Drought stress response in Jatropha curcas: Growth and physiology

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ContentslistsavailableatSciVerseScienceDirect

Environmental

and

Experimental

Botany

j o u r n al hom ep age :w w w . e l s e v i e r . c o m / l o c a t e / e n v e x p b o t

Drought

stress

response

in

Jatropha

curcas:

Growth

and

physiology

Helena

Sapeta

a

_{, J.}

_Miguel

_Costa

b,c

_{, Tiago}

_Lourenc¸

_o

a

_{, João}

_Maroco

d

_,

_Piet

_van

_der

_Linde

e

_,

M. Margarida

Oliveira

a,∗

a_Instituto_de_Tecnologia_Química_e_Biológica,_Universidade_Nova_de_Lisboa,_Genomics_of_Plant_Stress_Laboratory_(GPlantS_Lab),_Av._da_República,_2780-157_Oeiras,_Portugal_and_iBET, Apartado12,2781-901Oeiras,Portugal

b_Instituto_de_Tecnologia_Química_e_Biológica,_Universidade_Nova_de_Lisboa,_Plant_Molecular_{Ecophysiology,}_Av._da_República,_2780-157_Oeiras,_Portugal_and_iBET,_Apartado_12, 2781-901Oeiras,Portugal

c_Centro_de_Botânica_Aplicada_à_Agricultura,_Instituto_Superior_de_{Agronomia–UTL,}_Tapada_da_{Ajuda,1349-017}_Lisboa,_Portugal d_Grupo_de_Estatística_e_Matemática,_{ISPA-Instituto}_{Universitário,}_Rua_Jardim_do_Tabaco,_34,₁₁₄₉_-₀₄₁_Lisbon,_Portugal e_QUINVITA_NV,_Derbystraat_71,_B-9051_Ghent,_Belgium

a

r

t

i

c

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e

i

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Articlehistory: Received21May2012

Receivedinrevisedform14August2012 Accepted28August2012 Keywords: Purgingnut Droughtstress Re-watering Leafmorphology Leafgasexchange Photochemistry

a

b

s

t

r

a

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TolerancetodroughtremainspoorlydescribedforJatrophacurcasaccessionsfromdifferent geograph-icalandclimaticorigins.ToaddressthisissuewestudiedtheresponseoftwoJ.curcasaccessions,one fromIndonesia(wettropicalclimate)andtheotherfromCapeVerdeislands(semi-aridclimate).Potted seedlings(with71days)ofbothaccessionsweresubjectedtocontinuouswellwateredconditions (con-trol)ortoadroughtstressperiodfollowedbyre-watering.Tomimicnaturalconditionsinwhichdrought stressdevelopsgradually,stresswasimposedprogressivelybyreducingirrigation(10%reductionevery 2days,onaweightbase),foraperiodof28days,untilaﬁeldcapacityof15%(maximumstress)was achieved,followedbyoneweekunderwell-wateredconditions.Wemeasuredsoilandplantwater sta-tus,growthandbiomasspartitioning,leafmorphology,leafgasexchangeandchlorophyllaﬂuorescence. Bothaccessionsmaintainedhighleafrelativewatercontent(70–80%)evenatmaximumstress.Net pho-tosynthesis(An)wasnotaffectedbymildtomoderatestressbutitabruptlydroppedatseverestress.This

wasduetoreducedstomatalconductance,whichshowedearlierdeclinethanAn.Plantgrowth(stem

elongation,leafemergenceandtotalleafarea)wasreduced,minimizingwaterloss,butnosigniﬁcant differenceswerefoundbetweenaccessions.Droughtstressdidnotreducechlorophyllcontentsbutled toreducedchlorophylla/b.Bothaccessionsshowedfastrecoveryofbothstomatalandphotochemical parameterssuggestingagoodtolerancetowaterstress.BothJ.curcasaccessionsshowed a-dehydration-avoidantbehaviour,presentingatypicalwatersavingstrategyduetostrictstomatalregulation,regardless oftheirprovenance.

1. Introduction

Croplossesresultingfromabioticstressessuchasdroughtor

salinitycanreducecropyieldbyasmuch as50%(Boyer,1982;

ChavesandOliveira,2004).Climatechangesareexpectedto

exacer-batethissituationduetotheincreasingincidenceofmoreextreme

climateevents.Inthiscontext,plantswithimprovedperformance

Abbreviations: An,netphotosynthesis;C,controlconditions;Ci,internalCO2 concentration;CVi,CapeVerdeislandsaccession;DW,dryweight;E,transpiration rate;ETR,electrontransportrate;FC,fieldcapacity;gs,stomatalconductanceto watervapour;Ind,Indonesiaaccession;PPFD,photosyntheticphotonfluxdensity; R,re-wateringperiod;RWC,relativewatercontent;S,droughtstressconditions; S:R,shoottorootdrymassratio;SLA,specificleafarea;SWA,soilwateravailability; TLA,totalleafarea;WUEi,intrinsicwateruseefficiency(An/gs);PSII,photosystem IIoperatingefficiency.

∗ Correspondingauthor.Tel.:+351214469647;fax:+351214411277. E-mailaddress:mmolive@itqb.unl.pt(M.M.Oliveira).

undersub-optimal environmentalconditions, orwithincreased

tolerancetosevereabioticstress,arepotentialsourcesofgenes

forbreedingofagriculturalandforestrycrops.

