phellandrene and

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n seedlings of Alnus glutinosa without insects and well watered – control (�), under (�) or under drought stress (�). In herbivory treatments, five larvae of Monsomam flux density of 200 �mol m−2 s−1, air temperature of 28 ◦C and at ambient CO2

a. Each data point is the mean (±SE) of 6 independent replicate plants.

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Fig. 4. Emission time-courses (in pmol m−2 s−1) of total LOX products for non-treated plants (control), and plants fed by M. pulveratum larvae, and eitherwec

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ell-watered or drought-stressed. Means were separated by Tukey’s test and differ-nt letters indicate means that are statistically different with P < 0.05. Experimentalonditions as in Fig. 3.

E)-�-ocimene. Low emissions of terpenes (0.03 nmol m−2 s−1),lose to the baseline level, were observed in control plants (Fig. 5).n the first day after the larvae were added to the plants (day 1),o significant increase in monoterpene emissions was observedP = 0.13, Fig. 5). On day 2, the emission of monoterpenes fromhe drought-stressed larvae-infested plants increased drastically,eaching a level of 1547 ± 51 pmol m−2 s−1 while the emission fromell-watered infested plants were still low, and not significantlyifferent from controls (P = 0.32, Fig. 5). On the same day, themissions from non-infested drought-stressed plants increasedo a level of 752 ± 69 pmol m−2 s−1, being significantly differentP = 0.32) from control and drought-stressed larvae-infested plants.n day 3, the emissions of monoterpenes from drought-stressedlants had increased even more, and monoterpene emissionsrom well-watered infested plants were also significantly elevatedP < 0.01, Fig. 5). The differences among the treatments – themissions in drought-stressed infested plants higher than inhe two other treatments, and in well-watered infested plants

igher than in control plants – were maintained on the followingays (Fig. 5). On day 7, when the plants under drought weree-watered, the monoterpene emissions decreased to a value of

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ig. 5. Emission time-courses (in pmol m−2 s−1) of total monoterpene emissionser unit leaf area for control plants (non-herbivory treated, well-watered), andlants fed by M. pulveratum larvae, and either well-watered or drought-stressed.xperimental conditions as described in Fig. 3. and data presentation and statisticalnalysis as in Fig. 4.

erimental Botany 100 (2014) 55– 63 59

1070 ± 170 pmol m−2 s−1 and the monoterpene emission rateswere no longer significantly different among drought- and well-watered herbivory-stressed treatments (Fig. 5). The emissionfrom drought-stressed non-infected plants became also signif-icantly lower (257 ± 58 pmol m−2 s−1) than that from infestedwell-watered plants.

3.5. Emissions of herbivory-stress marker compounds(E)-ˇ-ocimene, DMNT and MeSA

Temporal variation in (E)-�-ocimene emissions was simi-lar to total monoterpene emissions with significant emissionselicited immediately after the start of feeding under droughtstress, and with a delay in well-watered plants. However, differ-ently from total monoterpenes, (E)-�-ocimene emission increasedcontinuously until reaching a maximum on day 4 in drought-stressed plants and on day 5 in well-watered plants (Fig. 6a).The maximum level of (E)-�-ocimene emission was much lower,30.6 ± 0.7 pmol m−2 s−1 in well-watered infested than in drought-stressed infested plants (44.6 ± 1.0 pmol m−2 s−1). Furthermore,differently from total monoterpenes that started to decrease indrought-stressed plants after re-watering and reached a similarlevel to that in well-watered plants on day 7, (E)-�-ocimene emis-sions reached a similar level in both infested treatments on day 6(cf. Figs. 5 and 6a).

The emission of nerolidol-derived homoterpene 4,8-dimethyl-nona-1,3,7-triene (DMNT) started on the first day when the larvaewere added (Fig. 6b). For the first two days (days 1 and 2) ofinsect treatment, the emissions were not statistically differentbetween the drought-stressed and well-watered plants (P > 0.5).The emission maximum was reached on day 2 in drought-stressedplants and on day 4 in well-watered plants. From this day on,the DMNT emissions from the well-watered herbivore-infestedplants exceeded the emissions from drought-stressed herbivore-infested plants (Fig. 6b). The emission of DMNT from plants underdrought stress alone was not statistical different from control plants(P > 0.5).

