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Environmental and Experimental Botany 71 (2011) 34–40

Contents lists available at ScienceDirect

Environmental and Experimental Botany

journa l homepage: www.e lsev ier .com/ locate /envexpbot

o plants remember drought? Hints towards a drought-memory in grasses

ulia Waltera,∗, Laura Nagyb,e, Roman Heinb,e, Uwe Rascherc, Carl Beierkuhnleinb,velin Willnerd, Anke Jentsche

Conservation Biology, Helmholtz Centre for Environmental Research- UFZ, Permoserstraße 15, 04318 Leipzig, GermanyBiogeography, Bayreuth University, 95440 Bayreuth, GermanyInstitute of Chemistry and Dynamics of the Geosphere (ICG), Phytosphere (ICG-3), Forschungszentrum Jülich GmbH, 52425 Jülich, GermanyLeibniz Institute of Plant Genetics and Crop Plant Research (IPK), Genebank, Satellite Collections North, D-23999 Malchow/Poel, GermanyGeoecology/Physical Geography, Institute for Environmental Science, University of Koblenz-Landau, Forststraße 7, 76829 Landau, Germany

r t i c l e i n f o

rticle history:eceived 1 July 2010eceived in revised form3 September 2010ccepted 22 October 2010

eywords:ecurrent drought stresslimate warming

a b s t r a c t

The frequency of extreme drought events is projected to increase under global climate change, causingdamage to plants and crop yield despite potential acclimation. We investigated whether grasses remainacclimated to drought even after a harvest and remember early summer drought exposure over a wholevegetation period. For this, we compared the response of Arrhenatherum elatius plants under a second,late, drought (they were pre-exposed to an early drought before), to plants exposed to a single, only late,extreme drought. Surprisingly, the percentage of living biomass after a late drought increased for plantsthat were exposed to drought earlier in the growing season compared to single-stressed plants, even afterharvest and resprouting after the first drought. Relative leaf water content did not differ between the twotreatments. Net photosynthesis was non-significantly reduced by 25% in recurrent drought treatment.

emory-effect

ater deficithlorophyll a fluorescencehenotypic plasticity

Maximum quantum efficiency (Fv/Fm) and maximum fluorescence (Fm) were reduced in plants that wereexposed to recurrent drought. These findings indicated improved photoprotection in double-stressedplants. Our results provide first hints towards a “drought memory” over an entire vegetation period,even after harvest and resprouting. However, the advantage of improved photoprotection might alsocause reductions in photosynthesis that could have adverse effects on crop yield under more severe or

longer droughts.

. Introduction

Droughts are often regarded as major threats to ecosystemsnder global climate change, as water stress limits crop yield morehan all other biotic and abiotic factors combined (Lambers et al.,008). The frequency and magnitude of regional drought periodsave been increasing since the 1970s, with an exacerbation of theituation projected for many parts of the world (Schar et al., 2004;renberth et al., 2003). In Europe, the Mediterranean and mid-

ontinental regions are expected to experience increased droughteriods, accompanied by heat waves, as witnessed during the sum-er drought of 2003 (Schar et al., 2004).

Abbreviations: Fm, maximum fluorescence yield of the dark adapted leaf;0, steady state fluorescence yield of the dark adapted leaf; Fv, variable fluores-ence yield of the dark adapted leaf; Fv/Fm, potential maximum quantum yield ofhotosystem II; Pn (�mol CO2 m−2 s−1), net photosynthesis; PPFD (�mol m−2 s−1),hotosynthetically active photon flux density; RWC (%), relative leaf water content.∗ Corresponding author. Tel.: +49 341 235 1654; fax: +49 341 235 1470.

E-mail address: Julia.walter@ufz.de (J. Walter).

098-8472/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.envexpbot.2010.10.020

© 2010 Elsevier B.V. All rights reserved.

Recent studies have investigated the impact of single droughtevents on ecosystems (Noormets et al., 2008), plant communi-ties (van Peer et al., 2004; Kreyling et al., 2008) or single species(Galle et al., 2007; Jentsch et al., 2009). However, the consequencesof recurrent drought events when compared to a single droughtevent on stress response and post-stress recovery are still not wellunderstood.

Theory predicts that abiotic stress reduces resilience of ecosys-tems as a response to a single stress event, resulting in deterioratedperformance and a total loss of resilience, when the system isaffected by recurrent stressful events (Scheffer et al., 2001). Onthe other hand, plants are able to acclimate when faced with abi-otic stress (Lambers et al., 2008; Bruce et al., 2007), revealingphenotypic plasticity as a response to environmental variability(Aubin-Horth and Renn, 2009). Short-term drought acclimationincludes modifications to pigment content and the Xanthophyllcycle (enhanced non-photochemical quenching) in order to pre-

vent photodamage (Munne-Bosch and Alegre, 2000; Jiang et al.,2005). Phenotypic plasticity can cause a “stress imprint” that mightfacilitate a fast and protective response to a recurrent stressfulevent. Bruce et al. (2007) mention epigenetic changes and accumu-lation of signalling proteins or transcription factors as mechanisms

d Experimental Botany 71 (2011) 34–40 35

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Table 1Accession numbers, countries of origin and geographical parameters of the sixprovenances included in the study.

