Photosynthetic limitations in leaves of young Brazilian Green Dwarf coconut palm

Available online at Environmental and Experimental Botany62(2008)195–204Photosynthetic limitations in leaves of young Brazilian Green Dwarf coconut(Cocos nucifera L.‘nana’)palm underwell-watered conditions or recovering from drought stressF´a bio P.Gomes a,∗,Marco A.Oliva b,Marcelo S.Mielke a,Alex-Alan F.de Almeida a,Helio G.Leite c,Leonardo A.Aquino ba Departamento de Ciˆe ncias Biol´o gicas,Universidade Estadual de Santa Cruz,Rod.Ilh´e us-Itabuna,km16,45662-000Ilh´e us,BA,Brazilb Departamento de Biologia Vegetal(DBV),Universidade Federal de Vi¸c osa(UFV),Av.PH Rolfs s/n,Vi¸c osa36571-000,MG,Brazilc Departamento de Engenharia Florestal(DEF),Universidade Federal de Vi¸c osa(UFV),Av.PH Rolfs s/n,Vi¸c osa36571-000,MG,BrazilReceived31July2007;accepted15August2007AbstractYoung plants of two Brazilian Green Dwarf coconut ecotypes from contrasting regions in relation to climate(UGD,from a hot and humid climate and JGD,from a hot and dry climate)were submitted to three consecutive drying/recovery cycles,under greenhouse conditions to determine the photosynthetic limitations encountered during the recovery phase of water deficiency.Three hypotheses were tested:(1)non-stomatal factors contribute to the incomplete recovery of net photosynthesis rate(A),(2)photochemical impairment is an important component of the non-stomatal limitations to A and(3)the two coconut ecotypes respond differently to the drought recovery.In each drying/recovery cycle,irrigation was suppressed until A reached zero.Then the plants were rewatered and the recovery was accompanied for8days.After4days of rewatering,CO2 and light response curve parameters were used to discriminate the stomatal and non-stomatal factors contributing to the incomplete recovery of A. Upon rewatering for4days,the pre-dawn leaf water potential of the stressed plants increased from−1.20to−0.15MPa on average.Only small differences were detected in the intercellular to atmospheric CO2concentration ratio but A did not recover completely to the control values.In both ecotypes,light and CO2-saturated A,carboxylation efficiency,maximum quantum yield of PSII and maximum electron transport rate were significantly lower in stressed plants in comparison with the well-watered ones.Relative stomatal limitation(L S)ranged from16to20%without significant differences between treatments and between ecotypes.The relative mesophyll limitation(L m)was significantly higher in JGD in cycle 1but this changed after repeated drought stress,decreasing to values significantly lower than those in UGD in the other two cycles.The results showed that after4days of rewatering stressed plant stomatal limitations were comparable to that of well-watered control plants(∼20%).However A did not recover completely after drought stress due to non-stomatal factors.Important differences between the ecotypes were identified.The ecotype JGD presented smaller L m,faster recovery of the apparent quantum efficiency of CO2assimilation,adjustment of some carbon balance components and signs of photosynthetic capacity acclimation after repeated drying/recovery cycles.©2007Elsevier B.V.All rights reserved.Keywords:Chlorophyll afluorescence;CO2response curve;Electron transport rate;Light response curve;Mesophyll limitation;Stomatal conductance1.IntroductionThe cultivation of dwarf coconut varieties(Cocos nucifera L.‘nana’)has emerged as an important activity in Brazil. This has increased the demand for information about growth, development and yield under different ecological conditions.