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Flora xxx (xxxx) xxx–xxx ContentslistsavailableatScienceDirect Flora journal homepage: www.elsevier.com/locate/flora Hungry and thirsty: Effects of CO and limited water availability on plant 2 ☆ performance Andries A. Temmea,b,⁎, Jin Chun Liua,c, Will K. Cornwella,d, Rien Aertsa, Johannes H.C. Cornelissena aDepartmentofEcologicalScience,VrijeUniversiteit,DeBoelelaan1085,1081HV,Amsterdam,theNetherlands bDepartmentofPlantBiology,UniversityofGeorgia,Athens,2502MillerPlantSciences,120Carltonstreet,GA,30602,USA cKeyLaboratoryofEco-environmentinThreeGorgesReservoirRegion,SchoolofLifeScience,SouthwestUniversity,Beibei,Chongqing,400715,China dEcologyandEvolutionResearchCentre,SchoolofBiological,Earth,andEnvironmentalSciences,UniversityofNewSouthWales,Kensington,2052,Sydney,Australia ARTICLE INFO ABSTRACT Editedby:HermannHeilmeier Carbondioxideandwaterarecrucialresourcesforplantgrowth.Withanthropogenicfossilfuelemissions,CO2 Keywords: availability is and has been increasing since the last glacial maximum. Simultaneously water availability is ElevatedCO2 expectedtodecreaseandthefrequencyandseverityofdroughtepisodestoincreaseinlargepartsoftheworld. Planttraits Howplantsrespondtothesetwochangeswillhelpinunderstandingplants’responsestoclimateofthefuture. Growth HerewesoughttounderstandhowdroughtaffectsplanttraitsresponsestoCO2andwhethertherearetrade-offs Biomassallocation inresponsivenesstolowandelevatedCO2anddrought.WegrewseedlingsofsevenC3annualsatpastlow Drought (160μll−1),ambient(450μll−1)andelevated(750μll−1)CO2.Ateachconcentrationplantsweresubjectedto Gasexchange well-wateredconditions(100%soilwateravailability,SWA),40%SWAor20%SWA.Wemeasuredbiomass AnnualC3herbs allocation,relativegrowthrate,tissueNconcentration,andgasexchange.Comparedtowell-wateredconditions plantsizewasanimportantelementintheabsoluteresponsetoSWAdecrease,i.e.thesmaller,slowgrowing specieswereunaffectedbydroughtatlowCO2.PlantsallocatedlessmasstoroottissueatlowCO2contrasting withincreasedrootmassfractionatlowerSWAatambientCO2.Acrossalltraitsmeasured,wefoundmostly additiveeffectsofCO2andwater.Asduetoclimatechangeregionsbecomemoredroughtpronetheseresults suggestCO2fertilizationwillnotcounteracttheeffectsofreducedwateravailability. 1. Introduction thefuturewere,areandwillbemultivariate.Thus,howtheavailability of CO2 and that of other resources interact is a key question for un- Plantgrowthislimitedbytheavailabilityoflight,nutrients,water derstandingthefullresponseofplantstoCO2. and carbon. In terms of carbon acquisition, the growth rate of plants Consideringthefulltrajectoryfrompastlow,tocurrent,tofuturehigh canbeseparatedintoamorphologicalandaphysiologicalcomponent CO2 concentrations aids in understanding plant response to CO2. (Evans, 1972). Several meta-analyses have shown that both morpho- Anthropogenic emissions will likely increase current carbon dioxide logical traits, such as specific leaf area (SLA; leaf area per dry mass), concentrationsfrom410μll−1toanestimated600–900μll−1bytheend leafmassfraction(LMF;leafmassperplantmass)androotmassfrac- ofthecentury(Meinshausenetal.,2011).Inplants’recentgeologicpast tion(RMF;rootmassperplantmass)andphysiologicaltraits,suchas CO2 levels haverisen from a >2 Myr period of low CO2, ≈180μl l−1 photosyntheticrate,nitrogenconcentrationandcarbonconcentration, duringthePleistoceneglacials(Hönischetal.,2009),totoday’s410μll−1 have a strong plastic response to elevated CO2 (Poorter and Navas, (Keelingetal.,2005)aftertheIndustrialRevolution.Inthelightofplants’ 2003;AinsworthandLong,2005;NorbyandZak,2011).Interestingly, evolutionary historyin lowCO2andthe currentfast shiftto highCO2, there is an even stronger response of these traits to low CO2, re- understandingplants’responsestowaterandcarbonavailabilityoverthe presentative of atmospheric composition in the distant past (Temme full range of CO2 could aid in predicting their responses to the