Accession no. Country Lat [◦ WGS84] Long [◦ WGS84]

GR 331 Germany 51.8 13.7GR 339 Poland 50.6 21.7GR 357 Germany 51.1 11

2.2. Aboveground biomass

Aboveground biomass was harvested on July 5th, 17 days afterthe first drought (D1 and C1) ended, and on October 9th, 21

Sept. 4th-19th3rd-18thJune

D1

C1

D2

C2

droughted

watered

J. Walter et al. / Environmental an

or such a “memory effect”. Persistent changes in gene expressionave been documented following a stressful event (Aubin-Horthnd Renn, 2009). Epigenetic modifications can be inherited throughitosis or even meiosis (Goh et al., 2003; Bird, 2007; Bruce et al.,

007; Verhoeven et al., 2010). Up to now, experiments investigat-ng these stress imprints have been mostly restricted to small timepans of less than 1 week (Bruce et al., 2007). Furthermore, studiesnvestigating plant performance and functional consequences, andot only underlying molecular mechanisms in response to recur-ent stress are rather rare. In woody communities, drought wasound to reduce resilience, rendering plants more vulnerable to aecurrent disturbance (Lloret et al., 2004; Mueller et al., 2005). Inrassland communities, mild droughts and warming did not leado an enhanced resistance or recovery to an extreme follow-uprought but rather to a larger decrease in green vegetation cover

n communities experiencing recurrent drought (Zavalloni et al.,008). It has also been shown that plants can reveal memory effectsfter frost events, leading to a deteriorated performance long afterrost stress was applied (Polle et al., 1996; Tahkokorpi et al., 2007;reyling et al., 2010).

The objective of our study was to identify differences in theffects of recurrent drought compared to a single drought on plantroductivity and the photosynthetic performance of potted indi-iduals of Arrhenatherum elatius (L.). A. elatius is a widely distributednd agriculturally important European perennial grass. It occursainly in moist, but not waterlogged habitats (Grime et al., 1988).ur explicit interest was on agriculturally important performancearameters (living biomass, photosynthesis, photoprotection) ofpotential long lasting drought memory, and not on elucidat-

ng underlying genetic or chemical mechanisms. We investigatedhe presence, and potential photosynthetic mechanisms, related to“long-term memory-effect” in grasses by imposing a recurrent

rought 8 weeks after an early drought. Between the first and theecond drought, plants had been harvested and resprouted. As aontrol we compared those double-stressed plants to plants thateceived a single drought only. We aimed at quantifying not onlyesistance to recurrent drought but also recovery of ecophysiolog-cal parameters in the early post-stress phase. In order to showhe generality of our results we included a variety of Europeanrovenances as a random factor in our experiment. Plants fromhese provenances have been found to be genetically distinct andhus potentially differing in phenotypic plasticity (Michalski et al.,010).

We hypothesized that plants experiencing recurrent droughtill not react differently to water deficit than plants experiencingsingle drought. Furthermore, grasses after recurrent drought willot recover faster or slower than grasses after single drought. This

s because drought acclimation that could buffer the adverse effectsf a recurrent drought is unlikely to persist after total abovegroundarvest and resprouting.

. Materials and methods

.1. Plant material and experimental setup

A. elatius plants from six different provenances from Germany,oland and Hungary were grown from seeds at the IPK Genebank,atellite Collections North on the Island of Poel in GermanyTable 1). In April 2007, 3-month-old individuals were planted intol bottomless tubes (20 cm in diameter) at the Ecological Botan-

cal Garden of the University of Bayreuth, Germany (49◦55′19′′N,1◦34′55′′E, 365 m asl). Tubes were embedded into the homoge-ized soil (loamy sand consisting of 82% sand, 13% silt, 5% clay to aepth of 80 cm). Plants were kept under natural ambient conditionsor 2 years and harvested twice per year. Furthermore, tubes were

