CSIRO PUBLISHING

www.publish.csiro.au/journals/fpb Functional Plant Biology, 2007, 34, 204–213

Photosynthetic responses of three C4 grasses of different metabolicsubtypes to water deficit

Ana E. Carmo-SilvaA,B,C,D, Ana S. SoaresA,C, Jorge Marques da SilvaA, Anabela Bernardes da SilvaA,Alfred J. KeysB and Maria Celeste ArrabacaA

ACentro de Engenharia Biologica and Departamento de Biologia Vegetal, Faculdade de Ciencias da Universidadede Lisboa, Campo Grande, 1749-016 Lisbon, Portugal.

BCrop Performance and Improvement Division, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK.CThese authors contributed equally to this work.DCorresponding author. Email: elizabete.carmo-silva@bbsrc.ac.uk

Abstract. C4 plants are considered to be less sensitive to drought than C3 plants because of their CO2 concentratingmechanism. The C4 grasses, Paspalum dilatatum Poiret (NADP-ME), Cynodon dactylon (L.) Pers (NAD-ME) and Zoysiajaponica Steudel (PEPCK) were compared in their response to water deficit imposed by the addition of polyethyleneglycol to the nutrient solution in which they were grown. The effects of drought on leaf relative water content (RWC), netphotosynthesis, stomatal conductance, carboxylating enzyme activities and chlorophyll a fluorescence were investigated.In C. dactylon the RWC was more sensitive, but the photosynthetic activity was less sensitive, to water deficit thanin P. dilatatum and Z. japonica. The decrease of photosynthesis in P. dilatatum under water deficit was not closelyrelated to the activities of the carboxylating enzymes or to chlorophyll a fluorescence. However, decreased activities ofribulose 1,5-bisphosphate carboxylase/oxygenase and phosphoenolpyruvate carboxylase, in addition to decreased stomatalconductance, may have contributed to the decrease of photosynthesis with drought in C. dactylon and Z. japonica. Thedifferent responses to water deficit are discussed in relation to the natural habitats of C4 grasses.

Additional keywords: C4 plants, drought, NADP-ME, NAD-ME, PEPCK.

Introduction

Temperature and water availability are two of the main factorsthat affect plant survival, productivity and distribution (Hatch1992). Global climate change is expected to increase thetemperature and aridity of many areas of the world (Petit et al.1999), and, hence, a knowledge of plant responses to water deficitis increasingly important. The more efficient use of water byC4 relative to C3 plant species (Long 1999) may make themmore competitive than C3 species under water deficit conditions(Sage et al. 1999). About one-quarter of the primary productivityon the planet and a large fraction of the primary productionconsumed by humans is derived from C4 crops and pasturegrasses (Sage 2004).

Decreased net photosynthetic rate (A) is generally an earlyeffect of decreased water availability. The decrease in A canbe the result of decreased CO2 supply as a result of stomatalclosure or of decreased efficiency of the photochemical andbiochemical processes (Lawlor and Cornic 2002). The relativecontribution of these mechanisms to the decrease of A underwater deficit has been much debated in the literature. Many ofthe studies on the response of photosynthesis to water deficithave been performed in C3 plants and suggest that stomatalclosure is primarily responsible for decreased A under moderate

water deficit, whereas metabolic or non-stomatal factors aremore important under severe stress conditions (Lawlor andCornic 2002). The stomatal limitations to photosynthesis in C4

plants may be less important than in C3 plants owing to theirability to concentrate CO2 in organelles and tissues containingRubisco (EC 4.1.1.39). Lal and Edwards (1996) showed thatin the NAD-malic enzyme (NAD-ME) species Amaranthuscruentus L., and in the NADP-malic enzyme (NADP-ME)species Zea mays L., photosynthesis was not affected undermoderate water deficit although the stomatal conductance (gs)decreased. However, under severe water deficit, when the gs