Jatrophacurcas(purgingnut)isasoft-woodyoil-seedbearing

plantoftheEuphorbiaceaefamilythathasemergedasapotential

sourceofbiodiesel(Fairless,2007).Thespecieshasbeendescribed

asdroughttolerantandcapableofgrowinginmarginalandpoor

soils(Heller, 1996;Fairless,2007; Divakaraet al.,2010).J.

cur-cashaspotentialtobecultivatedundersemi-aridand poorsoil

conditionswithoutcompetingwithfoodproductionforlanduse

(Fairless,2007;Divakaraetal.,2010).

OutsideMeso-America, itscentre oforigin, J.curcas haslow

geneticdiversity(Kumaretal.,2008a,b;Pamidimarrietal.,2008a,

2008b;Popluechaietal.,2009).However,phenotypicvariations

aredescribedforphysiologicalandbiochemicaltraits(Heller,1996;

Ginwaletal.,2004;Kaushiketal.,2007;Raoetal.,2008;Popluechai

etal.,2009).Recentstudiesalsosuggestthatphenotypevariation

isepigeneticallycontrolled(Popluechaietal.,2009;Yietal.,2010;

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Kanchanaketuetal.,2012).However,littleisknownabout

physio-logicaltraitsandtheadaptationcapacityofJ.curcastochangingand

moreadverseclimateconditions.Theseknowledgegapsrestrict

ourcapacitytoproperlyevaluateandpredicttheagronomic

perfor-manceofJ.curcasinresponsetoextremeenvironments,especially

intermsofgrowth(biomass)andseedyieldtraits.Untilnow,

selec-tionofJ.curcasgenotypeshasfocusedonoiltraitsinsteadofon

physiological traits,suchas carbon and wateruse efﬁciencyor

waterstressresponse.Thisisparticularlysurprisingifweconsider

thatthemajorcultivationareasofJ.curcasareoftencharacterized

byfrequentsoilwaterdeﬁcitandexcessivesalinity(Fairless,2007;

Divakaraetal.,2010).Therefore,improvedknowledgeonJ.

cur-casphysiologyisneededtosupportbreedingprograms,selecting

superiorgenotypesandoptimizationofcropmanagement.

In different crops, physiological responses to drought vary

withthegenotype(AnyiaandHerzog,2004;Bacelaretal.,2007;

Centrittoetal.,2009;Costaetal.,2012).Thestudyofstress/recovery

responsescontributestoabetterunderstandingoftheplant’s

abil-itytoadapttodifferentenvironmentsandclimaticconditions.The

recoveryisanimportantcomponentofplant’sresponse,andinthe

particularcaseofdrought,carbonbalancedependsnotonlyonthe

rateanddegreeofphotosyntheticdeclineunderwaterdepletion,

butalsoonthecapacityforphotosyntheticrecoveryafterwater

supply(Chavesetal.,2003;Flexas etal.,2004,2007;Miyashita

etal.,2005).

The ability of J. curcas to grow in marginal and dry soils

hasbeenpoorly explored.Several studieshave been published

describingplantperformance(biomassproductionand

partition-ing, plant–water relationships, leaf gas exchange and osmotic

adjustment)underconditionsoflimitedwateravailability(Maes

etal.,2009;Achtenetal.,2010;Pompellietal.,2010;Silvaetal., 2010a,b;Díaz-Lópezetal.,2012).Twostudieshavecomparedthe

behaviourofaccessionsfromdifferentprovenances(Maesetal.,

2009;Achtenetal.,2010).

Inordertobetterunderstandtheputativeimpactofthe

geo-graphicorigin onplant responsestowater stress,we followed

the drought/re-watering response of two J. curcas accessions

withdistinctgeographicalandclimaticprovenances(CapeVerde

islands–aridclimate,versusIndonesia–wettropicalclimate)under

semi-controlled conditions. Growth and morpho-physiological

responses of the two accessions along drought stress and

re-watering are described withfocus on growth morphology and

photosyntheticandstomatalresponses.

2. Materialandmethods

2.1. Plantmaterialandgrowingconditions

We tested two accessions of J.curcas from two distinct

cli-materegions,onefromawettropicalregionandtheotherfrom

anaridclimate.TheﬁrstaccessionwasoriginaryfromIndonesia

(Ind,GPScoord:S3◦ 14 28.82,E102◦5744.95)whilethe

sec-ondonewasfromCapeVerdeislands(CVi,GPScoord:N14◦ 57

16.11,W23◦3619.68).Seedsfrombothaccessionswereused

inCapeVerdeislandstoproducethemotherplantsfromwhich

weobtainedtheseedsusedinthiswork.Seedsweregerminated

incleansand,and10-day-oldhomogeneousseedlingswere

trans-plantedto7.5Lpotscontainingasoilmixtureofsand,peatandsoil

(3:1:1)andsupplementedwithacommercialfertilizer(Osmocote,

Scotts, Netherlands)(10.5g/pot)(N:P:K:Mg, 16:9:12:2.5).Plants

were grown in a climatic chamber with daily irrigation until

thebeginning of thetreatments. Experiments werecarried out

in agrowth chamber(3m_×6m_×2.8m) witha day/night

tem-perature of 28±2◦C/20±4◦C, and day/night relative humidity

of35±5%/75±5%.Aphotoperiodof12hwasused,withalight

intensityat plantlevel of 172±66␮mol photonsm−2s−1,

pro-videdbyamixtureofhigh-pressuresodium(SON/T-Agro400W,

Philips, Belgium) and metal-halide (Master HPI/T Plus 400W,

Philips,Belgium)lamps.Potswererandomlymovedeveryweek

tominimizepositioneffects.