GR 364 Poland 50 22.5GR 7260 Germany 51.3 12.4RCAT041661 Hungary 47.5 18.1

periodically weeded. In April 2009, tubes, including the soil, werearranged under a rain-out-shelter for the experiment. To avoid thelateral flow of water into the pots, they were placed on plates on aplastic sheet. The transparent rain-out shelter was left open at theside up to 80 cm, allowing air exchange near to the surface and thusavoiding greenhouse-effects. Plants were subjected to two differ-ent treatments and arranged in a completely randomized design:28 plants in the recurrent drought treatment (two plants died in the2 years before the experiment started) were subjected to an earlydrought in June 2009 (D1), whereby water was completely with-held for 16 days from June 3rd until June 18th. The same plants weresubjected to a later drought (D2), whereby water was withheldfor 16 days from September 4th to September 19th. The recurrentdrought treatment was compared to a single drought treatment: 27replicates (three plants died prior to the experiment) were wateredregularly every third day with 300 ml rain water (C1) while the firstdrought period was applied to double-stressed plants. C1 plantswere exposed to their first drought in September (C2), concomitantto the second drought of the recurrent drought treatment (see Fig. 1for an overview). Only in comparing plants subjected to a second,late drought (D2) to plants experiencing their first drought (C2) atthe same point in time, we could prevent confounding of poten-tial drought memory effects with seasonality or timing effects. Acomparison between the response to the first drought in June andthe response to the second drought in September is thus not validto investigate potential drought memory effects. We also did nothave a well-watered control in September, as we were interested ina potential “drought-memory”, which can only be investigated bycomparing single-stressed with double-stressed plants. To quantifyeffects of a single, early drought (D1), drought plants were com-pared to well-watered plants during the first drought period (C1).All plants were watered with the same amount of water (300 ml,every third day) until the onset of the experiment and in betweenthe two drought treatments.

Fig. 1. Overview on the experimental time course and on applied treatments. InJune, drought was applied for 16 days to D1 plants, while C1 plants were wateredregularly. All plants were watered after the 16th day until the onset of the latedrought in September. In September all plants (D2 and C2) were subjected to thedrought for 16 days and were watered after that.

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6 J. Walter et al. / Environmental and

ays after the second drought ended (D2 and C2). After this time,eversible drought damages should have been recovered. As wenly compare D2 with C2 plants and D1 with C1 plants, the dif-erence of 4 days in recovery time after drought does not have anffect on the results. Plants were cut 4 cm above the ground in ordero simulate common management techniques in meadows, sortednto living (green) and dead biomass. Dead biomass was defined as

ilted, brown plant parts that lost chlorophyll. Biomass was driedt 70 ◦C for 72 h and weighed. Percentage of dead biomass was cal-ulated as percentage of oven-dried, dead biomass in relation toverall oven-dried biomass of individual plants.

.3. Relative leaf water content (RWC)

Relative leaf water content was determined in the afternoon ofhe 13th day of the first and second drought treatment (June 15thnd September 16th), according to Barrs and Weatherley (1962).he second lowest leaf of each plant was cut, stored in a moistenedlastic bag for transport, and immediately weighed to determineresh weight (FW). Leaves were stored in distilled water at 4 ◦C overight and weighed the next morning to determine turgid weightTW). Afterwards leaves were dried at 70 ◦C and the dry weightDW) was determined.

RWC was calculated as:

WC (%) = FW − DWTW − DW

× 100

.4. Chlorophyll a fluorescence

Chlorophyll a fluorescence was recorded using a pulse-mplitude-modulated photosynthesis yield analyzer (PAM 2000nd Mini-PAM) (Waltz, Effeltrich, Germany) with a leaf clip holders described by Bilger et al. (1995). The second or third fullyxpanded leaves were measured on four different blades of onendividual. Four measurements per plant were averaged for fur-her analysis. We obtained predawn fluorescence values (between:00 and 4:00) at the end of the first drought treatment, through-ut the second drought period and throughout the early recoveryhase after the second drought. The maximum quantum efficiencyf photosystem II was calculated as Fv/Fm. Variable fluorescenceFv) and maximum fluorescence (Fm) were measured before dawn.v was calculated as Fm − F0, Fm being the maximum fluorescencef the dark adapted leaf after applying a saturating light pulse and0 being the steady state fluorescence yield of the dark adapted leafMaxwell and Johnson, 2000).

To enable a comparison between absolute fluorescence values, auorescence standard material was measured before and after eacheasuring cycle. Standard measurements were used to normalize

he fluorescence values obtained and to calibrate the two differentAMs in use. Predawn measurements of fluorescence at the darkdapted leaf allow drawing conclusions about underlying processeshich alter plant photosynthetic performance and about photoin-ibitory damage and non-photochemical quenching (Maxwell and

ohnson, 2000). Absolute F0 and Fm values were taken to separatehe effects of photodamage, becoming apparent with an increasef F0, from the effects of photoprotection related to enhanced non-hotochemical quenching, becoming apparent with a decrease inm (Osmond et al., 1993; Araus et al., 1998; Maxwell and Johnson,000).