was very low, the same authors found that the CO2 availablestarted to limit photosynthesis. Du et al. (1996) showed thatboth stomatal and non-stomatal effects caused decreased A insugarcane (NADP-ME) immediately after the imposition of thestress, but under severe drought metabolic limitations becamerelatively more important. Ghannoum et al. (2003) showed thatthe decrease in A was independent of ambient CO2 concentrationin two NADP-ME and two NAD-ME species suggesting thatmetabolic limitations were involved. Furthermore, Saccardyet al. (1996) for Z. mays, and Marques da Silva and Arrabaca(2004a) for Setaria sphacelata (Stapf) Clayton, both NADP-ME C4 species, showed that the contribution of stomatal

© CSIRO 2007 10.1071/FP06278 1445-4408/07/030204

Photosynthetic responses of C4 species to water deficit Functional Plant Biology 205

and non-stomatal limitations to the decrease in photosynthesisdepended on whether the stress was imposed rapidly or slowly. InZ. mays, the main contribution to the decrease of photosynthesisunder a slowly imposed stress was stomatal closure, whereasin a rapidly imposed stress the limitations were mainly non-stomatal (Saccardy et al. 1996). Conversely, Marques da Silvaand Arrabaca (2004a) found that in S. sphacelata non-stomatallimitations became more important when a slow dehydrationwas imposed, and stomatal limitations were more relevant whenstress was imposed rapidly.

The response of the activities of the carboxylatingenzymes Rubisco and phosphoenolpyruvate carboxylase (PEPC,EC 4.1.1.31) to water deficit is also a matter of controversy. Adecrease in PEPC activity with stress was observed in several C4

plants (Du et al. 1996; Marques da Silva and Arrabaca 2004b),but Foyer et al. (1998) found increased activity of this enzymein maize plants under moderate water deficit and Saccardyet al. (1996) found no change in the activity. Rubisco activitydecreased linearly with drought in sugarcane (Du et al. 1996),but in A. cruentus and maize plants, a decrease in activity wasonly observed under severe drought (Lal and Edwards 1996).Marques da Silva and Arrabaca (2004b) found an increase inRubisco activity in S. sphacelata plants when the dehydrationwas imposed slowly, but a decrease in activity in rapidly-droughted plants.

It is generally accepted that the photochemical apparatusof plants suffering from moderate water deficit is relativelyresistant to damage but is more susceptible under severe droughtconditions, especially at high irradiance (Havaux 1992; Lawlor2002; Lawlor and Cornic 2002). Studies on both C4 and C3

plants suggest that electron transport capacity can be in excessof the requirement for CO2 assimilation under water deficitconditions and that alternative electron sinks must becomeavailable (e.g. Loreto et al. 1995; Lu and Zhang 1998; Tezaraet al. 1999; Ghannoum et al. 2003).

The present study was undertaken to investigate the effect ofdrought on photosynthesis by three C4 grasses with differentdecarboxylating mechanisms: Paspalum dilatatum cv. Raki(NADP-ME), Cynodon dactylon var. Arizona Common (NAD-ME) and Zoysia japonica var. Zenith (phosphoenolpyruvatecarboxykinase, PEPCK). C. dactylon and Z. japonica are widelyused in golf courses (Beard 1982), whereas the major use forP. dilatatum is in pastures (Brown 1999). Water deficit wasrapidly induced (20–26 h) by the addition of polyethylene glycol4000 (PEG4000) to the nutrient solution. The responses to droughtby the three C4 grasses with respect to leaf water content, leafgas exchanges, Rubisco and PEPC activities, and chlorophyll afluorescence were investigated. It seemed likely that there may bedifferent responses to water stress in the three C4 subtypes. Thesedifferences might be related to the natural habitat in which eachsubtype originated and may give indications about their mostappropriate use for sports turf or pasture in dry regions.