future etal.,2013).However,environmentalchangesbothinthepastandin climateandatmosphericcomposition(TissueandLewis,2012). ☆Thisarticleispartofaspecialissueentitled:“Functionaltraitsexplainingplantresponsestopastandfutureclimatechanges”publishedatthejournalFlora254C, 2019. ⁎Correspondingauthorat:DepartmentofEcologicalScience,VrijeUniversiteit,DeBoelelaan1085,1081HV,Amsterdam,theNetherlands. E-mailaddress:[email protected](A.A.Temme). https://doi.org/10.1016/j.flora.2018.11.006 Received14March2018;Receivedinrevisedform21October2018;Accepted8November2018 0367-2530/ © 2018 Elsevier GmbH. All rights reserved. Please cite this article as: Andries A. Temme, et al., Flora, https://doi.org/10.1016/j.flora.2018.11.006 A.A.Temme,etal. Flora xxx (xxxx) xxx–xxx Carbonandwaterfluxesaretightlylinkedthroughthestomata.This ofsoilwateravailability(SWA). underpins the potential for interaction between CO2 and water availability. At elevated CO2, transpiration is reduced transiently due to 2. Materialandmethods closingstomata(AinsworthandRogers,2007)ordevelopmentallybya lower number, or density, of stomata (Haworth et al., 2013). Elevated We grew seedlings of 7 different C3 herbaceous species, including CO2 also reduces the nitrogen concentration in plants (Ainsworth and grasses(G),forbs(F)andN2-fixers(NF),atthreelevelsofcarbonand Long,2005).Oneofseveralmechanismshypothesizedtocausethisisa water supply in a fully factorial design. Species grown were Agrostis reducedfluxofwateracrosstherootsduetoreducedtranspiration(Feng capillaris L. (G), Clinopodium chinense (Benth.) Kuntze (F), Hemisteptia etal.,2015).Thusplantresponsestolimitwaterloss,byclosingstomata, lyrata(Bunge)Fisch.&C.A.Mey.(F),MedicagolupulinaL.(NF),Rumex canaffectperformanceatelevatedCO2indicatingthepotentialfortrade- chalepensis Mill. (F), Stellaria media (L.) Vill. (F) and Vicia sepium L. offsbetweenplantresponsestocarbonandwateravailability. (NF),i.e.asubsetofthespeciesinTemmeetal.(2015,2017).These Given the rapid increase in CO2 concentration in the atmosphere species,fromprovenancesintemperateEuropeandsubtropicalChina, andachangingclimate,alotofresearchhasbeendoneonhowplants wereselectedbasedonthebroadrangeinallocation,Nuptakestrategy respondtoelevatedCO2andhowwateravailabilityinteractswiththat andleaftraitstheyrepresented. response.Forexample,inforestplotscanopywaterusehasbeenfound Plantswere grown inthreecontrolled-environment walk-incham- to be elevated in response to elevated CO2 in developing stands, but bers (Reftech bv, Sassenheim, NL) at the Phytotron labs at Utrecht reduced in establishedstands(Warren et al., 2011). Inclimate cham- University,TheNetherlands,atwhichwekeptCO2atlow,ambientand bers,theCO2fertilizationeffect hasbeenfoundtobe relativelylarge highlevelrespectivelyfollowingTemmeetal.(2015,2017).CO2inthe underdryconditions(PoorterandPérez-Soba,2001).Incontrast,inthe lowchamberwaskeptatalow160μll−1byscrubbingambientairof field drought tolerance is not necessarily increased. In a desert en- CO2downtotargetlevelwithamolecularsieve(PG1500L,CMCIn- vironment,elevatedCO2washypothesisedtoincreaseproductivitydue struments GmbH, Eschborn). The ambient CO2 chamber was not di- toincreasedwateruseefficiency.However,after10yearsofelevated rectlycontrolledforCO2.Concentrationtherewasfoundtobe450μl CO2 productivity and community composition remained unaltered l−1,likelyduetothechambersbeingsituatedinsideanofficebuilding (Newingham et al., 2013; Smith et al., 2014). Here the strong water andnearamajorroad.TheelevatedCO2chamberwaskeptat750μl limitationledtonostimulationbyelevatedCO2. l−1 by adding fossil fuel derived CO2 from pressurized canisters to Whilethemajorityofresearchhasbeendoneonpredictingplants’ ambient air ventilating the chamber. CO2 levels were continuously response to future environmental conditions, understanding plants’ monitored(GMP343,VaisalaGmbH,Bonn)withscrubbercapacityand response to conditions of the past might provide