Methyl salicylate (MeSA) emission from plants under droughtstress was strongly increased on day 1 of the insect treatment andremained at the high level (on average 63 ± 17 pmol m−2 s−1) untilthe plants were re-watered on day 6, after which the MeSA emis-sions first decreased to 14 ± 5 pmol m−2 s−1 on day 7 and then tothe background level on day 8 (Fig. 6c). The same trend in MeSAemission was evident in plants under drought stress alone with amaximum emission of 40 ± 17 pmol m−2 s−1 on day 1. MeSA emis-sions of well-watered insect-infested plants did not significantlydiffer from the control plants (Fig. 6c).

3.6. Changes in the emission blends

Larval feeding had a great impact on the emitted VOC con-tent and composition in both well-watered and drought-stressed A.glutinosa plants (Fig. 7a and b). During the experiment, the compo-sition of the VOC bouquet did not change in control plants (MonteCarlo test, P > 0.05). In the case of drought stress, the plant emis-sion blend changed already on the first day of drought (D0 inFig. 7b). Moreover, on the next day (D1 in Fig. 7b), feeding of lar-vae for only 2 h was sufficient to cause considerable changes inthe emissions of A. glutinosa plants (Monte Carlo test, P < 0.01).The bouquet of drought-stressed plants changed again after the

plants were re-watered (Monte Carlo test, P < 0.01). During thefirst days of infection in well-watered plants (from W-1 to W1,Fig. 7b), the emission bouquet of well-watered plants was similar tothat in control plants (Monte Carlo test, P > 0.05), and considerable

60 L. Copolovici et al. / Environmental and Exp

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Fig. 6. Temporal variations in monoterpene (E)-�-ocimene (a), homoterpene4,8-dimethylnona-1,3,7-triene (DMNT) (b) and benzenoid methyl salicylate (c)eaE

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missions (in pmol m−2 s−1) from A. glutinosa control plants (well-watered, non-fed)nd plants fed by M. pulveratum larvae and either well-watered or drought-stressed.xperimental conditions and treatments as in Fig. 3 and data presentation as in Fig. 4.

odifications were discovered from the second day on (day W2,ig. 7b, Monte Carlo test, P < 0.05).

. Discussions

.1. Attractiveness and palatability of water-stressed vs.ell-watered plants to herbivores

Plant photosynthesis and growth rates are strongly reducedy drought due to stomatal closure, and our results are consis-ent with these general patterns (Chaves et al., 2009, 2011; Flexasnd Medrano, 2002; Centritto et al., 2011). Stopping watering of

rought-sensitive species A. glutinosa reduced photosynthesis andtomatal conductance by more than 80% during the 7 days ofrought treatment (Fig. 3). Olfactometer experiments indicatedhat the M. pulveratum larvae preferred the well-watered plants

erimental Botany 100 (2014) 55– 63

compared with the drought-stressed plants (Fig. 2a). The drought-stressed plants were apparently also less palatable as suggested bymore than four-fold greater amount of leaf area consumed by herbi-vores in well-watered plants (Fig. 2b). This result is in agreementwith other studies demonstrating that the insects perform better onhealthy plants (Huberty and Denno, 2004, 2006) and might suggestinduction of enhanced investment in defense metabolites such astannins and polyphenols in drought-stressed plants. However, paststudies in deciduous trees have actually suggested that droughtleads to reduced phenolic compound concentrations (Shure et al.,1998; Thomas and Schafellner, 1999), but the data are limited.We suggest that more work is needed to analyze modifications ininvestment in secondary metabolites in drought-stressed plants.

4.2. Herbivory effects on photosynthetic characteristics

Previous studies have shown that net assimilation rate and sto-matal conductance can be stimulated, suppressed or not affectedby the herbivore feeding (Copolovici et al., 2011; Kerchev et al.,2012; Woolery and Jacobs, 2011). In our study, well-watered plantswith only herbivore stress maintained the photosynthetic ratesand stomatal conductance at the same level as control plantsthrough the entire experiment (Fig. 3a and b). Leaf damage dueto herbivory typically results in suppressed photosynthesis in theimmediate vicinity of the damaged leaf parts, but also in reducedphotosynthesis in surrounding areas due to free water evapora-tion and desiccation stress (Aldea et al., 2006; DeLucia et al., 2008).Thus, constancy of net assimilation rates and stomatal conduc-tance suggests a compensatory response. Such a compensatoryresponse may result from alteration of the strength of the source-sink relationships with increasing the absolute demand of sinks forcarbohydrates (Copolovici et al., 2011). Recovery of photosyntheticrates of drought-stressed insect-fed plants to the level before thedrought stress suggests that if there was compensatory photosyn-thesis, it occurred similarly in drought-stressed and well-wateredplants.