Materials and methodsPlant material and water deficit inductionPlants of Paspalum dilatatum Poiret cv. Raki (NADP-ME),Cynodon dactylon (L.) Pers var. Arizona Common (NAD-ME) and Zoysia japonica Steudel var. Zenith (PEPCK)

were grown in a controlled environment chamber undera PPFD of 500 µmol m−2 s−1, a photoperiod of 16 h andtemperatures of 25/18◦C (day/night). Light was providedby Halolux Ceram 64476KL incandescent lamps, FluoraL58W/77 and L30W/77 fluorescent lamps and Powerstar HQI-BT-400W/D sodium vapour lamps, all from Osram (OsramGmbH, Munich, Germany). Seeds were soaked in deionisedH2O at room temperature for 1 h, and sown on 8–16 mmdiameter expanded clay balls (‘growrocks’, Maxit – ArgilasExpandidas, SA, Avelar, Portugal) almost covered with 2 Lof deionised H2O in 3-L pots. After germination, four seedlingswere kept per pot and the deionised water was replaced withnutrient solution, containing 6 mM KNO3, 6 mM Ca(NO3)2,1.5 mM MgSO4, 1 mM NaH2PO4, 10 µM MnSO4, 1 µM CuSO4,1 µM ZnSO4, 50 µM H3BO3, 100 µM NaCl, 50 nM (NH4)6MO7O24

and 110 µM Fe-EDTA (Hewitt 1966). The nutrient solution wasreplenished as needed to the top layer of the expanded clay ballsand completely renewed once a week.

Water stress was induced by including various concentrationsof PEG4000 in the nutrient solution supplied to the plants.The water potential (�w) of the nutrient solutions wasdetermined with a C-52 chamber connected to a HR33 DewPoint microvoltmeter (Wescor Inc., Logan, UT, USA). Astandard curve was established with NaCl solutions of differentconcentrations. Stress was induced 6 weeks after sowing forP. dilatatum and C. dactylon, and 12 weeks after sowing forZ. japonica. Gas-exchange and fluorescence measurements weremade near the beginning of the light period 20–26 h after theinduction of stress. The leaf relative water content (RWC) ofeach sample was determined according to Catsky (1960), by theequation RWC = 100 (FW – DW)/(TW – DW), where FW, DWand TW are the fresh, dry and turgid weight, respectively.

Gas-exchange measurementsGas exchanges were measured at the widest part of young fullyexpanded leaves, equidistant from the two ends, of control anddehydrated plants. Measurements of net photosynthetic rate(A), transpiration rate (E) and stomatal conductance to watervapour (gs) were made using an LCA-2 portable infrared gasanalyser fitted with a PLC(B) broad leaf Parkinson chamber(The Analytical Development Co. Ltd, Hoddesdon, UK). Themeasurements were made on the plants inside the growthchamber at ambient CO2 concentration (370 ± 20 µL L−1), aPPFD of 500 µmol m−2 s−1, at 25 ± 2◦C and a relative humidityof 35–40%. After each measurement, the fresh weight and thearea of leaf enclosed were determined (portable area meter LI-3000, Li-Cor Inc., Lincoln, NE, USA). The net photosyntheticrate was calculated according to von Caemmerer and Farquhar(1981) and the transpiration rate according to Long and Hallgren(1985, 1993). The instantaneous water use efficiency (WUE) wascalculated as A/E.

Rubisco and PEPC activitiesLeaf samples similar to those used for in vivo measurements,were collected at the same time as gas exchanges were measuredand quickly frozen. The frozen samples were added to a coldmortar containing quartz sand, 1% (w/v) Polyclar AT and 20or 30 mL g−1 tissue of ice-cold extraction medium containing50 mM HEPES-KOH pH 7.3, 1 mM EDTA, 5% (w/v) PVP25, 6%