2.2. Treatments

Potted71-day-oldplantsofthetwoaccessionsweresubjected

todroughtstress(S)orcontinuouslygrownunderwell-watered

conditions(C,control).Droughtstresswasimposedbysubjecting

plantstoagradualdecreaseofsoilwateravailability(10%reduction

every2daysonaweightbase)foramaximumperiodof28days

(S),followedbya7-dayre-wateringperiod(R).

2.3. Parameters

2.3.1. Soilandplantwaterstatus

Soil water availability (SWA) was calculated as

SWA=[(Pot weight− Minimum Pot Weight)/(Maximum Pot

Weight−MinimumPotWeight)]×100.Minimumpotweightwas

consideredtobepot’sweightcontainingatotallydrysoilmixture.

Forthismeasurementsoilwasspreadinaﬁnelayerandair-dried

intheglasshouseuntilaconstantweightwasachieved.Maximum

potweightwasmeasuredwhensoilwasatﬁeldcapacity(FC).

Leaf relative water content(RWC) wasdeterminedat

maxi-mumstress(day28)andattheendoftheexperiment(day35).

Forthispurpose,sixleafdiscs(Ø=19mm)werecollectedforeach

plantfromthethreeyoungestexpandedleaves(2discsperleaf).

Five plants were analysed per treatment. RWC was calculated

accordingtoBarrsandWeatherly(1962)usingtheformula:RWC

(%)=[(FW−DW)/(TW−DW)]×100, where FW,DWandTWare

fresh,dryandturgidweight,respectively.

2.3.2. Growthandmorphology

Stemgrowthcharacteristicswereweeklymonitoredby

mea-suringlength(fromsubstratesurfacetotheapicalmeristem)and

diameter(atthebase)andbycountingthenumberofleaveslonger

than2cm.Therelativeincreaseinstemlength,stemdiameterand

numberofleaveswascalculated.Freshweightofleaves,rootsand

shootswasdeterminedattheendoftheexperiment(day35).Dry

weight(g)wasdeterminedafterdryingplantmaterialat70◦C(until

aconstantweightwasachieved).Shoottorootdrymassratio(S:R)

wascalculated.Thespeciﬁcleafarea(SLA)wascalculatedasleaf

areaperunitdrymass(cm2_g−1_)._Total_leaf_area_(TLA)_was

deter-minedwithacolourimageanalysissystem(WinDIAS2,Delta-T

Devices,Cambridge,UK).

2.3.3. Leafgasexchange,ChlaﬂuorescenceandChlcontent

Leafgasexchangewasassessedwithaportable infraredgas

analyzer (LI-6400; LI-COR Inc., Lincoln, USA) equipped with a

ﬂuorometer (LI-6400-40, LI-COR Inc., Lincoln, USA). Before the

experiments,lightresponsecurveswereperformed5–11hafter

photoperiodinitiation,usingfullyexpandedleavesof71days-old

plantsundergoodillumination.Weusedablocktemperatureof

28◦C,withCO2 concentrationat400ppmandanairﬂowrateof

500␮mols−1.Photosyntheticphotonﬂuxdensity(PPFD)was

grad-uallydecreasedfrom2000to0(2000;1750;1500;1200;900;700;

500;250;100;70;50and0␮molphotonm−2s−1)inordertoavoid

limitationofphotosynthesisathighlightduetoinsufﬁcient

stoma-talopening,causedbytheinitiallowerlightintensities(Singsaas

etal.,2001).A2–3minacclimationperiodwasperformedbetween

themeasurementsofthe12lightlevels.

Along the experiments, we monitored net photosynthesis

(An, ␮mol CO2 m−2s−1), transpiration (E, mol H2O m−2s−1),

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Fig.1.SoilwateravailabilityalongtheexperimentfortwoJatrophacurcas acces-sionsfromIndonesia(Ind)andCapeVerdeislands(CVi),continuouslygrownunder well-wateredcontrolconditions(C)orsubjectedtodroughtstress(S)for28days followedbya7-dayre-wateringperiod.Backgroundcolourindicates:nontomild stress(100–70%FC)(grey);mildtomoderatestress(70–30%FC)(lightgrey);severe stress(30-15%FC)(darkgrey);nostress(dashed).Thearrowatday28indicates re-wateringstart.Valuesaremeans.Barsrepresent±SE(n=5).

and internal CO2 concentration (Ci, ppm). Data was

periodi-cally collected from the youngest fully expanded leaf (5–9h

afterphotoperiod initiation), never extending a total period of

90min.Block temperaturewas setat 28◦C, light intensitywas

set at 300␮mol photons m−2s−1 (based on the light curves

responsesFig.3)andCO2concentrationwaskeptat400ppmwith

an air ﬂow rate of 500␮mols−1. Intrinsic water use efﬁciency

(WUEi=An/gs,␮molCO2mol−1H2O)andinstantaneous

carboxyla-tionefﬁciency(An/Ci,␮molCO2m−2s−1ppm−1)werecalculated

aswell.BysimultaneouslymeasuringChlaﬂuorescencewecould

alsoevaluatethephotosystemIIoperatingefﬁciency(PSII),which

translatesphotochemistryefﬁciency(Gentyetal.,1989;Maxwell

and Johnson, 2000). PSII operating efﬁciencywas calculated as

(Fm’−Fs)/F’m (Genty et al., 1989). Fs and Fm’ represent the

steady state and maximum ﬂuorescence measuredunder light

adapted conditions. We estimated the electron transport rate

(ETR=PSII×PPFD×0.5×0.84,␮molelectronm−2s−1)(Bjökman

andDemmig,1987),where thePPFDis thephoton ﬂuxdensity

ofphotosynthetically activeradiation(400–700nm),0.84 isthe

assumedlight absorbanceofthesample, and0.5is thefraction

ofexcitationenergydivertedtothephotosystemII.Thecontents

(mgg−1DW)ofchlorophylla(Chla)andchlorophyllb(Chlb)were

determinedaccordingtoLichtenthaler(1987)atmaximumstress

(day28)andattheendoftheexperiment(day35).Threeleafdiscs

(Ø=19mm)werecollectedforeachplantfromthethreeyoungest

expandedleaves(1discperleaf).Fiveplantswereanalysedper

treatment.Absorbancewasmeasuredwithaspectrophotometer

(DU-70Spectrophotometer,Beckman,USA)at663.2and646.8nm.