.5. Leaf gas exchange

The net CO2 assimilation rate (Pn) and transpiration were mea-ured at midday (between 11:30 and 13:30), when drought stresshould be at its maximum, due to high temperature and irradiance.

rimental Botany 71 (2011) 34–40

It was measured on the second, fully developed leaf of each plantusing a gas-exchange system (Li-6400, Li-Cor, Lincoln, NE, USA)equipped with a CO2 cartridge to adjust and maintain constantCO2 of 400 �mol mol−1 air within the leaf cuvette. Gas exchangemeasurements were conducted on clear days without clouds tomaintain constant PPFD. After reaching steady-state photosynthe-sis, data were logged. The leaf area was estimated simultaneouslyby measuring the leaf width and later on used to correct values fornet photosynthesis, as leaf blades did not fill the whole leaf cuvette.Gas exchange was measured under ambient light conditions at theend of the first drought period, in the early and late drought periodand in the early recovery phase after the drought.

2.6. Statistical analysis

To determine significant differences between single and recur-rent drought treatments, analyses of variance were performed forall variables for each sampling date. We defined “treatment” as afixed factor. “Provenance” was a random factor in this experiment,as provenances were chosen randomly out of a larger popula-tion of provenances, and as we were not interested in the specificprovenances, but in the whole population of our plants (Dormannand Kühn, 2009). We examined the residuals against fitted plotsand normal qq-plots prior to each analysis to test whether theassumptions for ANOVA, homogeneity of variances and normality,could be met (Faraway, 2006). If this was not the case, data werepower-transformed (fluorescence data), log-transformed (abso-lute biomass data) or arcsin-transformed (relative water content)accordingly.

All statistical analyses were performed using R 2.11.0 (RDevelopment Core Team, 2010). For mixed effect models we usedthe software package nlme (Pinheiro et al., 2008).

3. Results

3.1. Effects of the first drought (D1)

Temperature and precipitation data during the experimentalperiod in June are shown in Fig. 2a. Early drought treatment (D1) inJune significantly reduced the relative leaf water content measuredat the end of the drought treatment compared to the well-wateredcontrol (C1) by around 22% (P < 0.001; Fig. 3a).

Early drought treatment (D1) had no effect on the total above-ground biomass when compared with the well-watered controltreatment (C1) (data not shown). However, drought reduced thepercentage of living biomass significantly by around 15% (P = 0.03;Fig. 3b). On the last day of early drought treatment (D1) thephotochemical efficiency (Fv/Fm) of plants under drought was sig-nificantly reduced compared to the well-watered control (P = 0.004;Fig. 3c).

Plants under early drought treatment (D1) exhibited reducednet photosynthesis by 58% on the last day of the early droughttreatment (P < 0.001; Fig. 3d).

3.2. Effects of recurrent drought events (D2) compared to a singledrought event (C2) in September

3.2.1. Temperature, relative leaf water content and productivityin the second drought period

Average daily temperatures during the experimental period in

September ranged between 10 ◦C and 20 ◦C (Fig. 2b). The maximumtemperature was exceptionally high with 25.5 ◦C on the 6th day ofthe experiment in September and lowest on the 11th day of theexperiment with 12.3 ◦C. The withholding of water was reflectedin the relative water content of the leaves, which was reduced to

J. Walter et al. / Environmental and Experimental Botany 71 (2011) 34–40 37

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Fig. 3. Effects of the first drought (D1) in June (dark grey) compared to well-wateredcontrol plants (C1) (light grey) on relative water content of leaves (a), percentage ofliving biomass (b), maximum quantum efficiency Fv/Fm, measured predawn (c), andnet photosynthesis Pn, measured during midday (d). Relative leaf water content (a)was measured 3 days before the end of the drought treatment, maximum quantumefficiency Fv/Fm (c) and net photosynthesis Pn (d) were recorded on the last day

with significant reductions in Fv/Fm (14th and 17th day of exper-iment) revealed that the decrease of Fv/Fm in plants subjected torecurrent drought can be explained by a decrease in Fm ratherthan by an increase in F0 (Table 2). Two days before rewatering, Fm

Re

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ig. 2. Course of daily maximum temperature (grey squares), daily average temper-ture (black circles) and precipitation (dark grey bars) at the study site during thexperimental periods in June (a) and September (b). Vertical black line indicates thetart of the drought treatment, grey dashed line indicates when rewatering started.

round 65% but did not reveal any differences between single andecurrently dried plants (D2 vs. C2: P = 0.38, Fig. 4a).

Plants in single and recurrent drought treatments produced anlmost equal amount of overall (living and dead) abovegroundiomass (1.47 g in recurrent drought treatment (D2) and 1.49 g iningle drought treatment (C2)). However, the percentage of livingiomass, was increased significantly by 7% in plants subjected toecurrent drought (D2) compared to plants experiencing their firstrought (C2) (P = 0.048) (Fig. 4b). Total living biomass was increasedy 10% in recurrent drought treatment, although this effect did notrove to be significant (0.91 g in recurrent drought treatment and.82 g in single drought treatment, P = 0.18).