206 Functional Plant Biology A. E. Carmo-Silva et al.

(w/v) PEG4000, 10 mM DTT, 1% (v/v) protease cocktail inhibitor(Sigma, St Louis, MO, USA) and 0.5% (v/v) Triton X-100.After grinding to produce a fine suspension the homogenatewas centrifuged for 20 s at 15 800g at room temperature. Thesupernatant was used immediately for measuring the activitiesof Rubisco and PEPC. Rubisco initial activity (Vi) was assayedat 25◦C by 14CO2 incorporation into acid-stable products bythe method of Parry et al. (1997) with some modifications. Thereaction was started by the addition of 50 µL of extract to 550 µLof assay medium containing 50 mM HEPES-KOH pH 8.0, 30 mM

MgCl2, 10 mM NaH14CO3 (0.05 µCi µmol−1) and 0.5 mM RuBP(Sigma). The reaction was stopped after 60 s by the additionof 100 µL of 2 N HCl. The mixture was completely dried at60◦C. The residue was resuspended in 1 mL of distilled waterand mixed with 8 mL of scintillation liquid (OptiPhase ‘HiSafe’3, Fisher Chemicals, Loughborough, UK). Radioactivity due to14C in the acid-stable products was measured by scintillationcounting (LS 7800, Beckmann Instruments Inc., Fullerton, CA,USA). PEPC physiological activity (Vphysiol) was measuredspectrophotometrically (UV500, Unicam Ltd., Cambridge, UK)in a continuous assay at 340 nm and 25◦C according toBakrim et al. (1992) with some modifications. The reactionmixture (1 mL) consisted of 50 mM HEPES-KOH pH 7.2,10 mM MgCl2, 10 mM NaHCO3, 2.5 mM PEP (Sigma), 12units of MDH (Sigma) and 20 µL extract. The reactionwas started by the addition of 0.2 mM (final concentration)NADH (Sigma).

Each value presented for the activities of Rubisco and PEPCis the mean of three replicate measurements on the sameextract.

Chlorophyll a fluorescence measurementsChlorophyll a fluorescence measurements were made on themiddle part of a young fully expanded leaf. Whenever necessary,leaves of Z. japonica were uncurled before the measurement.Photochemical parameters were measured at room temperature(25◦C) and at the ambient atmospheric concentration of CO2

with a pulse amplitude modulation fluorometer (PAM-210,Heinz Walz GmbH, Effeltrich, Germany) operated by DA-TEACH 1.01 software, 1997 (Heinz Walz GmbH). Leaves werefirst dark-adapted for 4 min. The minimum fluorescence levelwas determined with a measuring light of 0.04 µmol m−2 s−1.The maximum fluorescence level was measured on dark-adaptedleaves by a saturating light pulse of 4000 µmol m−2 s−1 for 0.9 s.The leaf was then continuously illuminated for 5 min with lightof 590 µmol m−2 s−1. The steady-state value of fluorescencewas recorded and a second saturating pulse was imposed todetermine the maximum fluorescence level in the light-adaptedstate. The actinic light was then removed and the minimumfluorescence level in the light-adapted state was determined after3 s illumination with far-red light. The maximum photochemicalefficiency of PSII reaction centres of dark-adapted leaves(Fv/Fm) was calculated according to Kitajima and Butler(1975), the effective quantum yield of PSII electron transport(�PSII) according to Genty et al. (1989), and the photochemicalquenching coefficient (qP) and non-photochemical quenchingcoefficient (qN) according to Schreiber et al. (1986). For qP andqN calculations the minimum fluorescence level was taken intoaccount as proposed by Horton and Bowyer (1990).

Statistical analysisRegression analysis was applied to assess the relationshipbetween pairs of parameters with either the Statistical Packagefor Social Sciences (SPSS) 12.0, 2003 (SPSS Inc., Chicago,IL, USA) or the Statistical Package GenStat 8.2, 2005(Lawes Agricultural Trust, Rothamsted Experimental Station,Harpenden, UK). Models were selected that best explainedthe results on the basis of an F test of probability (P < 0.05)(Zhar 1996). Because of the greater variability obtained on thedata relating instantaneous WUE to RWC for P. dilatatum, noregression was applied but grouped data were compared witha T-test, with the SPSS program (P < 0.05). All the absolutevalues and percentages presented in the text were calculated fromregression equations presented with each figure.