Chla/bwasalsocalculated.

2.4. Statistics

DatacollectedweresubjectedtoAnalysisofVariance(ANOVA)

usingthe statistical software SPSSStatistics (v20, SPSSan IBM

company,Chicago,USA).Meancomparisonwascarriedoutusing

Tukey’s multiplecomparison test using the statistical software

Fig.2. Effectofdroughtstressandre-wateringon(A)stemdiameter,(B)stemlength and(C)leafnumber(valuesrepresentthe%ofincrease,relativetoday0)measured fortwoaccessionsofJatrophacurcasfromIndonesia(Ind)andCapeVerdeislands (CVi),continuouslygrownunderwellwateredcontrolconditions(C)orsubjected todroughtstress(S)for28daysfollowedbya7-dayre-wateringperiod.Arrowsat day28indicatere-wateringstart.Valuesaremeans.Barsrepresent±SE(n=5).

packageSIGMAPLOT11.0(SystatSoftwareInc.,Chicago,USA).

Sig-niﬁcantresultswereassumedforp≤0.05.

3. Results

3.1. Soilandplantwaterrelations

Soilwater availabilitydecreased 10%every2days alongthe

imposed drought period reaching a minimum of 15% by day

28 (maximumstress)forboth accessions(Fig.1).Based onthe

observeddecreaseofSWAwehave consideredtheexistenceof

threemajorsoil waterdeﬁcitcategories:(1) nontomildstress

(100–70%FC;day0–9);(2)mildtomoderatestress(70–30%FC;day

10–20)and(3)severestress(30–15%FC,day21–28).Regardingleaf

RWC(Table1),wefoundthatCVimaintainedahigherwater

con-tentthanInd,inbothcontrolandmaximumstressconditions.Both

accessionsatmaximumstressshowedhigherRWCthanthe

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Table1

Leafrelativewatercontent(RWC)andwatercontent(leaf,stem,rootandtotal)measuredfortwoJatrophacurcasaccessionsoriginalfromIndonesia(Ind)andCapeVerde islands(CVi),continuouslygrownunderwell-wateredconditions(Control)ordroughtstress(Stress)for28daysfollowedby7daysofre-watering.RWCwasmeasuredat maximumstress(day28)andattheendoftheexperiment(day35).Leaf,stemandrootwatercontentweredeterminedatday35.Valuesaremeans±SE(n=5).Different letterswithinthesamecolumnindicatesigniﬁcantdifferencesaccordingtotheTukey’stest(p<0.05).

LeafRWC(%) Watercontent(%H2O)

Maximumstress Endofrecovery Endofrecovery

Accession Leaf Stem Root Total

Ind Control 66.4±0.6b 72.4±1.8a 94.4±0.1a 83.9±1.7a 85.1±0.3ab 89.5±0.4a

Stress 73.5±2.6ab 71.9±3.6a 93.9±0.9a 85.9±0.7a 86.2±1.1a 89.6±0.9a

CVi Control 75.6±2.5ab 75.7±1.3a 94.2±0.5a 86.1±0.5a 85.8±0.3a 90.0±0.5a

Stress 82.9±3.3a 69.9±1.1a 92.9±0.5a 85.3±0.3a 83.1±0.6b 88.2±0.4a

p-value 0.002 0.348 0.296 0.393 0.020 0.194

wereobservedbetweentreatmentsoraccessions(p=0.348).We

foundhighlevelsofwatercontentinleaves,stemsandrootswith

valuesequalorabove80%(Table1).Moreover,wefoundsigniﬁcant

differencesbetweenaccessionsforrootwatercontent(p=0.02).

RootsfromIndplantshadsigniﬁcantlyhigherwatercontentthan

rootsfromCViplantsafterthere-wateringperiod(83.1against

86.2%,forCViandIndrespectively).

3.2. Growthandmorphology

Therelativeincreaseofstemdiameterwasnotmuchaffectedby

drought(p=0.048)(Fig.2A).Valueswereidenticalinboth

acces-sionsandnodifferenceswerefounduntilday14oftheexperiment

(p=0.122).Fromday21onwards,theIndcontrolplantspresented

largerstems(p=0.036)compared totheotherplants.Although

fromday21onwardsstemdiameterenlargementwasarrested,

forbothstressedandnon-stressedplants,atmaximumstress(day

28)thearrestwasmorepronouncedfortheIndstressedplants

whencomparedwithitscontrol(p=0.007).Droughtarrestedstem

lengthandleafnumberrelativeincrease(p<0.001)(Fig.2BandC).A

reductionwasobservedfortherelativeincreaseofstemlengthand

leafnumberfromday7onwards(p=0.008andp=0.028,

respec-tively),withstemelongationandleafemergencebeingarrestedat

severestress(30–15%SWA,days21to28).Nevertheless,stem

elon-gationwasrapidlyresumedafterre-watering(Fig.2B,day28–35).