clues about possible CO2 supply adjusted accordingly. While handling plants in the low physiological or morphological constraints in response to future con- chamber,exhaledbreathwascapturedusingagasmaskconnectedwith ditions. Low CO2 has profound impacts on plant traits and plant per- anairtightbaginordertolimitCO2levelsrising. formance, including a strong reduction in biomass and growth rate, Individualsweregerminated fromfield-collectedseedsonwetfilter higherspecificleafarea(SLA,i.e.thinnerorlessdenseleaves),larger paperorsandandtwotothreedaysaftergerminationindividualswere leaf mass fraction (leaf mass per plant mass) and strongly increased transferredtoexperimentalconditions.Individualsweretransplantedto nitrogenconcentration(GerhartandWard,2010;Temmeetal.,2013, 400ml pots, one individual per pot, containing coarse river sand (con- 2015,2017;Becklinetal.,2014).Leaftraitsareadjustedinsuchaway tainingtraceamountsofclayandsilt)tofacilitatingrootwashing.Wedid as to move towards the resource acquisitive end of the across-species notexpectpotsize(i.e.volumeavailableforrooting)toplayasubstantial leafeconomicspectrum(Wrightetal.,2004;Reich,2014)asexpressed roleinplantresponsetoCO2anddrought.Plantswereexpectedtoremain by higher leaf mass fraction, higher SLA and higher nitrogen content below 1g dry weight L−1 soil volume during the experiment (Poorter (Temme et al., 2017). However, this suite of traits is associated with etal.,2012)andthesoilwatercontentatwhichdroughteffectsbecome lesserdroughttolerance(Halliketal.,2009;Ouédraogoetal.,2013). apparentisnotrelatedtopotsize(RayandSinclair,1998).Wegrew6–8 Thisstronglysuggeststhattherecouldbeatrade-offbetweenadapta- individualsperspecies/treatmentcombination. tionstolowversushighCO2andthosetodrought. ExperimentalconditionsweresimilartothoseinLiuetal.(2016). Owingtothetechnicalhurdlesassociatedwithgrowingplantsatlow Lightwassetto≈350μmolm−2s-1witha10-hphotoperiod,at21°C CO2, only limited studies have assessed drought effects on plant func- during day, 18°C at night and 70% relative air humidity. Up to the tioningatpastlowCO2(GerhartandWard,2010).Plantperformanceat development of the first leaf, pots were watered three times per day dryconditionsinlowCO2showscontrastingresponsesdependingonthe frombelowbywaterautomaticallyflowingoverthebench.Afterthat speciesandtheexperiment.Forexample,Sequoiatreeshadgreaterxylem individualswereseparatedintothreeequalsizegroups,oneofwhich hydraulicfailure,increasedmortalityandreduceddefensivecompounds waskeptatfullwatersupply(100%soilwateravailability,SWA)and atlowCO2(Quirketal.,2013).Yet,inPhaseolusvulgarisdroughttoler- two of which were subjected to drought (40% SWA and 20% SWA). ance was increased at low CO2 due to improved xylem functioning Three times per week (Mo–Wed–Fri) all drought treatment pots were (MedeirosandWard,2013).Moreover,thesmallersizeofplantsatlow weighedtothenearest0.1gandwaterwasaddedbacktotargetSWA CO2 might prove beneficial during periods of reduced precipitation as level. To prevent nutrient limitation 50ml of full Hoagland solution theytakeuplesswaterandmaydepleteagivenwatersupplymoreslowly (6mM KNO3, 4mM Ca(NO3)2, 2mM NH4H2PO4, 1 μM KCl, 25 μM withinalimitedsoilvolume(Liuetal.,2016). H3BO3, 2 μM MnSO4, 2 μM ZnSO4, 0.1 μM CuSO4, 0.1 μM In general plants balance resource uptake such that growth is (NH4)6Mo7O24,20μMFe(Na)EDTA)wereaddedtoeachpotthreetimes equally limited by all resources (Bloom and Mooney, 1985; Chapin perweek.Damagetofreshlygerminatedindividualswaspreventedby etal.,1987).Hereweaimtorevealoverallpatternsofplanttraitsand slowly increasing the concentration from 25% to full Hoagland as growth performance response to CO2 and water availability regimes, plantsgrewuntilfulldevelopmentofthefirstleaf. addressing: (1) how CO2 concentration from past to future and soil Aftertheopeningofthefirstleaf(t0),abaselineharvest(wt0)was wateravailabilityinteracttoaffectplantperformanceand(2)whether