.2.2. Photosynthetic parametersWith progressive drought stress, Fv/Fm in the single and recur-

ent drought treatment declined, reaching a minimum for theouble-stressed plants (D2) on the 14th day of the experiment, 2ays before rewatering (Fig. 5a). Single-stressed plants (C2) alreadyeached minimal quantum efficiency on the 11th day of the droughtnd Fv/Fm values rose again after that. The loss of leaf water andhotochemical efficiency under extreme drought was reflected indecline of net photosynthesis by more than 60% compared to nethotosynthesis before the drought treatment started (D2 and C2)Fig. 5b).

Grasses under recurrent drought (D2) showed lower maximumuantum efficiency compared to plants exposed to a single droughtC2), from the eleventh day of the experiment until the end of

easurements, 10 days after the onset of rewatering (Fig. 5a).

his reduction was significant on the last day of measurementsnder the drought (14th day of the experiment, P = 0.05) and onhe first day after rewetting (17th day of the experiment, P = 0.02)Fig. 5a). During drought and in the post-drought recovery phase,lants subjected to single (C2) and recurrent drought (D2) did

of the drought treatment. Total aboveground harvest was conducted 17 days afterthe end of the first drought treatment and the percentage of dead biomass (dryweight) (b) was calculated. Means and SE are shown, asterisks indicate significance(*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).

neither differ significantly regarding net photosynthesis, nor tran-spiration (transpiration data not shown). However, on the 14thday of the experiment, net photosynthesis of grasses subjected torecurrent drought (D2) was 25% lower compared to grasses beingsubjected to their first drought (C2), but this effect was not signif-icant (P = 0.11).

A closer look at fluorescence parameters on both of these days

C2 D2C2 D2

Fig. 4. Leaf water status at the end of the second drought period 13 days after waterhad been withheld (a), and percentage of living biomass for the second harvest, 3weeks after the end of the drought treatment (b). Means ± 1 SE are shown, asteriskindicates significance of difference (*P ≤ 0.05).

38 J. Walter et al. / Environmental and Expe

Rewatering

34

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-2s

-1]

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25 22 19 16 13 10 7 4 1

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m

C2- singleD2- recurrent

Drought

a

b

Fig. 5. Course of maximum quantum efficiency Fv/Fm, measured predawn (a) andnet photosynthesis, measured during midday (b) in A. elatius subjected to recurrentor single drought before and during the drought phase and rewatering in September.Dark grey dashed line indicates the start of the drought, black dashed line indicatesthe end of the drought and the start of rewatering in September. Means ± 1 SE aresd

wFrrwdgveh9

TMtt(

hown, asterisk indicates significance of difference between single and recurrentrought treatments on single days (*P ≤ 0.05).

as reduced by around 20% (14th day of the experiment, P = 0.07).0 was non-significantly reduced by around 6% (P = 0.57). Fm waseduced by around 10% on the first day of measurements afterewetting (17th day of the experiment, P = 0.3) as opposed to F0,hich was reduced by only 0.5% in plants subjected to recurrentrought (P = 0.94) (Table 2). Fv/Fm of both treatments recoveredradually after the drought treatment ended, reaching pre-droughtalues 10 days after rewatering had started, on the 26th day of thexperiment (Fig. 5a). One week after rewetting, net photosynthesisad been almost completely restored, showing reductions of only

% compared to pre-drought values.

able 2aximum fluorescence (Fm) and steady state fluorescence (F0) 2 days before rewa-

ering (14) and 1 day after rewatering (17) in the recurrent and single droughtreatment during the experimental period in September. Means ± 1 SE are shownn = 5).

Day of experiment

14 17

Fm

Single 0.696 ± 0.0031 0.793 ± 0.0026Recurrent 0.553 ± 0.0030 0.718 ± 0.0025

F0

Single 0.157 ± 0.0001 0.170 ± 0.0001Recurrent 0.148 ± 0.0001 0.169 ± 9.9e-5

rimental Botany 71 (2011) 34–40

4. Discussion

This study investigated, whether A. elatius plants of six mid- andeastern European provenances can remember drought stress overan entire vegetation period even after a harvest. We hypothesizedthat plants would not show different performance under recurrentdrought. This hypothesis was not confirmed, as grasses respondedconsistently different in recurrent drought as compared to a sin-gle drought, indicating enhanced photoprotection. Surprisingly,this effect persisted after total aboveground biomass harvest andregrowth. The observed changes in reaction to recurrent droughtare not in accordance with findings indicating reduced resistanceor resilience after having already been exposed to drought stressbefore (Lloret et al., 2004; Zavalloni et al., 2008). However, thesewere conducted under field conditions and Zavalloni et al. (2008)investigated community responses. Thus, different outcome of theexperiments are not surprising. The findings are in accordance withreported stress imprint effects or stress memory (Bruce et al., 2007).However, to our knowledge, no study has already provided evi-dence that grasses do remember drought stress even after a harvestand can exhibit improved performance in the face of repeated abi-otic stress over such a long duration. Stress imprint and acclimationwere previously mostly reported to last for “several days” (Bruceet al., 2007).