∗Corresponding author.E-mail address:gomes@uesc.br(F.P.Gomes).Two ecotypes of the Brazilian Green Dwarf coconut cultivar have been largely used for the planting of new areas.Brazil-ian Green Dwarf from Una(UGD)is cultivated in a hot and humid environment in the southeastern Bahia State.Rainfall indexes are high,air relative humidity is above80%and mean temperature is between24and27◦C(Almeida and Santana, 1999).Brazilian Green Dwarf from Jiqui(JGD)is cultivated in a hot and dry environment in Rio Grande do Norte State. Temperatures are high(maximum of33◦C and minimum of 23◦C),pluviometric precipitation is low(<650mm in1year)0098-8472/$–see front matter©2007Elsevier B.V.All rights reserved. doi:10.1016/j.envexpbot.2007.08.006196F.P.Gomes et al./Environmental and Experimental Botany62(2008)195–204and average air relative humidity is69%(Carmo Filho et al., 1991).Coconut is frequently exposed to soil and atmospheric drought because it is a perennial palm with long productive life span(Rajagopal and Kasturi Bai,2002;Gomes et al.,2002). Short-term responses of coconut to water stress such as low stomatal conductance to water vapour(g s)and leaf water poten-tial(ΨW)with negative consequences for the net photosynthesis rate(A)and transpiration rate(E)have been demonstrated (Repellin et al.,1994,1997;Rajagopal and Kasturi Bai,2002; Passos et al.,1999).However,the contribution of non-stomatal factors to the impairment of A,mainly during the drought recov-ery period,has not been investigated.Therefore,it is important tofind useful characteristics such as drought recovery ability for use in drought tolerance breeding programs and coconut production in drought-prone areas.Drought stress effects on photosynthesis have been well demonstrated.Consistent information has been reported on water deficit effects on the relative stomatal and non-stomatal limitations to photosynthesis during stress conditions(Escalona et al.,1999;Flexas and Medrano,2002;Flexas et al.,2002; Lawlor,2002;Lawlor and Cornic,2002;Tezara et al.,2002; Grassi and Magnani,2005;Monteiro and Prado,2006;Zhou et al.,2007).However,most of these works have focused on stress responses such as soil moisture depletion.Studies concerning photosynthetic rate recovery from drought stress exist and most of them show incomplete recovery of photosynthesis immedi-ately after rewatering(Calbo and Moraes,2000;Oliveira et al., 2002;Souza et al.,2004;Miyashita et al.,2005;Sarker et al., 2005).However,the contribution of stomatal and non-stomatal factors to photosynthesis impairment and its incomplete recov-ery upon rewatering has not been fully determined and has therefore been debated for a long time(Lawlor and Cornic,2002; Galm´e s et al.,2007).Stomatal closure is often considered as an early physiological response to water deficit,which results in decreased A,through limited CO2availability in the mesophyll(Cornic,2000).How-ever,there is strong evidence that photosynthetic processes in the mesophyll such as Rubisco activity,RuBP regeneration,ATP supply,electron transport rate(J)and light capture efficiency in the photosystems are impaired as water stress increases(Flexas and Medrano,2002;Lawlor,2002;Lawlor and Cornic,2002; Grassi and Magnani,2005;Zhou et al.,2007).Growing plants with a constant level of water stress is an artificial situation because in nature the soil water con-tent continually changes.This can be observed in humid climates,where the soil water content and the water poten-tial decreases steadily after a rain until the soil is rewetted by another rain(Kramer and Boyer,1995).Thus,the present study aimed to determine the photosynthetic limitations of two Brazilian Green Dwarf coconut ecotypes during the recov-ery phases of simulated drying/recovery cycles in the soil. Three