Alongtheexperimentalperiodstemdiametervariedbetween1.2

and1.8cm andstemlengthvariedbetween36and50cm (data

notshown).Thenumberofleavesvariedbetween13and20(data

notshown).Nodifferenceswereobservedbetweentreatmentsat

theendoftheexperimentforleaves(p=0.150),stem(p=0.356),

roots (p=0.516) ortotal biomass (p=0.446) (Table 2).Similarly

nodifferencesexistedbetweenaccessionsortreatmentsfor S:R

(p=0.221)(Table2).Wefoundhowever,marginaldifferencesfor

TLA(p=0.054)andSLA(p=0.068).Droughtledtoareductionof

TLAforbothaccessions.MoreovertheCViplantspresenthigherleaf

areabothincontrolanddroughtconditions.Despitethehigh

vari-ationbetweenSLAvalues,nospeciﬁctrendwasobservedbetween

provenancesorimposedtreatments.

3.3. Leafgasexchange,ChlaﬂuorescenceandChlcontent

Net photosynthesis response to increasing light was

sim-ilar for both accessions (Fig. 3A). For both accessions An

increasedrapidlyasPPFDincreasedto250␮molphotonm−2s−1

(the linear phase), slowly increasing thereafter to a

maxi-mumof 1200␮molphotonm−2s−1 (the light saturationpoint),

and remaining constant during light intensity increase up

to 2000␮molphotonm−2s−1 (Fig. 3A). The light

compen-sation point was estimated to be approximately 1.6 and

1.2␮molphotonm−2s−1 (for the Ind and the CVi accessions,

respectively).TheinternalCO2rapidlydecreaseduntilanirradiance

levelof250␮molm−2s−1 (Fig.3B),slowlyincreasingafterwards

untilthemaximumlightlevelwasreached(2000␮molm−2s−1).

TheCiresponsepatternwasidenticalforbothaccessions,although

from1200␮molm−2s−1theIndaccessionpresentedslightlylower

Civalues.

Valuesandvariation ofnetphotosynthesisalongthe

experi-mentweresimilarinbothaccessions(Fig.4A).An valuesunder

droughtweresimilartothoseobservedforcontrolplantsuntilday

20,decliningthereafter.Bothaccessionspresentedafastdecrease

ofAnunderseverewaterdeﬁcit(30–15%ofSWA,days23–28),with

areductionof77%and78%inIndandCViplantsrespectivelyat

maximumstress(SWAof15%)ascomparedtocontrol(p=0.009).

ValuesofAnforcontrolvariedbetween8and12␮molCO2m−2s−1

inbothaccessions.Stomatalconductancetowatervapourhada

differentpatternfromAnasitshowedanearlierdecrease(Fig.3A

andB).Stomatalconductancewasreducedby42%(inInd)and33%

(inCVi)atday11(SWAof60%)whileatmaximumstress(15%SWA,

reachedbyday28),thereductionwasof96%and99%respectively

ascomparedtothecontrol(p<0.001).Thepronounceddecrease

ofgsandthemaintenanceofnetphotosynthesis,causedastrong

reductioninCi,fromday11to23(60–30%ofSWA),withminimum

valuesofabout180ppm(datanotshown).Anidenticalreduction

patternwasobservedforleaftranspiration(E)(datanotshown).

Increased WUEi for stressed plants was observed since day

11 (Fig. 4E). Instantaneouscarboxylation efﬁciency (An/Ci) also

increasedinbothaccessionsuntilday23(Fig.4F)butthemarked

reductionin An at severestress,decreased An/Citovalues close

to0byday26oftheexperiment.ConcerningChlaﬂuorescence

parameters,weobservedanabruptreductionofthePSIIoperating

efﬁciency(PSII)sinceday26(20%ofSWA)untilday28(maximum

stress)(Fig.4C).IdenticalbehaviourwasobservedforETR(Fig.4D).

Regardingrecoveryafterre-watering,leafgasexchange (An,gs)

andphotochemical(PSIIandETR)parametersrevealedacomplete

recoverywithin24h(day29)(Fig.4).WUEiandinstantaneous

car-boxylationefﬁciency(Fig.3EandF)ofwaterstressedplantsalso

fullyrecoveredtocontrolvaluesafter3daysofre-watering.

Atmaximumstress(day28),ahighercontentofChlaandChlb

appearedtooccurinstressedplants,althoughthiswasnot

sup-portedbystatisticalanalysis.Thistendencywasespeciallyevident

fortheIndaccession(Fig.5AandB).AlthoughtheincreaseofChlb

wasnotsigniﬁcant,itresultedinasigniﬁcantreductionoftheChla/b

(p=0.005)(Fig.5C).Afterthe7-dayre-wateringperiodweobserved

thefullrecoveryofChla/bvaluesandnodifferencesexistedbetween

stressedandcontrolplantsofbothaccessions.

4. Discussion

We have characterized J. curcas physiology and growth in

responsetowaterstressusingseedlingsoftwoaccessions

orig-inating from contrasting climatic conditions. We found no, or

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Table2

Effectofdroughtstressandre-wateringonbiomass(DW)partitioning,totalleafarea(TLA,cm2_),_speciﬁc_plant_leaf_area_(SLA,_cm2_g−1₎_and_shoot_to_root_dry_mass_ratio_(S:R) oftwoJatrophacurcasaccessionsfromIndonesia(Ind)andCapeVerdeislands(CVi),continuouslygrownunderwell-wateredconditions(Control)ordroughtstress(Stress) for28daysfollowedby7daysofre-watering.Measurementswereperformedattheendoftheexperiment(day35).Valuesaremeans±SE(n=5),comparisonwithinthe samecolumnwasperformedwithTukey’stest.