4.3. Elicitation of LOX products

Damage of A. glutinosa leaves by M. pulveratum larvae elicitedcharacteristic emissions of lipoxygenase pathway compounds(LOX) with the maximum emission rates already achieved inthe first day of the feeding (Fig. 4). Induction of LOX emissionsby herbivory treatment is in agreement with numerous studiesdemonstrating enhanced emissions in herbivore-fed plants.

The emissions under water limitation were much higher despitelower degree of herbivory damage (Figs. 2b and 4). Thus, a clearpriming effect of drought was found on LOX emissions. This evi-dence suggest that a given level of herbivory stress was “moresevere” in the case of drought-stressed plants. Provided that LOXare likely released in response to early signaling events includingformation of reactive oxygen species (ROS) and electrolyte leak-age at the site of insect damage (Arimura et al., 2011; Bruinsmaet al., 2010; Wu and Baldwin, 2009) it might be that drought-drivenformation of ROS (Chaves et al., 2009) sensitized the LOX response.

4.4. Induction of terpenoid emissions

Leaves of A. glutinosa plants do not emit terpenes (monoter-penes, sesquiterpenes, or terpenoid derivatives), and do notaccumulate any of these compounds under non-stressed condi-tions (Copolovici et al., 2011) and this was confirmed by very low,

close to the detection limit, emission of terpenoids in control plants.Insect feeding resulted in induction of moderately high monoter-pene emissions from well-watered and drought-stressed plants,but monoterpene emissions were induced later that LOX emissions.

L. Copolovici et al. / Environmental and Experimental Botany 100 (2014) 55– 63 61

Fig. 7. Loading (a) and score (b) plots of principal component analysis (PCA) based on the time-courses of emitted volatiles (in pmol m−2 s−1) for non-treated plants (C), andplants fed by M. pulveratum larvae, and either well-watered (W) or drought-stressed (D). In the score plot, each symbol represents a mean bouquet of 6 replicate plants(numbers after the treatment symbol denote the day of the experiment), while in the loading plot, the numbers represent different volatiles as following: 1 – 1-hexanol; 2 –2 mphe1 T; 17

2 ith th

Aiea

iahedeNbd

4

msonDtaptSsa2ph

�iMhsetem

-(E)-hexenal; 3 – 3-(Z)-hexenol; 4 – 3-(Z)-hexenyl acetate; 5 – bornyl acetate; 6 – ca2 – (Z)-�-ocimene; 13 – �-phellandrene; 14 – �-pinene; 15 – �-pinene; 16 – DMN0 – nonanal. In the loading plot, the amount of the emitted compound increases w

nalogous induction of monoterpene emissions has been observedn several lepidopteran larvae feeding experiments (Copolovicit al., 2011; Holopainen et al., 2013; Schaub et al., 2010; Trowbridgend Stoy, 2013).

As with LOX emission, monoterpene emissions were highern drought-stressed plants, and the emissions increased faster,gain suggesting that drought primed the plants to more sensitiveerbivory response (Gouinguene and Turlings, 2002; Takabayashit al., 1994). Such enhancement of monoterpene emissions byrought has been demonstrated in constitutively monoterpene-mitting species (Blanch et al., 2009; Llusià and Penuelas, 1998;ogués et al., 2012; Ormeno et al., 2007; Penuelas et al., 2007),ut to our knowledge such a drought enhancement has not beenemonstrated for induced emissions.

.5. Induction of stress-specific compounds

Three key compounds implicated in herbivore response, acycliconoterpene (E)-�-ocimene, homoterpene DMNT, and methyl

alicylate were apparently regulated differently than either LOXr other monoterpenes (cf. Figs. 4–6). Differently from LOX oron-stress-specific monoterpenes, emission of (E)-�-ocimene andMNT were characterized by a maximum indicating downregula-

ion in these emissions at later stages of herbivory. Furthermore,lthough these emissions were induced earlier in drought-stressedlants, the differences among the treatments were much smallerhan the differences in either LOX or ubiquitous monoterpenes.uch similarity in the emissions is in agreement with our previoustudy, where no herbivory “dose” effect was found for DMNT and

weak effect was observed for (E)-�-ocimene (Copolovici et al.,011), suggesting that emissions might serve as signals for theresence of herbivores, but are not indicative of the number oferbivores.