hypotheses were tested:(1)non-stomatal factors con-tribute to photosynthetic impairment in plants recovering from drought stress;(2)photochemical impairment is an important component of the non-stomatal limitations to photosynthe-sis;(3)the two coconut ecotypes are distinct with relation to drought recovery behavior,in accordance with their geographic distribution.2.Materials and methods2.1.Plant material and experimental conditionsTwo Brazilian Green Dwarf ecotypes were used in the experiments:Brazilian Green Dwarf from Una(UGD)and Brazilian Green Dwarf from Jiqui(JGD).UGD is cultivated in the Una municipality of southeastern Bahia State(15◦15 S, 39◦05 W,105m asl),whose predominant climate is the A f type of the K¨o ppen climatic classification system.It is a hot and humid environment,with annual pluviometric precipita-tion above1300mm,homogeneously distributed during the year and without a dry season(McKnight and Hess,2004). JGD is cultivated in the Rio Grande do Norte State(05◦47 S, 35◦12 W,30m asl),a contrasting semi-arid hot climate of the B sh type of the K¨o ppen system.This type has a defined and severe dry season,low pluviometric precipitation and average annual temperature of27.5◦C(McKnight and Hess,2004). The plants were cultivated in150L pots in a soil/sand mix-ture(1:2),under greenhouse conditions at the Universidade Federal de Vic¸osa,Brazil(20◦45 S,42◦52 W,648m asl).The average air temperature and solar irradiance inside the green-house were measured daily with a non-linear thermistor and a LI-200SA pyranometer sensor,respectively.Both sensors were connected to a datalogger LI-1200Data Set Recorder(LI-COR Inc.,USA).Relative humidity was measured with a“harp”of human hair sensor type thermohygrograph calibrated weekly using a ventilated wet bulb psycrometer.The plants were irri-gated daily for12months to maintain the soil water content close tofield capacity.All essential nutrients were supplied, following the leaf and soil analyzes and the specific crop require-ments(Magat,2003).The drought treatment was initiated when the plants were1year old by suspending the irrigation until A approached zero,which was considered the point of maxi-mum stress(PMS).Stressed plants were then rewatered for8 days.This procedure was repeated two times,giving a total of three successive drying/recovery cycles.Twenty plants(12 stressed and8well-watered controls)were organized in a com-pletely randomized design for each ecotype.The plants of each ecotype were split into two unequal groups since a great variation of measured values in drought stressed plants was expected,in spite of the rigorous selection of the plants.The term cycles(1–3)was used to indicate the4th day after rewa-tering.2.2.Leaflet water statusAt the point of maximum stress and in the middle of the recovering phase(after rewatering for4days),pre-dawn leaf water potential(ΨPD)was measured at4:30a.m.in one leaflet per plant of four to six randomly selected plants per treatment with a pressure chamber Skye SKPM1400 (Skye Instruments Ltd.,UK).All measurements were done on leaflets opposite to those used for gas exchange andfluo-F.P.Gomes et al./Environmental and Experimental Botany62(2008)195–204197rescence measurements,following the methodology described by Milburn and Zimmermann(1977).The base of the lam-ina was trimmed with a sharp knife after detaching the leaflet to free the cylindrical midrib.The leaflet was then rolled (keeping the adaxial surface outermost to avoid breaking the rib)and sealed into the pressure chamber.The pressure was increased slowly(0.02MPa s−1)with compressed nitro-gen until the sap was visible at the exposed base of the midrib.2.3.Gas exchange measurementsThe