Biomass(g) TLA(cm2₎ _SLA_(cm2_g−1₎ _S:R

Accession Leaf Stem Root Total

Ind Control 14.7±1.7 10.6±1.6 6.1±0.7 31.4±4.0 3510±349 241.2±5.4 2.7±0.1

Stress 11.1±0.7 9.6±1.3 5.2±1.0 25.9±2.8 2808±99 254.1±8.3 3.0±0.2

CVi Control 13.3±0.7 9.4±0.6 5.9±0.3 28.7±1.7 3645±202 257.3±14.0 2.6±0.1

Stress 12.7±0.5 12.2±1.1 6.7±0.7 31.6±2.1 3129±101 247.2±3.4 2.9±0.1

p-value 0.150 0.356 0.516 0.446 0.054 0.068 0.221

responsestodroughtandontherecoveryfollowingre-watering.

ThisagreeswithpreviousstudiesforJ.curcaswheregrowth

con-ditionsappearedtohavelargerinﬂuenceonplantperformancein

responsetostressthantherespectivegeneticbackground(Maes

etal.,2009;Achtenetal.,2010).Inourexperimentalconditionsnet

photosynthesiswasonlysigniﬁcantlyreducedwhenSWAdropped

below 30%. Meanwhile, stomatal conductance towater vapour

showedtobequitesensitivetosoilwateravailabilityandastrict

stomatalregulationinJ.curcaswasevidentbyreducedgssinceday

11.ThemaintenanceofAnvaluesundermoderatestressresulted

inincreasedWUEi.Thiswaterconservationstrategycanjustifythe

maintenanceofhighleafrelativewatercontentsevenundersevere

stress(15%of SWA).It isalsopossiblethattheosmotic

adjust-mentcapacityhascontributedtomaintainahighwatercontentof

tissues,aspreviouslysuggestedbySilvaetal.(2010b)forJ.curcas.

4.1. Morphologicaladaptationstodrought

Photosynthesisandgrowth(biomassproduction)arethe

pri-maryprocessestobeaffectedbydrought(Chaves andOliveira,

2004).Inourstudygrowthreductionwasobservedunder

mod-erateand severestress.Thissuggests thateven atreduced soil

wateravailability,J.curcasplantsareabletogrow.Ourresultsagree

withtheﬁndingsofAchtenetal.(2010)whohaveobservedthat

waterwithholdwouldarrestgrowthbutmaintainingplantsatlow

SWA(40%)wouldallowthemtocontinuegrowing,althoughata

slowerratethanfullyirrigatedplants.Accordingtoourdata,the

imposedstressperiodwasnotlongenoughtocauseany

signif-icantdifferencesinbiomassatharvest.Nodifferenceswerealso

foundinbiomasspartition.However,droughtresultedina

reduc-tionoftotalleafareainbothaccessionsattheendoftheexperiment

(Table2,p=0.054).Reductionofcanopysizeisawaterconservation

mechanism that allows plants to minimize transpiration area

(Boyer,1970).Attheendofourexperiments,SLAvaluesranged

between240and260cm2_g−1_{independently}_of_the_treatment_or

accession(Table2).ThisdiffersfromobservationsbyDíaz-López

etal.(2012)whofoundanincreaseinSLAfrom200to300cm2_g−1

inJ.curcas.Itispossiblethattheabsenceofmarkedplasticitywe

observedrelatestoaconstitutivelyhigherSLAinthetwotested

accessionsascomparedtothosestudiedbyDíaz-Lópezetal.(2012).

Moreover,andcontrarytoDíaz-Lópezet al.(2012),our

experi-mentsincludedare-watering/recoveryperiod,whichcouldhave

contributedforthedifferentresults.

ReductioninS:RhasbeendescribedforJatrophainresponseto

drought(Díaz-Lópezetal.,2012).However,inthewaywehave

imposed waterdeﬁcit, wedidnot observethis effect,probably

becauseplantacclimationmayhaveoccurredwithproportional

reductionsinplantsizeor/andnopronouncedaerialgrowtharrest,

ascanbeassumedbytheabsenceofsigniﬁcantbiomassdifferences

betweencontrolandstressplantsatharvest.

4.2. Leafgasexchange,ChlﬂuorescenceandChlcontent

Leafrelativewatercontentwaskeptstablealongthetrial,and

evenat maximumstress,whichsuggestsa rapidadjustmentof

plantstoavoid waterloss.Similarﬁndingshavebeendescribed

forJ.curcasgrownunderdrought(Silvaetal.,2010a,b;Díaz-López

etal.,2012)andsaltstress(Silvaetal.,2012).However,Arcoverde

etal.(2011)haveshowedthatunderseverewaterstressleafRWC

wasmoderatelyreduced.Reductionofstomatalconductancewas

notrelatedtoadecreaseinleafwatercontent(Pompellietal.,2010;

Silvaetal.,2010a;Arcoverdeetal.,2011),suggestingthatstomata

arerespondingtootherfactorssuchashormones(e.g.abscisicacid

(ABA)withorigininthedryingroots)(WilkinsonandDavies,2002;

Fig.3.LightresponsecurvesfortwoJatrophacurcasaccessionsfromIndonesia(Ind)andCapeVerdeislands(CVi):(A)Netphotosynthesis(An)and(B)intercellularCO2 concentration(Ci).Valuesaremeans±se(n=5).