performed. Plants were then grown for three more weeks (t1) after there are trade-offs in the growth responsiveness to CO2 – including which a final harvest (wt1) was done. Relative growth rate (RGR) plantsize–andtodrought. duringtheexperimentwascalculatedfollowingHoffmannandPoorter We sought to answer these questions by experimentally growing (2002)asRGR=[Ln(wt1)-Ln(wt0)]/(t1-t0).Atfinalharvestwecounted seedlings of seven annual C3 herbaceous species broadly ranging in dead individuals and measured live plants for root and shoot fresh responsivenesstoCO2anddifferinginspecificleafareaandleafmass weight and leaf area and fresh weight of a single representative leaf. fractionatpastlow,ambientandfuturehighCO2andatabroadrange Therepresentativefull-grownleafwasscanned(CanonLiDe110)and 2 A.A.Temme,etal. Flora xxx (xxxx) xxx–xxx measuredusingImageJv1.47forSLA(mleaf2gleaf−1dryweight).Plant SWA.LowerSWAledtoincreasedrootmassfraction(RMF)atambient materialwasovendriedfor>48hat70°Cafterwhichroot,shootand CO2butnotatloworhighCO2.Additionally,CO2increasefromlowto leaf dry mass were determined. Leaves were subsequently removed ambientconcentrationsubstantiallyincreasedRMF(AppendixFig.1). fromstems,orotherstem-liketoughtissueincludingpetioles,andstem Leaf mass fraction was significantly increased at low CO2 but only dry mass determined. From these mass data we calculated leaf mass underseverewaterlimitation(AppendixFig.2). fraction (LMF, gleaf gplant−1), root mass fraction (RMF, groot gplant−1) The scaling relationship between log-transformed leaf biomass and andstemmassfraction(SMF,gstemgplant−1).Leafmaterialwasground root biomass did not differ between water treatments, but there was a up in a ball mill (MM200, Retsch, Haan, DE) and leaf C and N con- difference in elevation between CO2 levels (Fig. 3). Standardized major centrationdeterminedbyflashcombustionusingaPerkinElmer2400 axis(SMA)regressionshowedasimilarslopebetweenCO2levelswhenall CHNSanalyzer(ThermoScientific,Rodana,IT). watertreatmentswerepooled.Thescalingrelationshipbetweenleafand For a subset of species at ambient and elevated CO2 leaves were rootbiomassatambientandhighCO2wasthesame,withplantsathigh largeenoughtoallowthatadaypriortoharvestwecouldmeasuregas CO2beingmovedalongthecommonaxesindicatingthestimulatingeffect exchange using a LI-6400 (LICOR, Nevada, USA). Gas exchange was of CO2 on plant biomass. However, the scaling slope at low CO2 had a carriedoutfollowingTemmeetal.(2017).Briefly,onefullydeveloped higherinterceptthanatambientandhighCO2(p<0.01)(Fig.3).Thus leafperindividualwasplacedtoacclimateinthecuvette,setsimilarto foragivenleafbiomassplantsgrownatlowCO2hadalowerrootbiomass. growthconditions,fortwominutesinthelight(red-bluelightsource, LeaftraitswereaffectedbyCO2andSWAaswell.Specificleafarea LI-6400-02B).Theareainsidetheleafcuvettewasmarkedandremoved (SLA) was strongly affected by CO2 with species increasing their SLA atfinalharvest.Whenleavesdidnotfillthetotalcuvettearea(6cm2) from ambient and high to low CO2 (Appendix Fig. 3). SWA only af- the portion of the leaf that could be placed inside the cuvette was fectedSLAatlowCO2wheredroughtledtoplantshavingalowerSLA. scannedusingaCanonLiDe110scanner.Leafareawasthenmeasured Nitrogen concentration per unit mass (N%) was strongly affected by usingImageJv1.47.Netarea-basedphotosynthesis(Anet)andrespira- lowCO2withhigherNlevelsatlowerCO2(p<0.001),whileitwasnot tion rates (R) and stomatal conductance (gs) were then calculated by affectedbySWA(AppendixFig.4). usingthecorrectareainthegasanalyserequations. LeafgasexchangedataunderreducedSWAcouldonlybeobtained DataanalysisandstatisticswerecarriedoutusingRversion3.12(R from plants grown at ambient and high CO2. Leaves of individuals at Core Team, Vienna, Austria) as in Temme et al. (2015). To limit the low CO2 and 20%–40% SWA were too small to fit the LiCOR 6400 effectofpseudoreplication(onlyoneclimatechamberperCO2level), cuvette,sotheinteractionofCO2andSWAcouldnotbetestedatlow