Under severe drought, grasses experiencing recurrent droughtshowed reduced maximum quantum efficiency Fv/Fm (Fig. 5a).This was mainly related to reductions in maximum fluorescence,indicating enhanced dissipation of light energy to prevent photo-damage (Maxwell and Johnson, 2000). Osmond et al. (1993) andAraus et al. (1998) suggest that a correlation of reduced Fv/Fm

with an increase of F0 can be interpreted as chronic photoin-hibitory damage due to the degradation of the D1 protein in thereaction centers. By contrast, a constant F0 and decreasing Fm

values, as in our study, point towards photoinhibition related toenhanced non-photochemical quenching via the Xanthophyll cycleand thus indicate photoprotection (Araus et al., 1998). Another pos-sible explanation of decreased Fv/Fm caused by reduced Fm valuesmight be a reduction of chlorophyll. Unfortunately, we did notmeasure chlorophyll content in our study. However, a reductionof chlorophyll can be considered as a feature of acclimation, asit reduces the possibility of photodamage because of an excessof energy (Munne-Bosch and Alegre, 2000). Net photosynthesisdid not reveal any differences between grasses under recurrent(D2) and single drought (C2), but showed a trend towards lowerphotosynthesis in plants receiving recurrent stress under extremedrought (Fig. 5b). This is in accordance with the reduced pho-tochemical efficiency in plants recurrently experiencing drought(D2).

The results of the aboveground biomass support the hypoth-esis of enhanced photoprotection of double-stressed plants, asthe percentage of living biomass was significantly increased inplants experiencing their second drought, although total above-ground biomass or total living biomass were not significantlyaltered.

Plants can adapt to drought by enhancing root growth. How-ever, relative water content of the leaves was not significantlyenhanced in plants experiencing recurrent drought, indicating thatthe observed results can not be explained by changes in rootbiomass or improved water uptake mechanisms.

Ecophysiological measurements did not reveal any consis-tent differences between recurring (D2) and single drought (C2)

in the post drought recovery phase. Maximum quantum effi-ciency 2 days after rewetting in grasses subjected to recurringdrought was significantly lower compared to grasses subjectedto their first drought. This was more likely related to increasedstress levels under drought rather than a lower recovery rate.

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J. Walter et al. / Environmental an

ome studies indicated that recovery rate depends on experi-nced stress level (Miyashita et al., 2005; Resco et al., 2009).he significantly lower percentage of dead biomass in plants sub-ected to recurring drought can be a sign of quicker recovery,ut is more likely to be a sign of improved photoprotection, asiscussed above. In accordance to other studies, the recovery ofcophysiological parameters was quite fast, almost reaching pre-tress levels after about 1 week (Galle et al., 2007; Galmes et al.,007).

We did not elucidate underlying molecular or biochemicalechanisms for acclimation in this study, as we were interested

n the effects or recurrent drought on agricultural relevant perfor-ance parameters. Thus, we can only hypothesize about potential

ong-lasting acclimation processes in the grasses. The observedhenotypic plasticity could be either explained by belowgroundynamics or by long-lasting changes in gene expression, renderinghe plants more permissive to react quickly to recurrent stress, e.g.pigenetic processes (Aubin-Horth and Renn, 2009; Molinier et al.,006; Bird, 2007; Bossdorf et al., 2008). Verhoeven et al. (2010)ecently showed that stress induces changes in methylation pat-erns and that these patterns are heritable. An investigation of thehanges in methylation patterns as a response to drought and aink of observed methylation patterns to stress response are veryromising. Furthermore, we could only investigate six provenancesf A. elatius plants, which originated mainly from areas in Europeith quite similar climatic conditions. Other, more different prove-ances were not surviving in sufficient replicates for our study.evertheless, an extension of our experiment to other provenancesnd plant groups seems promising, as they may reveal differ-nt acclimation patterns and therefore also different responses toecurrent drought.