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Fig.4.Effectofdroughtstressandre-wateringon(A)netphotosynthesis(An),(B)stomatalconductancetowatervapour(gs),(C)photosystemIIoperatingefficiency(PSII), (D)electrontransportrate(ETR);(E)intrinsicwateruseefficiency(An/gs)and(F)instantaneouscarboxylationefficiency(An/Ci),measuredfortwoaccessionsofJatrophacurcas fromIndonesia(Ind)andCapeVerdeislands(CVi),continuouslygrownunderwell-wateredcontrolconditions(C)orsubjectedtodroughtstress(S)for28daysfollowedbya 7-dayre-wateringperiod.MeasurementsweredoneadjustingtheLI-CORblocktemperatureto28◦_C,_light_intensity_to₃₀₀_␮mol_photons_m−2_s−1_and_CO₂_{concentration}_to 400ppm.InEandF,valuesforstresstreatmentondays26or/and28werenotregisteredduetolackofsensitivityoftheequipment.Arrowsatday28indicatere-watering start.Valuesaremeans.Barsrepresent±SE(n=5).

Chavesetal.,2003)topreventwaterlossbytranspiration.

More-over,thereisstrongevidencethatgsrespondstosoilwatercontent

ratherthanleafwatercontent/turgor(Jones,1985,1992).

How-ever,furtherstudiesareneededtotesttheinﬂuenceofsignalling

moleculesandtheirroleinthistightstomatalcontrol(e.g.treating

leavesofwellirrigatedplantswithABA).Inlinewithprevious

ﬁnd-ings,ourdatasupportsthatthemainstrategyofJ.curcastoendure

droughtstressisviaastrictstomatalcontrol(Pompellietal.,2010;

Silvaetal.,2010a,b;Díaz-Lópezetal.,2012).Thisalsoholdstruefor

otherEuphorbiaceaespeciessuchasRicinuscommunis(Sausenand

Rosa,2010),HeveabrasiliensisandManihotesculenta(El-Sharkawy, 2007).

Interestingly, despite the tight stomatal regulation that we

observedundermildandmoderatewaterstress,stomatal

limita-tionstophotosynthesisonlyoccurredunderseverestress(30–15%

SWA).Althoughstomatalclosureisafastmechanismthatreduces

waterlossinthisspecies,increasingtheresistanceofCO2

diffu-sionintothemesophyll,carboxylationefﬁciencyisonlyaffected

aftera reduction ofstomatalconductance of approx.50% (days

23–26,Fig.4BandF).Thisshowstheresilienceofthe

photosyn-theticapparatustodroughtandpointstoCO2limitationbeingthe

mainfactorresponsibleforthedecreaseofAn.Thisalsosuggests

thatthisspeciescanbegrownundermildtomoderatewaterdeﬁcit

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Fig.5. Effectofdroughtstressandre-wateringonchlorophyll(Chl)content.(A)Chla content,(B)Chlbcontentand(C)Chla/bratiomeasuredforleavesoftwoJatropha curcasaccessionsfromIndonesia(Ind)andCapeVerdeislands(CVi)forplants con-tinuouslygrownunderwell-wateredcontrolconditions(C)orsubjectedtodrought stress(S)for28days,followedbya7-dayre-wateringperiod(R).Samplingpoints wereperformedatmaximumstress(day28)andattheendofthe7-day recov-eryperiod(day35).Valuesaremeans.Barsrepresent±SE(n=5).Differentletters abovetheSE-barindicatesigniﬁcantdifferencesbetweenbothtreatmentsaccording toTukey’stest(p<0.05).

andwithoutasigniﬁcantreductionofbiomass,duetoanincreased

WUEi.The highwateruseefﬁciencyis a directconsequence of

thedecreaseingspriortothedecreaseinAn,whichisatypical

responseobservedinotherspecieswhensubjectedtomilddrought

stress(Medranoetal.,2010)andalsoforthecloselyrelatedManihot

sp.(Euphorbiaceaefamily)(El-Sharkawy,2007).However,several

authors(Silvaetal.,2010a,b;Pompellietal.,2010;Díaz-Lópezetal.,

2012)reportedasynchronizedreductionofstomatalconductance

andnetassimilation.Inourexperimentalconditions,weobserved

higherWUEi under mild tomoderatewater deﬁcits. Moreover,

WUEiwashigherinstressedplantswithSWAvaluesbetween60

and20%(Fig.4E),whichiscontrastingtotheresultsofDíaz-López

etal.(2012),whoobservedthatreducingsoilwatercontentto50%

decreasedwateruseefﬁciency.

PSII decrease occurred after the net assimilation decrease,

reinforcing that stomatal limitations have primarily limited An

ratherthanphotochemistry.ReducedCO2 supplyisexpectedto

negativelyaffecttheCalvincycle,whichinturnwouldlimitPSII

efﬁciency.ThesequentialdeclinesofAnandPSIIunderdrought

havebeendescribedintheliterature(ChavesandOliveira,2004),

andarethoughttobecausedbythecloselinkagebetweenthe

activityofETR/PSIIandphotosynthesis(Gentyetal.,1989).

Under severe drought stress leaf chlorophyll contents often

declineduetochlorophylldegradation(Martínez-Ferrietal.,2004;

Jaleeletal.,2009;Anjumetal.,2011).Thishasalsobeenreported

forJ.curcas(Pompellietal.,2010).However,inthepresent

experi-mentalconditionswefoundnoreductioninChlaorChlbcontents,

notevenatmaximumstress(Fig.5AandB).Infact,someincrease

wasobservedwhichhoweverwasnotsigniﬁcant.Anincreasein

Chla/bratioduringdroughthasbeenreportedforseveralspecies

(e.g.Guerfeletal.,2009;Liuetal.,2011),however,inJ.curcaswe

observedareductionoftheChla/bratiosduringdrought(Fig.5C,

maximumstress).Pompellietal.(2010)alsofoundareductionof

theChla/bratioforJ.curcasalthoughthiswasaccompaniedbya

decreaseintotalChlcontent,especiallyofChla.Inourexperiments,

thereductionofChla/bratiowasduetoanincreaseinChlbcontents.