we took the individual species mean responses as replicates in all CO2. In absolute terms photosynthesis (Anet) was stimulated by in- analysesontraitresponsestoCO2andsoilwatercontent(SWA).Trait creasingCO2fromlowtoambient.AtambientandhighCO2reducing responses to CO2 and SWA were viewed both in absolute terms and SWAledtolowerAnet(p<0.05)(4a).Stomatalconductance(gs)was relative to ambient CO2. For relative responses trait values were loge notaffectedbyCO2thoughplantsdidshowastrongdecreaseingsat transformed prior to analysis. This approach has the benefit that a lowerSWA(Fig.4b).Anetandgscombinedinintrinsicwateruseeffi- halving or a doubling in trait value from ambient CO2 has the same ciency (iWUE, Anet/gs) being significantly higher at reduced water transformeddifference.TraitresponsestoCO2andSWAweretestedvia availability,thoughmoresoatambientthanhighCO2(Fig.4c). two-way ANOVA, as shown in Appendix table 1. Within each CO2 or SWA level differences between groups were tested by a Tukey test 3.2. Trade-offsinbiomassresponsetoCO2andwater correctedformultiplecomparisons.Associationbetweentraitsandtrait responses were tested via Standardized major axis (SMA) regression Trade-offs in response to CO2 and soil water availability were not (Wartonetal.,2012). readily apparent. At low CO2 there was no relationship between the extent at which carbon starvation decreased biomass compared to 3. Results ambient CO2 and the effect of reduced soil water availability on biomass compared to that at 100% SWA at low CO2. Thus, plants that PlantswerestronglyimpactedbydroughtatallCO2levels.Atthe could cope well with drought stress were not affected differently by lowest levelofsoilwateravailability(20% SWA)some individualsof reduced carbon concentration(Appendix Fig. 5 a). Speciesthat were most species died, except in Agrostis capillaris and Vicia sepium. For stimulated more by elevated CO2 also tended to be more affected by ClinopodiumchinenseandMedicagolupulinathisonlyhappenedatlow reducedSWA(AppendixFig.5b).Therewasasignificantrelationship CO2,forRumexonlyathighandlowCO2,forStellariaonlyatambient betweenplantbiomassandbiomassreductionduetodrought(Fig.5, CO2andforHemisteptiaatallCO2levels. R2=0.68, p<0.001). Thus, in absolute and relative terms larger plants were more affected by drought than smaller plants. Taken to- 3.1. DroughtandCO2effectsonplantperformanceandtraits getherthissuggeststhatthelargeplantsthatwerestimulatedmostby elevatedCO2werethemostnegativelyaffectedbydrought. Increased carbon availability from 160μl l−1 to 450μl l−1 and 750μll−1CO2resultedinlargeroverallplantbiomassatwell-watered 4. Discussion and moderately dry water levels. CO2 concentration and SWA inter- actedinsuchamannerthatatverylowCO2concentration(160μll−1) There are many potential interactive effects between carbon gain increased drought had no significant effect on biomass (Fig. 1a). Re- and water loss. We investigated how CO2 affected plant performance lative to ambient CO2 the effect of decreased CO2 and increased CO2 fromwell-wateredtoseverelydroughtedconditionsandiftherewere wasthesameatallSWAlevels(Fig.1b).ResponsetoCO2wasvaried trade-offsinthegrowthresponsivenesstoCO2versusthattodrought.In acrossspeciesresultinginlowpowertodetectdifferences.Withaless agreementwithearlierworkwefoundthatplantgrowthandbiomass stringentmultiplecomparisonpenaltytherelativeeffectofCO2(com- were strongly reduced at low CO2 (Gerhart and Ward, 2010) and sti- pared to ambient) was significantly reduced biomass at low CO2 and mulatedby highCO2(PoorterandNavas,2003).Droughtled toasi- significantlyincreasedbiomassatelevatedCO2andwell-wateredcon- milar relative reduction of plant biomass at all CO2 levels. However, ditions. Relative growth rate (RGR, g g−1d−1) was affected by CO2 due to the carbon fertilisation effect of increasing CO2, plants accu- comparabletoplantbiomass.BetweenlowCO2andhighCO2RGRwas