It has been demonstrated that increased emission of (E)--ocimene in herbivore-damaged plants of Medicago truncatula

s associated with accumulation of transcripts of corresponding. truncatula (E)-�-ocimene synthase (MtEBOS), indicating that

erbivore feeding increases volatile release by inducing terpeneynthase gene expression (Navia-Gine et al., 2009). Herbivore-

licited (E)-�-ocimene emissions have been shown to occur viahe plastidial 2-C-methyl-d-erythritol 4-P (MEP) pathway (Arimurat al., 2000) while the emissions of DMNT likely occur throughevalonate pathway (Bartram et al., 2006). Both isoprenoid

ne; 7 – para-cymene; 8 – 3-carene; 9 – limonene; 10 – linalool; 11 – (E)-�-ocimene;– geranyl acetone; 18 – 6-methyl-5-heptene-2-one; 19 – methyl salicylate (MeSA);e distance from the origin of the coordinate system.

synthesis pathways are upregulated under herbivory (Arimuraet al., 2000; Bartram et al., 2006). Earlier induction of these twocompounds in drought-stressed plants furthermore emphasizesthe priming effect of drought, possibly involving upregulation ofthe two isoprenoid synthesis pathways.

The most interesting result is the strongly enhanced MeSAemissions in drought-stressed plants during herbivory. It has beenshown that oxidative stress can induce premature senescence ofmature leaves and salicylic acid may be involved in the regulationof this process (Elizabeth Abreu and Munné-Bosch, 2008; Munné-Bosch and Penuelas, 2003). On the other hand, the genes which areregulated by salicylic acid are defense-related genes, and they canparticipate in plant responses to biotic and abiotic stresses (Morriset al., 2000). Methyl salicylate is commonly elicited in responseto fungal pathogens (Cardoza et al., 2002) and attacks by sap-sucking herbivores such as aphids or whiteflies (Li et al., 2006;Zarate et al., 2007), but it may also be elicited downstream the cas-cade of jasmonic acid induced responses after chewing herbivoreattacks (Cardoza et al., 2002; Dicke et al., 1999; Rodriguez-Saonaet al., 2001). Thus, MeSA emissions following the start of herbivoryin drought-stressed plants reflect interactions among defense path-ways that clearly deserve further study.

We did not study tritrophic interactions, but alteration ofvolatile blends by drought stress might serve as an important sig-nal for enemies of herbivores as well. The natural enemies of theherbivores use different chemical compounds that can be detectedat long distances from attacked plants (see for reviews Dicke andBaldwin, 2010; Dicke and Loreto, 2010). Differences in the emis-sion blend between well-watered and drought-stressed plants, inparticular the apparent repellence to herbivores of the odor ofdrought-stressed plants may be informative of the quality of preyfor the parasitoids.

5. Conclusions

These data provide evidence that A. glutinosa plants exposed tomild drought prior to herbivore attack become primed to subse-quent different type of stress. In this context, alder trees becomeless sensitive to herbivory by the larvae of M. pulveratum. Also,

changes in the emission blend by drought that clearly changethe attractiveness of plant odor may play an important role intritrophic interactions. These data collectively suggest that abioticstresses can strongly interact with biotic stresses and such stress

6 nd Exp

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A

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B

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B

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B

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2 L. Copolovici et al. / Environmental a

nteractions can play a major role in determining the ultimateegree of insect damage as well as alter tritrophic interactions.

cknowledgements

Funding for this study has been provided by the Estonianinistry of Science and Education (Institutional Grant IUT-8-

), Estonian Science Foundation (Grant 9089), the Europeanommission through the European Regional Fund (the Centerf Excellence in Environmental Adaptation) and the Europeanesearch Council (advanced Grant 322603, SIP-VOL+), and Roma-ian National Authority for Scientific Research, CNCS – UEFISCDIproject number: PN-II-RU-TE-2011-3-0022). We thank Kaleviuukool (Lääne-Virumaa, Kiltsi, Estonia) for A. glutinosa plants.

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