leaf gas exchange was evaluated4days after rewa-tering using attached leaflets at the middle of the rank two leaf(counting from the top,taking the spindle as zero) of three tofive different plants randomly selected at each time from each treatment.The measurements were performed from8:30to11:ing a Portable Photosynthesis Sys-tem LI-6400(LI-COR Biosciences Inc.,Nebraska,USA) equipped with an artificial irradiance source6400-02B Red-Blue.Light response curves were created with eight levels of pho-tosynthetic photonflux density(PPFD)(0,50,100,200,400, 800,1200and1600␮mol photon m−2s−1)in decreasing order, with2–3min intervals between each reading.The CO2flux was adjusted to maintain a concentration of380␮mol mol−1inside the chamber.The CO2response curves were measured under a PPFD of 1200␮mol photon m−2s−1,since it was preliminarily shown to be higher than the photosynthesis light saturation intensity for all measured leaves.Different CO2concentrations were obtained from the12g CO2cartridges and automatically controlled by the LI-COR6400CO2injector device(6400-01CO2injec-tor,LI-COR,USA).The measurement sequence initiated at a 50␮mol mol−1in chamber CO2concentration,increasing to 100,200,380,600,800and1000␮mol mol−1at3–4min inter-vals(Escalona et al.,1999).These rapid A/C i curves were chosen since the objective was to measure the CO2-driven changes in A and not in g s,whose response would be slower.The values of A were stable(CV<1%)3–4min after each in chamber CO2 concentration change.The intercellular CO2concentration(C i)was calculated from the A,g s and E values(von Caemmerer and Farquhar,1981). The intercellular to atmospheric CO2concentration ratio(C i/C a) was calculated from the C i and C a values of the A versus PPFD curves when PPFD>800␮mol photon m−2s−1.The leaf chamber(6cm2)temperature was maintained at26◦C and the leaf-to-air water vapour pressure deficit(VPD)was on average 1.0±0.2kPa during the measurements.The relative stomatal limitation(L S)was calculated from A versus C i curves,as L S=100×(A0−A)/A0,where A and A0 are the photosynthetic rate at ambient C a(≈380␮mol mol−1) and at C i=C a,respectively(Farquhar and Sharkey,1982). The relative mesophyll limitation(L m)was calculated as L m=100×(A c−A S)/A c,where A c and A S are CO2-saturated A(C i=800␮mol mol−1)of control and stressed leaves,respec-tively(Jacob and Lawlor,1991).2.4.Chlorophyll afluorescence measurementsInstant light response curves offluorescence were mea-sured simultaneously on the same attached leaflets(n=3–5)used for the gas exchange measurements,using the light-curveprogram of the Mini PAMfluorometer(Walz,Effeltrich,Ger-many)(Rascher et al.,2000).The leaflets where dark adaptedfor30min before the light curve runs,when the actinic lightintensity was increased from0to1600␮mol m−2s−1in ninesteps of30s.Apparent electron transport rate of PSII(J)was estimated as J=ΦPSII×PPFD×a×0.5,whereΦPSII is the effective quantum yield of PSII and a is the fraction ofincident PPFD absorbed by the leaflet,assumed to be0.84(Ehleringer,1981)and the factor0.5assumes equal excitationof both PSI and PSII(Laisk and Loreto,1996).ΦPSII was cal-culated as(F m−F/F ,where F is thefluorescence yield of the light-adapted sample and F m is the maximum light-adaptedfluo-rescence yield when a saturating light pulse of800ms of duration (intensity>5000␮mol m−2s−1)is superimposed on the prevail-ing environmental light levels.The maximum quantum yield of PSII,[F v/F m=(F m−F0)/F m)]was calculated using the so called dark-adapted parameters F0and F m(obtained after the first saturating pulse of the light curves)where F0is the basalflu-orescence yield measured after the illumination of a dark adapted