mulatedmorebiomassinabsolutetermsathigherCO2levels.Thus,the significantly increased at well-watered and moderately dry levels absolute effect of drought was in fact greater at higher CO2 because (Fig.2a).SWAandCO2didnotshowasignificantinteractionforRGR. plants could grow larger at well-watered conditions. This shows that BiomassallocationtorootsandleaveswasaffectedbybothCO2and plantbiomassappearstobeakeyelementintheresponsivenesstosoil 3 A.A.Temme,etal. Flora xxx (xxxx) xxx–xxx Fig.1.PlantbiomassinrelationtoCO2 concentration and soil water availability(SWA)in(a)absolutetermsand (b)relativetoambientCO2.Greydots indicate species average biomass (n=4–6perspecies),greybarsdenote standard error. Letters denote Tukey post-hoc tests of difference between SWA levels (lowercase) and difference between CO2 levels (uppercase). In panel(b)insteadofcomparingCO2le- velsthedifferencefromzero,noeffect, istested.Insetshowstwo-wayANOVA of CO2 and SWA and their interaction (see Appendix Table 1 for full model output).ns:notsignificant,*:p<0.05, **:p<0.01,***:p<0.001. wateravailability(SWA)atpastlow,ambientandfuturehighCO2. 4.1. Interactiveeffectsofcarbonandwater ComparedtoambientCO2wefoundnoincreaseinCO2stimulation ofplantbiomassunderdryconditions,whichcontrastswithanearlier study(PoorterandPérez-Soba,2001),butisconsistentwithsomefield resultsfromaridecosystems(Smithetal.,2014).Howeverwedidfind whentakingsizeintoconsideration,thatplantsthatgrewlargerathigh CO2andamplewaterweremoreaffectedbyreducedSWA(Fig.5),as Liuetal. (2016)foundforthegrassAvenasativaandtheforbCheno- podiumalbum.Moreover,lowerSWAatelevatedCO2reducedtheeffect ofCO2fertilizationonbiomassproduction.WhenSWAislowitseems thatexcessavailablecarboncannotbeusedandthestimulatingeffect ofelevatedCO2disappears.Furthermore,wefoundthattheallometric relationship between leaf and root biomass was different at low CO2 fromthatatambientandhighCO2(Fig.3).Foragivenleafmass,plants hadlessrootmassatlowCO2.Moreextremedroughtthaninthisstudy could exacerbate negative effects on growth at low CO2 as low root mass allocation is linked to poor drought tolerance (Zwicke et al., 2015). 4.2. Trade-offsinresponsivenesstoCO2andwater Fig.3.Allometricrelationshipbetweenleafandrootbiomass atlow(160μl l−1),ambient(450μll−1),andhigh(750μll−1)CO2.Linesindicatepropor- Inthisshort-termstudywefoundnoclearevidencefortrade-offsin tional(log10)scalingslopeofleafbiomasstorootbiomassateachCO2con- centrationbasedonSMAregression.***:p<0.001. the responses to water versus to CO2 (Appendix Fig. 5). Species that couldtoleratelowerSWAdidnotresponddifferentlytoeitherelevated orreducedCO2.Howeverwithmorespeciesorlongerdroughtduration Fig.2.Plantrelativegrowthrate(RGR) in relation to CO2 concentration and soilwateravailability(SWA)in(a)absolutetermsand(b)relativetoambient CO2.Greydotsindicatespeciesaverage RGR (n=4–6 per species), grey bars denote standard error. Letters denote Tukey post-hoc tests of difference be- tweenSWAlevels(lowercase)anddif- ference between CO2 levels (uppercase).Inpanel(b)insteadofcomparing CO2levelsthedifferencefromzero,no effect, is tested. Inset shows two-way ANOVAofCO2andSWAandtheirin- teraction(seeAppendixTable1forfull model output). ns: not significant, *: p<0.05,**:p<0.01,***:p<0.001. 4 A.A.Temme,etal. Flora xxx (xxxx) xxx–xxx Fig.4.Effectofsoilwateravailability(SWA)andCO2concentration,on(a)netphotosynthesis(Anet),(b)stomatalconductance(gs)and(c)intrinsicwateruse efficiency(iWUE).Greydotsindicatespeciesaveragevalue(n=4–6perspecies),greybarsdenotestandarderror.LettersdenoteTukeypost-hoctestsofdifference betweenSWAlevels(lowercase)anddifferencebetweenCO2levels(uppercase).Insetshowstwo-wayANOVAofCO2andSWAandtheirinteraction(seeAppendix Table1forfullmodeloutput).ns:notsignificant,*:p<0.05,**:p<0.01,***:p<0.001. becauseresourcesweremoreevenlydistributed(Alietal.,2015).Field experimentscombiningCO2anddroughtwouldbeanexcellentwayto see if plants exhibit the same response to water as modelled for nutrients;andhow,asinourstudy,plantsizemodulationofdroughtef- fectsaffectsthisresponse. 