. Conclusion

To conclude, our study indicates that grasses under droughtetain a long-lasting stress imprint that facilitates a faster andore protective response towards a recurrent drought. Grasses

eing subjected to recurring drought showed improved photopro-ection. However, under more intense or frequent drought events,he reduction of photochemical efficiency and thus photosynthe-is, could lead to a loss of productivity (Murchie et al., 2009). This isndicated in our study by a trend towards a lower photosyntheticate under extreme drought. However, further studies have to elu-idate acclimation processes on a molecular and biochemical levelore deeply, and relate them to functional parameters. Long-term

cclimation mechanisms still have to be scrutinized for a betternderstanding of plant responses to a changing climate, and to beble to make projections and recommendations for adaptation tolimate change.

cknowledgements

Many thanks to all interns, who assisted with the measure-ents, in particular Inés Pastor. We thank Prof. W. Beyschlag and

un. Prof. Christiane Werner-Pinto of the University of Bielefeld androf. J. Tenhunen of the University of Bayreuth for providing us withheir PAMs. Many thanks to Dr. H. Auge of the Helmholtz Centre fornvironmental Research in Halle, for providing us with the LI-6400.hanks to Reinhold Stahlmann and several technical assistantsor their help. This work was kindly supported by the Helmholtz

mpulse and Networking Fund through the Helmholtz Interdisci-linary Graduate School for Environmental Research (HIGRADE).hanks to the anonymous reviewers for helping to improve theanuscript.

rimental Botany 71 (2011) 34–40 39

References

Araus, J.L., Amaro, T., Voltas, J., Nakkoul, H., Nachit, M.M., 1998. Chlorophyllfluorescence as a selection criterion for grain yield in durum wheat underMediterranean conditions. Field Crop. Res. 55, 209–223.

Aubin-Horth, N., Renn, S.C.P., 2009. Genomic reaction norms: using integrative biol-ogy to understand molecular mechanisms of phenotypic plasticity. Mol. Ecol.18, 3763–3780.

Barrs, H.D., Weatherley, P.E., 1962. A re-examination of relative turgidity techniquefor estimating water deficits in leaves. Aust. J. Biol. Sci. 15, 413–417.

Bilger, W., Schreiber, U., Bock, M., 1995. Determination of the quantum efficiency ofphotosystem II and nonphotochemical quenching of chlorophyll fluorescencein the field. Oecologia 102, 425–432.

Bird, A., 2007. Perceptions of epigenetics. Nature 447, 396–398.Bossdorf, O., Richards, C.L., Pigliucci, M., 2008. Epigenetics for ecologists. Ecol. Lett.

11, 106–115.Bruce, T.J.A., Matthes, M.C., Napier, J.A., Pickett, J.A., 2007. Stressful “memories” of

plants: evidence and possible mechanisms. Plant Sci. 173, 603–608.Dormann, C.F., Kühn, I., 2009. Angewandte Statistik für die biolo-

gischen Wissenschaften, second ed. URL: http://www.ufz.de/data/deutschstatswork7649.pdf (June 29th, 2010).

Faraway, J.J., 2006. Extending the Linear Model with R-Generalized Linear, MixedEffects and Nonparametric Regression Models. Chapman and Hall/CRC, BocaRaton.

Galle, A., Haldimann, P., Feller, U., 2007. Photosynthetic performance and water rela-tions in young pubescent oak (Quercus pubescens) trees during drought stressand recovery. New Phytol. 174, 799–810.

Galmes, J., Medrano, H., Flexas, J., 2007. Photosynthetic limitations in response towater stress and recovery in Mediterranean plants with different growth forms.New Phytol. 175, 81–93.

Goh, C.H., Nam, H.G., Park, Y.S., 2003. Stress memory in plants: a negative regulationof stomatal response and transient induction of rd22 gene to light in abscisicacid-entrained Arabidopsis plants. Plant J. 36, 240–255.

Grime, J.P., Hodgson, J.G., Hunt, R., 1988. Comparative Plant Ecology. A FunctionalApproach to Common British Species. Unwin Hyman, London.

Jentsch, A., Kreyling, J., Boettcher-Treschkow, J., Beierkuhnlein, C., 2009. Beyondgradual warming: extreme weather events alter flower phenology of Europeangrassland and heath species. Global Change Biol. 15, 837–849.

Jiang, C.D., Li, P.M., Gao, H.Y., Zou, Q., Jiang, G.M., Li, L.H., 2005. Enhanced photopro-tection at the early stages of leaf expansion in field-grown soybean plants. PlantSci. 168, 911–919.

Kreyling, J., Wenigmann, M., Beierkuhnlein, C., Jentsch, A., 2008. Effects of extremeweather events on plant productivity and tissue die-back are modified by com-munity composition. Ecosystems 11, 752–763.

Kreyling, J., Beierkuhnlein, C., Jentsch, A., 2010. Effects of soil freeze–thaw cyclesdiffer between experimental plant communities. Basic Appl. Ecol. 11, 65–75.

Lambers, H., Chapin III, F.S., Pons, T.L., 2008. Plant Physiological Ecology, second ed.Springer, New York.

Lloret, F., Siscart, D., Dalmases, C., 2004. Canopy recovery after drought dieback inholm-oak Mediterranean forests of Catalonia (NE Spain). Global Change Biol. 10,2092–2099.