sample with a measuring light(<0.1␮mol m−2s−1,λ>650nm) and F m is the maximumfluorescence yield of a dark adapted sample obtained following a saturating light pulse(Roh´a cek, 2002).2.5.Leaflet pigments concentrationConcentration of pigments per area was determined in dimetylsulphoxide(DMSO)extracts of leaflet discs(col-lected from the leaflet used for gas exchange measurements) following Hiscox and Israelstam(1979),with some modi-fications.After the incubation of three leaf discs(0.5cm) with5mL of CaCO3-saturated DMSO at65◦C for48h,the absorbance of extracts was read in a Hitachi U-2000double-beam spectrophotometer(Hitachi Instruments Inc.,Danbury, CT),and the total chlorophyll and carotenoids were calculated using the equations proposed by Wellburn(1994)for DMSO extracts.2.6.Modeling and statisticsNon-linear regression for exponential equations was used to estimate the photosynthetic parameters.For A versus PPFD curves the following equation was used(Iqbal et al.,1997):A=A maxPAR1−exp−αPPFDA maxPAR−R d,where A maxPAR is the light-saturated photosynthetic rate,αthe apparent quantum yield and R d is the dark respiration rate(esti-mated as the y-intercept of a linear regression of thefirst points of A versus PPFD curves).That exponential model was recently demonstrated as suitable for this purpose in dwarf coconut (Gomes et al.,2006).For A versus C i curves the following198F .P .Gomes et al./Environmental and Experimental Botany 62(2008)195–204Table 1Average (S.E.)maximum (T max ),minimum (T min )and mean (T mean )air temper-ature,solar irradiance (SI)and relative humidity daily values measured inside the greenhouse during the drying/recovery cyclesCycle 1Cycle 2Cycle 3T max (◦C)30.8(0.5)a 31.4(0.3)a 29.2(0.4)b T min (◦C)19.4(0.2)a 19.2(0.2)a 17.3(0.2)b T mean (◦C)23.7(0.2)a 23.8(0.2)a 21.8(0.2)b SI (MJ m −2)7.5(0.4)a 7.0(0.2)a 4.3(0.2)b RH (%)76.7(1.2)a73.7(0.8)a73.5(1.4)aMeans followed by the same letter are not different by Tukey’s test (P ≤0.05)equation was used (Escalona et al.,1999):A =A maxCO 2×(1−exp(−k ×(C i −ΓCO 2)),where A maxCO 2is the CO 2-saturated photosynthetic rate,k a constant of proportionality and ΓCO 2is the compensation point for CO 2.Light respiration rate (R L )and carboxylation effi-ciency (ε),respectively the y -intercept and the slope of A versus C i ,were estimated using a linear regression of the first points of the A versus C i curves (Escalona et al.,1999).The equa-tion used for J versus PPFD was the same as A versus PPFD,where A and A maxPAR were replaced by J and J max ,respec-tively (Rascher et al.,2000).The photosynthetic parameters were compared using a linear model identity test (Cunia et al.,1973),whose procedure was extrapolated for non-linear mod-els.For each individual replicate,the compensation irradiance values (Γlight ,x -intercept),estimated using a linear regres-sion of the first points of the A versus PPFD (Escalona et al.,1999),ΨPD ,L m ,L S ,pigments,F 0and F v /F m were submit-ted to an ANOV A and,when indicated,to the Tukey’s mean comparison test,at 5%probability.All mathematical proce-dures were performed using the software Statistica (Statsoft Inc.).3.ResultsThe drought period lasted 44,35and 35days in cycles 1–3,respectively.Except the relative humidity,all the microclimatic variables inside the greenhouse were reduced in cycle 3,as compared to cycles 1and 2(Table 1).3.1.Gas exchange and water relationsThe progressive soil water depletion led to decreases in ΨPD to average values of −1.22±0.04MPa at the point of maximum stress (Fig.1).Upon rewatering,the mean ΨPD value increased to −0.15±0.01MPa on the 4th day of recovery,although the values of the stressed plants were still significantly