4.3. Recommendationsandexperimentalconsiderations The role plant size had in modulating the effect of SWA is likely influencedbyourdroughtscenario.Droughteffectscanbeinvestigated usingmanydifferentscenarios(Tardieu,2012;O’Gradyetal.,2013;He andDijkstra,2014).Hereweachieveddroughtstressexperimentallyby subjectingplantstoreducedwateraddition.SWAwaskeptat20%and 40% of fully watered conditions by adding water to the desired level threetimesaweek.LocallyinthesoilSWAwastheninevitablyhigher thantheaverageSWA.Forsmallplants,theseshorttimeperiodswhere they had access to water may have been enough to maintain functioning. Aslarger plantsneed more watertheydraw down soilwater supply more quickly and as such are more affected by reduced precipitation(Liuetal.,2016). Longertermdroughtcombinedwithcarbonstarvationasinthepast could have had more detrimental effects on plant growth and performance (Hartmann et al., 2013). At high CO2 changes in plant mor- Fig.5.Effectofplantsizeatwell-wateredconditionsonthelevelofbiomass phologyandphysiologycouldalsomodulatetheeffectsoflongerterm r(4ed5u0cμtlioln−1d)uaendtohisgohil(w75a0teμrlal−va1i)laCbOil2i.tyLin(SeWsiAn)diactatleowSM(A16r0egμrlesl−si1o)n,baemtwbieeennt hdrigohugShLtA(SapnedrrylowandroLootvme,a2ss01fr5a)c.tCioOn2(sTteamrvmateioentlaela.d,s20to15p)lawnthsicwhitihs biomass and biomass effect at reduced SWA at low CO2 (red), ambient CO2 oppositetowhatwouldbebeneficialunderdroughtconditions(Hallik (green) and high CO2 (blue). Dashed lines: non-significant slope. ***: et al., 2009; Ouédraogo et al., 2013). Thus longer dry periods could p<0.001.(Forinterpretationofthereferencestocolourinthisfigurelegend, showtrade-offsas,inthelongterm,therecouldbeoppositeresponses thereaderisreferredtothewebversionofthisarticle). to CO2 and water. Moreover, long term periods of elevated CO2 and environmental stress could lead to evolutionary adaptations different these trade-offs, if present, may become apparent. Plant species with fromtheplasticresponsetorapidshiftsinCO2,aswasshownfornat- high growth rates are stimulated more by elevated CO2 (Cornelissen uralCO2vents(Onodaetal.,2009). et al., 1999; Poorter and Navas, 2003) and more affected by reduced OurresultscorroborateearlierfindingsthatherbaceousC3species, CO2 (Temme et al., 2015). Traits associated with high drought toler- when grown at elevated CO2, are not more drought tolerant than at ance(highRMF,lowSLA)arenegativelyassociatedwithplantgrowth ambientCO2(MedeirosandWard,2013)andthattherelativeeffectof rates(Reich,2014).Potentiallyplantswithreducedgrowthratesdueto droughtisnotgreateratloworhighCO2(Wardetal.,1999).However, adaptationstopoorwaterconditionscouldresultinareducedstimu- the results from an earlier study with the tree Sequoia sempervirens lationbyelevatedCO2atwell-wateredconditions. (Quirk et al., 2013) are markedly different from our andprevious re- From a competition point of view, the greater stimulation of fast sultsbasedonherbaceousspecies.ThisstudyfoundthatlowCO2ledto growerswithtraitsbeneficialatlowlevelsofdroughtcouldhelpthem increased drought stress in the form of greater mortality and slower in outcompeting drought tolerant slow growers at sufficient water growth. Though it should be noted that plants’ different first appear- supply.However,inamodelthatincorporatesnutrientuseandcapture ance in the geological record and thus different past experience with it was found that elevated CO2 led to increased coexistence between fluctuating CO2 levels on geological timescales could impact their re- species due to reduced competitive ability and increased evenness sponse to varying CO2 this does raise the question if availability of 5 A.A.Temme,etal. Flora xxx (xxxx) xxx–xxx water and carbon affects (slow growing) woody plants species differ- andmorphologicalresponsesofstomatatoelevated[CO2]invascularplants. entiallyfrom(fast-growing)herbaceousplants. Oecologia171,71–82. He,M.,Dijkstra,F.A.,2014.Droughteffectonplantnitrogenandphosphorus:ameta- analysis.NewPhytol.204,924–931. 