Weobservedthatafterre-wateringChla/bwasfoundtoincrease

instressedplantsuptothecontrolvalues,suggestingthatreduction

ofChla/b ratioisa speciﬁcresponsetodrought.Itisknownthat

Chla/bratioisinverselyrelatedtotheantennasize(Tanakaetal.,

2001).Althoughlargeantennasareparticularlydescribedforlow

lightconditions,itmightbeinterestingtoinvestigatetheirputative

correlationwithChla/bcontentinresponsetodroughtstressinJ.

curcas.

4.3. Recoveryfollowingre-watering

Therecoveryofphotosyntheticratesafterwaterstressdepends

ontheintensityofstressimposition.Afteramoderatestressitcan

befastandcomplete,butafterseverestressrecoveryof

photosyn-thesiscanlastfromdaystoweeksandsometimesisnevercomplete

(Boyer,1971; Miyashitaet al.,2005;Flexaset al.,2006;Chaves etal.,2009,2011).Inourtrial,plantsofbothaccessionsneeded

onlyonedaytorecovertonormalfunctioningandreachcontrol

levels,despiteaseverereductioninleafgasexchangeand

photo-systemIIoperatingefﬁciencyatmaximumstress(Anreductionto

77%,gsto98%andPSIIto65%).Suchrecoveryefﬁciencysuggests

thattheimposedwaterstresscausednopermanentdamagetothe

leafphotochemicalsystem.Italsoshowsthehighresilienceofboth

accessionstodroughtstressasdescribedforotherspecies(Chaves

etal.,2009,2011).

Theabsenceofmarkeddifferencesbetweenthetwoaccessions

studiedcouldbeexplainedbythelowgeneticdiversityobserved

inJ.curcasoutsidetheirMeso-Americancentreoforigin(Sunetal.,

2008;BashaandSujatha,2007;Ranadeetal.,2008;Pamidimarri

etal.,2009).Moreover,thefactthatinourstudythemother-plants

fromwhichseedswereharvestedweregrowingside-by-sidein

CapeVerdemustalsobeaccountedfor,sinceatepigeneticlevelthis

couldhaveresettheIndonesiaseed-derivedmaterialtoCapeVerde

climatic conditions. Such putative re-adjustment could

attenu-ateputativedifferencesindroughtstressresponsebetweenboth

accessions.Althoughfurtherstudiesareneededtotestthis

hypoth-esis, it isinteresting to verifythat there is increasingevidence

thatepigeneticsplaysarelevantroleinJ.curcasphenotypic

diver-sity(Popluechaietal.,2009;Yietal.,2010;Kanchanaketuetal.,

2012).TheﬁndingsofKanchanaketuetal.(2012)areparticularly

interesting,sincetheseauthorsshowed,bymethylation-sensitive

ampliﬁedfragmentlengthpolymorphism(MS-AFLP),that

popu-lationswithlowgeneticdiversityhavehighepigeneticvariability

andapopulationofJ.curcasgrowinginsalineareashasspeciﬁc

DNAmethylationpatterns.

Fieldtrials withJ.curcas are still neededto evaluate

physi-ological and agronomicalbehaviour andassess yield undersoil

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accessions from the centre of origin where a higher genetic

diversity is available. These studies are particularly

rel-evant for the ongoing breeding programs of J. curcas,

designed to achieve improved oilproduction in drought-prone

environments.

5. Conclusions

ThetwoaccessionsofJ.curcasherecompared,originatingfrom

CapeVerdeislandsand Indonesia,showednomarked

morpho-physiologicaldifferencesinresponsetodroughtstress.Ourresults

showthat,underagradualreductionofsoilwateravailabilityalong

a28daysperiodfollowedbyoneweekrecovery,plantsofboth

accessionsareabletomaintainaﬁnalbiomasssimilartoplants

grown underwellirrigated conditions. Net photosynthesiswas

maintainedduringwaterdeﬁcit,droppingfastonlyafter30%of

SWAwasreached,duetoreducedstomatalconductance.Drought

stressdidnotreducechlorophyllcontents butledtodecreased

chlorophylla/b.Furthermore,thisspecieshasshowntoimprove

itswateruseefﬁciencyalongmildtomoderatestresskeepinga

reasonablegrowthundersoilwaterconditionsupto30%ofSWA

andresistingseveredroughtwithagoodrecoverycapacityafter

re-watering.OurresultssupporttheideathatJ.curcasisappropriate

forcultivationinareaswithlimitedwateravailability.

Acknowledgments

The present study was funded by Fundac¸ão para a

Ciên-cia e a Tecnologia (FCT) (projects PTDC/AGR-GPL/101435/2008

and PEst-OE/EQB/LA0004/2011).JM Costa and TLourenc¸owere

also supported by FCT through grants SFRH/BPD/34429/2006

(JMC)andSFRH/BPD/34943/2007(TL).Theauthorswouldliketo

acknowledgeMariadaGrac¸aPalha(InstitutoNacionaldeRecursos

Biológicos,Oeiras,Portugal)formakingavailablethecolourimage

analysissystem,andJacintaCampofortechnicalsupporttothese

analyses.Prof.ManuelaChavesisalsogratefullyacknowledgedfor

valuableinputsandcriticalreviewofthemanuscript.Theauthors

wouldstillliketothankthereviewersfortheirtimeandvaluable

comments.

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