lower (more negative)in cycles 1and 2,when compared to the irrigated plants (Fig.1).There were not significant differences in ΨPD between ecotypes.A and g s ,measured at the ambient CO 2concentration (C a =380␮mol mol −1)and saturating irradiance (PPFD =1200␮mol m −2s −1)had not completely recovered 4days after rewatering (Table 2).A and g s of UGD water-stressed plants were respectively 37and 58%lower than their controls in cycle 1(Table 2).In JGD,these values were 43and 68%,respectively.In JGD the effect of drought stress on A was pro-gressively reduced with the imposition of successive cycles.This was indicated by the decrease in water-stressed plants compared to well-watered ones.The genotypic difference was even clearer in g s .While the reduction in g s was nearly constant in UGD (58,41and 53%for cycles 1–3,respectively),a marked increase in the speed of g s recovery was observed in JGD after the suc-cessive drying/recovery cycles (68,47and 19%reduction for cycles 1–3,respectively),the differences between the control and stressed not being significant in cycle 3.Despite the low g s ,only small C i /C a reductions (<8%)were observed,which were significant for UGD in cycle 3and for JGD in cycles 1and 2(Table 2).3.2.PPFD and CO 2response curve parametersThe A maxPAR and J max values were significantly lower in stressed plants after 4days of rewatering.A reduction of 27and 17%was obtained in cycle 1and 14and 11%in cycle 3for UGD and JGD,respectively (Table 3).A progressively minor effect of the imposed drying/recovery cycles was observed for J max in the two ecotypes,but the values of the stressed plants still were significantly reduced (46%in UGD and 32%in JGD)at the end of the experiment (Table 3).αwas significantly reduced in the stressed UGD plants in the three cycles,but the effect of drought was also progressively reduced with the imposition of the other cycles (Table 3).Fig.1.Pre-dawn leaf water potential measured in well-watered (WW)and water-stressed (WS)plants of Brazilian Green Dwarf coconut from Una (UGD)and Jiqui (JGD)at the point of maximum stress (PMS)and on the 4th day of recovery (R).Bars represent means (n =4–6)and *significant (P ≤0.05)difference between WW and WS,by the F -test.Error bars represent the S.E.F.P.Gomes et al./Environmental and Experimental Botany62(2008)195–204199 Table2Net photosynthesis(A),stomatal conductance(g s)and intercellular to ambient CO2concentration(C i/C a),measured under saturating irradiance (PPFD=1200␮mol m−2s−1)and ambient CO2concentration(C a=380␮mol mol−1)in two Green Dwarf coconut ecotypes cultivated under well-watered conditions (R WW)and on the4th day of recovery from three drying/recovery cycles(R WS)Cycle1Cycle2Cycle3R WW R WS%R WW R WS%R WW R WS% A(␮mol m−2s−1)UGD11.8(0.4)a7.4(0.5)b3714.0(0.3)a11.5(0.4)b1813.1(0.1)a9.9(0.4)b24 JGD12.3(0.5)a7.0(0.4)b4313.8(0.4)a10.9(0.4)b2112.9(0.2)a11.8(0.2)b9 g s(mol m−2s−1)UGD0.26(0.02)a0.11(0.01)b580.34(0.06)a0.20(0.02)b410.45(0.02)a0.21(0.03)b53 JGD0.28(0.02)a0.09(0.01)b680.34(0.02)a0.18(0.02)b470.36(0.01)a0.29(0.03)a19 C i/C aUGD0.75(0.01)a0.68(0.02)a90.76(0.03)a0.71(0.01)a70.82(0.01)a0.74(0.02)b10 JGD0.76(0.01)a0.63(0.01)b170.77(0.01)a0.68(0.02)b120.78(0.02)a0.77(0.02)a1 These are mean(S.E.)values from three tofive replicates.UGD,Brazilian Green Dwarf from Una;JGD,Brazilian Green Dwarf from Jiqui;%,reduction percentage compared to control.For each variable and ecotype,different letters indicate significant(P≤0.05)differences between R WW and R WS,by the F-test.Only small,non-significantαreductions were observed inJGD.Non-significant reductions in R d were observed for thetwo ecotypes,although a marked R d decrease(from6%in cycle1to54%in cycle3)was observed in stressed JGD plants withthe imposition of the repeated cycles(Table3).