5. Conclusion Hoffmann,W.A.,Poorter,H.,2002.Avoidingbiasincalculationsofrelativegrowthrate. Ann.Bot.80,37–42. Hönisch,B.,Hemming,N.G.,Archer,D.,Siddall,M.,McManus,J.F.,2009.Atmospheric Taken together these results paint a picture of limited interactive carbondioxideconcentrationacrossthemid-Pleistocenetransition.Science324, effects of CO2 and water availability. Across all traits measured, we 1551–1554. Keeling,C.D.,Piper,S.C.,Bacastow,R.B.,Wahlen,M.,Whorf,T.P.,Heimann,M.,Meijer, foundmostlyadditiveeffectsofCO2andwater.However,itshouldbe H.A.,2005.AtmosphericCO2and13CO2exchangewiththeterrestrialbiosphereand noted that the already stressed plants at low CO2 did not experience oceansfrom1978to2000:observationsandcarboncycleimplications.In: extrastressduetoreducedwateravailability.Asduetoclimatechange Ehleringer,J.R.,Cerling,T.E.,Dearing,M.D.(Eds.),AHistoryofAtmosphericCO2 andItsEffectsonPlants,Animals,andEcosystems.SpringerVerlag,NewYork,pp. regions become more drought prone these results suggest CO2 fertili- 83–113. zationwillnotcounteracttheeffectsofreducedwateravailability. Liu,J.-C.,Temme,A.A.,Cornwell,W.K.,vanLogtestijn,R.S.P.,Aerts,R.,Cornelissen, J.H.C.,2016.Doesplantsizeaffectgrowthresponsestowateravailabilityatglacial, modernandfutureCO2concentrations?Ecol.Res.31,213–227. Authorcontributions Medeiros,J.S.,Ward,J.K.,2013.Increasingatmospheric[CO2]fromglacialtofuture concentrationsaffectsdroughttoleranceviaimpactsonleaves,xylemandtheirin- tegratedfunction.NewPytologist199,738–748. AAT, JCL, WKC, RA and JHCC conceived and designed the study, Meinshausen,M.,Smith,S.J.,Calvin,K.,Daniel,J.S.,Kainuma,M.L.T.,Lamarque,J.-F., AATandJCLgrewplantsandcollectedthedata,AAT,JCL,RA,JHCC Matsumoto,K.,Montzka,S.A.,Raper,S.C.B.,Riahi,K.,Thomson,A.,Velders,G.J.M., analysed and interpreted the data. AAT drafted the manuscript with Vuuren,D.P.P.,2011.TheRCPgreenhousegasconcentrationsandtheirextensions from1765to2300.Clim.Change109,213–241. substantialrevisionbyRAandJHC.Allauthorsagreeonthefinaltext. Newingham,B.A.,Vanier,C.H.,Charlet,T.N.,Ogle,K.,Smith,S.D.,Nowak,R.S.,2013. AATtakesresponsibilityfortheintegrityoftheworkasawhole,from Nocumulativeeffectof10yearsofelevated[CO2]onperennialplantbiomass componentsintheMojaveDesert.Glob.Chang.Biol.19,2168–2181. inceptiontofinishedarticle. Norby,R.J.,Zak,D.R.,2011.Ecologicallessonsfromfree-airCO2enrichment(FACE) experiments.Annu.Rev.Ecol.Evol.Syst.42,181–203. Acknowledgements O’Grady,A.P.,Mitchell,P.J.M.,Pinkard,E.A.,Tissue,D.T.,2013.Thirstyrootsand hungryleaves:unravellingtherolesofcarbonandwaterdynamicsintreemortality. NewPhytol.200,294–297. We would like to thank R.C.E. de Man and R.A.M. Welschen for Onoda,Y.,Hirose,T.,Hikosaka,K.,2009.DoesleafphotosynthesisadapttoCO2-enriched theiraidandadviceingrowingandharvestingthemanyplantsgrown environments?AnexperimentonplantsoriginatingfromthreenaturalCO2springs. NewPhytol.182,698–709. during this experiment; and Utrecht University for hosting us during Ouédraogo,D.Y.,Mortier,F.,Gourlet-Fleury,S.,Freycon,V.,Picard,N.,2013.Slow- this experiment. We would like to thank the reviewers for their con- growingspeciescopebestwithdrought:evidencefromlong-termmeasurementsina tropicalsemi-deciduousmoistforestofCentralAfrica.J.Ecol.101,1459–1470. structive comments. This study was financially supported by grant Poorter,H.,Navas,M.-L.,2003.PlantgrowthandcompetitionatelevatedCO2:onwin- 142.16.3032oftheDarwinCentreforBiogeosciencestoRA.JHCCand ners,losersandfunctionalgroups.NewPhytol.157,175–198. JCL benefitted from Grant CEP-12CDP007 by the Royal Netherlands Poorter,H.,Pérez-Soba,M.,2001.ThegrowthresponseofplantstoelevatedCO2under non-optimalenvironmentalconditions.Oecologia129,1–20. 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