Maxwell, K., Johnson, G.N., 2000. Chlorophyll fluorescence—a practical guide. J. Exp.Bot. 51, 659–668.

Michalski, S.G., Durka, W., Jentsch, A., Kreyling, J., Pompe, S., Schweiger, O., Willner,E., Beierkuhnlein, C., 2010. Evidence for genetic differentiation and divergentselection in an autotetraploid forage grass (Arrhenatherum elatius). Theor. Appl.Genet. 120, 1151–1162.

Miyashita, K., Tanakamaru, S., Maitani, T., Kimura, K., 2005. Recovery responses ofphotosynthesis, transpiration, and stomatal conductance in kidney bean follow-ing drought stress. Environ. Exp. Bot. 53, 205–214.

Molinier, J., Ries, G., Zipfel, C., Hohn, B., 2006. Transgeneration memory of stress inplants. Nature 442, 1046–1049.

Mueller, R.C., Scudder, C.M., Porter, M.E., Trotter, R.T., Gehring, C.A., Whitham, T.G.,2005. Differential tree mortality in response to severe drought: evidence forlong-term vegetation shifts. J. Ecol. 93, 1085–1093.

Munne-Bosch, S., Alegre, L., 2000. Changes in carotenoids, tocopherols and diter-penes during drought and recovery, and the biological significance of chlorophyllloss in Rosmarinus officinalis plants. Planta 210, 925–931.

Murchie, E.H., Pinto, M., Horton, P., 2009. Agriculture and the new challenges forphotosynthesis research. New Phytol. 181, 532–552.

Noormets, A., McNulty, S.G., DeForest, J.L., Sun, G., Li, Q., Chen, J., 2008. Droughtduring canopy development has lasting effect on annual carbon balance in adeciduous temperate forest. New Phytol. 179, 818–828.

Osmond, C.B., Ramus, J., Levavasseur, G., La Franklin, Henley, W.J., 1993. Fluorescencequenching during photosynthesis and photoinhibition of Ulva-Rotundata Blid.Planta 190, 97–106.

Pinheiro, J., Bates, D., DebRoy, S., Sarkar, D., and the R Core team, 2008. nlme, Linearand Nonlinear Mixed Effects Models. R Package Version 3.1-89.

Polle, A., Kröniger, W., Rennenberg, H., 1996. Seasonal fluctuations of ascorbate-related enzymes: acute and delayed effects of late frost in spring on antioxidative

systems in needles of Norway Spruce (Picea abies L.). Plant Cell Physiol. 37,717–725.

R Development Core Team, 2010. R: A Language and Environment for StatisticalComputing. R Foundation for Statistical Computing, Vienna, Austria. ISBN:3-900051-07-0, URL: http://www.R-project.org.

4 Expe

R

S

S

T

0 J. Walter et al. / Environmental and

esco, V., Ewers, B.E., Sun, W., Huxman, T.E., Weltzin, J.F., Williams, D.G., 2009.Drought-induced hydraulic limitations constrain leaf gas exchange recoveryafter precipitation pulses in the C-3 woody legume, Prosopis velutina. NewPhytol. 181, 672–682.

char, C., Vidale, P.L., Luthi, D., Frei, C., Haberli, C., Liniger, M.A., Appenzeller, C., 2004.The role of increasing temperature variability in European summer heatwaves.

Nature 427, 332–336.

cheffer, M., Carpenter, S., Foley, J.A., Folke, C., Walker, B., 2001. Catastrophic shiftsin ecosystems. Nature 413, 591–596.

ahkokorpi, M., Taulavuori, K., Laine, K., Taulavuori, E., 2007. After-effects ofdrought-related winter stress in previous and current year stems of Vacciniummyrtillus L. Environ. Exp. Bot. 61, 85–93.

rimental Botany 71 (2011) 34–40

Trenberth, K.E., Dai, A., Rasmussen, R.M., Parsons, D.B., 2003. The changing characterof precipitation. Bull. Am. Met. Soc. 84, 1205–1217.

van Peer, L., Nijs, I., Reheul, D., de Cauwer, B., 2004. Species richness and suscep-tibility to heat and drought extremes in synthesized grassland ecosystems:compositional vs physiological effects. Funct. Ecol. 18, 769–778.

Verhoeven, K.J.F., Jansen, J.J., van Dijk, P.J., Biere, A., 2010. Stress-induced DNA methy-

lation changes and their heritability in asexual dandelions. New Phytol. 185,1108–1118.

Zavalloni, C., Gielen, B., Lemmens, C.M.H.M., de Boeck, H.J., Blasi, S., van den Bergh,S., Nijs, I., Ceulemans, R., 2008. Does a warmer climate with frequent mild watershortages protect grassland communities against a prolonged drought? PlantSoil 308, 119–130.