ΓPAR was notaffected by the drying/recovery treatments,although a decreas-ing trend was also observed,which was more pronounced inJGD than in UGD(Table3).The water deficit led to significant A maxCO2reductions inthe two ecotypes(Table4).With the imposition of the cycles,the A maxCO2reductions in the recovering plants decreased from35to18%in JGD but stayed at26–29%in UGD(Table4).εalso did not recover completely,but significant reductions were observed in cycle1,for the two ecotypes,and in cycle3for UGD.Significant drought effects were observed on R L in UGD during cycle1.There were not significant water deficit effects onΓCO2for the two ecotypes(Table4).3.3.Stomatal versus non-stomatal limitationsL S ranged from16to19%in irrigated UGD plants and from 16to18%in JGD ones(Table5).Non-significant L S increases were observed in stressed JGD plants in all three cycles(Table5). From cycles1to3,L m decreased from32to19%in JGD,butTable3Light response curves parameters estimated for two Green Dwarf coconut ecotypes cultivated under well-watered conditions(R WW)and on the4th day of recovery from three drying/recovery cycles(R WS)Cycle1Cycle2Cycle3R WW R WS%R WW R WS%R WW R WS% A maxPAR(␮mol m−2s−1)UGD14.9(0.2)aA10.9(0.3)bA2715.2(0.2)aA12.7(0.2)bA1615.2(0.2)aA13.0(0.2)bA14 JGD13.8(0.2)aB11.4(0.2)bA1714.2(0.2)aB11.4(0.2)bB2014.3(0.2)aB12.7(0.2)bA11α(␮mol mmol−1)UGD54.6(2.0)aA35.6(1.8)bA3554.2(1.9)aA42.9(1.7)bA2155.0(2.0)aA45.4(1.9)bA17 JGD54.5(1.8)aA49.9(1.9)aB847.8(2.0)aB42.1(2.1)aA1253.9(1.9)aA50.6(2.0)aA6 R d(␮mol m−2s−1)UGD0.77(0.14)aA0.72(0.14)aA60.80(0.16)aA0.57(0.14)aA290.60(0.15)aA0.46(0.15)aA23 JGD0.64(0.18)aA0.60(0.15)aA60.62(0.27)aA0.34(0.23)aA450.74(0.18)aA0.34(0.21)aA54ΓPAR(␮mol m−2s−1)UGD16.1(1.7)aA21.7(3.2)aA+3516.7(1.4)aA15.1(2.1)aA1013.4(3.0)aA10.7(2.2)aA20 JGD13.9(0.2)aA13.8(1.3)aA115.1(1.8)aA11.5(5.0)aA2415.4(2.5)aA10.5(0.9)aA32 J max(␮mol m−2s−1)UGD94.2(2.3)aA42.2(1.8)bA55104.1(2.9)aA82.8(2.4)bB20104.3(2.6)aA56.3(1.5)bB46 JGD94.8(3.4)aA42.0(1.9)bA56106.7(1.6)aA87.9(1.5)bA18110.2(4.0)aA75.2(2.2)bA32 These are mean(S.E.)values from three tofive replicates.A maxPAR,light-saturated photosynthetic rate;αapparent quantum yield;R d,dark respiration rate;ΓPAR, light compensation point;J max,light-saturated apparent electron transport rate;UGD,Brazilian Green Dwarf from Una;JGD,Brazilian Green Dwarf from Jiqui;%, reduction or increment(+)percentage compared to control.For each parameter,lower case letters indicate significant differences between R WW and R WS within each ecotype and capital letters indicate significant differences among ecotypes within each treatment,by the F-test(P≤0.05).200F.P.Gomes et al./Environmental and Experimental Botany62(2008)195–204Table4CO2response curves parameters estimated for two Green Dwarf coconut ecotypes cultivated under well-watered conditions(R WW)and on the4th day of recovery from three drying/recovery cycles(R WS)Cycle1Cycle2Cycle3R WW R WS%R WW R WS%R WW R WS%A maxCO2(␮mol m−2s−1)UGD19.0(0.5)aB14.1(0.5)bA2623.5(0.7)aA16.7(0.5)bB2921.8(0.5)aA15.4(0.3)bB29 JGD24.3(0.8)aA15.7(0.9)bA3524.6(0.9)aA20.8(0.7)bA1520.9(0.5)aA17.1(0.5)bA18ε(mmol m−2s−1/cm3m−3)UGD53.2(3.3)Ab41.1(2.7)bA2364.1(4.1)aA54.1(4.1)aA1659.9(2.0)aA52.2(2.0)bA13 JGD66.5(3.7)aA47.7(5.0)bA2867.2(3.4)aA65.5(3.7)aA365.0(5.3)aA60.0(5.1)aA8 R L(␮mol m−2s−1)UGD 3.65(0.34)aA 2.67(0.30)bA27 3.57(0.42)aA 3.33(0.43)aA7 3.38(0.22)aA 3.01(0.21)aA11 JGD 4.01(0.39)aA 2.81(0.51)aA30 3.94(0.36)aA 3.97(0.38)aA+1 3.66(0.57)aA 3.52(0.53)aA4ΓCO2(␮mol mol−1)UGD71.2(3.5)aA68.1(4.7)aA458.4(3.7)aA65.3(3.7)aA+1259.8(3.2)aA60.9(3.0)aA+2 JGD62.1(4.7)aA63.2(7.2)aA+260.5(3.8)aA61.7(3.9)aA+259.5(4.1)aA61.0(3.8)aA+3These are mean(S.E.)values from three tofive replicates.A maxCO2,CO2-saturated photosynthetic rate;ε,carboxylation efficiency;R L,light respiration rate;ΓCO2,CO2compensation point;UGD,Brazilian Green Dwarf from Una;JGD,Brazilian Green Dwarf from Jiqui;%,reduction or increment(+)percentage compared to control.For each parameter,lower case letters indicate significant differences between R WW and R WS within each ecotype and capital letters indicate significant differences among ecotypes within each treatment,by the F-test(P≤0.05).did not vary(18–23%)in UGD.The ecotype JGD showed a significantly higher L m in cycle1when compared to UGD,the inverse being observed for the other two cycles(Table5).3.4.Pigments,F v/F m and F0Drought stress led to reductions in the leaflet chlorophyll (Chl)concentration per unit area,which were only signifi-cant for UGD(Fig.2).Significant Chl content reduction was observed for JGD in cycle2.Except for UGD in cycle3,only small non-significant drought stress effects were observed on carotenoids(Fig.2).The average F v/F m for all measurements was0.83±0.01in the control plants.Significant reductions of this parameter were observed in the stressed plants of the two ecotypes in cycles1and2.The F0values increased in the stressed plants of the two ecotypes,although significant differences were observed only in cycle1(Fig.2).4.DiscussionConsidering the small changes in the microclimatic variables from cycles1to2,their different duration may be attributed to the incomplete recovery of g s and to a faster stomatal closure with the imposition of the next cycle.On the other hand,the signifi-cant air temperature and solar irradiation decreases from cycles 2to3(Table1)should have been responsible for a larger cycle 3duration.However,A approached zero at the same time in the two cycles,which may be due to the direct effect of high residual abscisic acid(ABA)levels on the guard-cells,to drought-related changes in the guard-cell function and in the hydraulic conduc-tivity,which are only slowly reversed,or to a combination of these factors(Tardieu et al.,1996;Comstock,2002;Sperry et al.,2002;Buckley,2005;Miyashita et al.,2005).The leaflet ABA concentration was shown to increase in drought stressed as well as in drought recovering coconut plants(Gomes,2006), which could explain the incomplete g s recovery.In water-stressed plants,a rapid increase of A upon rewatering indicates that the basis for both biochemical and photochem-ical mechanisms is not impaired by drought,leading to the conclusion that the decreases in A are the result of stomatal clo-sure(Cornic,2000).The present results showed that A and g s were not fully recovered after4days of rewatering,despite the almost complete recovery of C i/C a(Table2).Even though stom-atal closure seemed to be the main factor limiting A in youngTable5Relative stomatal(L S)and mesophyll(L m)limitation to photosynthesis in two Green Dwarf coconut ecotypes cultivated under well-watered conditions(R WW)and on the4th day of recovery from three drying/recovery cycles(R WS)Cycle L S(%)L m(%)UGD JGD UGD JGDR WW R WS R WW R WS116.8(1.2)aA16.2(2.0)aA18.2(1.1)aA22.0(0.4)aA23.5(1.3)B31.9(0.3)A 219.4(1.2)aA19.2(0.6)aA18.1(0.9)aA19.0(0.4)aA18.1(0.6)A11.0(1.2)B 315.5(0.4)aA17.4(1.1)aA16.4(0.5)aA19.6(0.8)aA23.4(0.6)A18.7(0.8)B These are mean(S.E.)values from tow to three replicates.UGD,Brazilian Green Dwarf from Una;JGD,Brazilian Green Dwarf from Jiqui;lower case letters(only for L s)indicate significant differences between R WW and R WS within each ecotype and capital letters indicate significant differences among ecotypes within each treatment,by the F-test(P≤0.05).。