Results

Water relations

There was a second-degree polynomial relationship between thewater potential (�w) of the nutrient solution and the PEG4000

concentration (Fig. 1). Up to a PEG concentration of 25% (w/v)the �w decreased by only 1 MPa but from 25 to 40% the furtherdecrease was almost 5 MPa. Therefore, the concentration of PEGwas not a useful basis for indicating the imposed water stress.Fig. 2 shows that the leaf RWC of each of the three grass specieswas differently affected by the water potential of the nutrientsolution. A small decrease in �w (from −0.05 to −1.5 MPa)led RWC values to decrease to 35% in C. dactylon but onlyto 78% in P. dilatatum and 86% in Z. japonica. For lower �w

values a gradual decrease of RWC was found for P. dilatatumand Z. japonica, but for C. dactylon the RWC appeared to reacha minimum at −2 MPa. At the lowest water potential (−5 MPa)C. dactylon had the lowest RWC and Z. japonica the highest. Theleaf RWC was used as a reference to compare the physiologicalresponses to water stress of the three grasses.

The three species showed different morphological responsesto water deficit. The leaves of P. dilatatum and C. dactylon wiltedand shrank when RWC started to decrease, whereas the leaves ofZ. japonica curled longitudinally. In all three species there wasa decrease in leaf area with stress (data not shown), which was

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Fig. 1. Effect of different PEG4000 concentrations on the water potentialof the nutrient solution (�w) (R2 = 0.989). Each point corresponds to themean ± s.e. of three independent samples.

Photosynthetic responses of C4 species to water deficit Functional Plant Biology 207

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Fig. 2. Effect of nutrient solution water potentials (�w) on the leaf relativewater content (RWC) of Paspalum dilatatum (◦, y = 29.66 + 79.29 e0.332x ,R2 = 0.741), Cynodon dactylon (�, y = 29.51 + 145.9 e2.147x , R2 = 0.852)and Zoysia japonica (N , y = 95.45 + 6.52 x, R2 = 0.627). Results aremean ± s.e. of 4–46 independent samples.

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y = 2.867 + 0.062 x y = –0.630 + 0.076 x

Fig. 3. Net photosynthetic rate (A) (a–c), stomatal conductance to water vapour (gs) (d–f) and instantaneous water use efficiency (WUE) (g–i) atdifferent leaf relative water contents (RWC) in the leaves of Paspalum dilatatum (a, R2 = 0.658; d, R2 = 0.641; g), Cynodon dactylon (b, R2 = 0.818;e, R2 = 0.529; h, R2 = 0.189) and Zoysia japonica (c, R2 = 0.720; f, R2 = 0.633; i, R2 = 0.186). Measurements were performed at 25 ± 2◦C, atatmospheric concentrations of CO2 and O2 and at a PPFD of ∼500 µmol m−2 s−1. Each data point represents a different sample.

greatest for Z. japonica because of the longitudinal curling. Dueto the difficulty in determining leaf area accurately in Z. japonica,all the parameters were expressed on a DW basis.

Gas-exchange parameters

The net photosynthetic rate (A, Fig. 3a–c) in fully hydratedleaves was higher in C. dactylon (around 50 µmol min−1 g−1

DW) than in P. dilatatum and Z. japonica (around30 µmol min−1 g−1 DW). A decreased nearly to zero inP. dilatatum and Z. japonica as the RWC fell to 60%, but thedecrease was less pronounced in C. dactylon. A decreased by69% in P. dilatatum, 51% in Z. japonica and by just 27% inC. dactylon, as the RWC decreased from 100 to 80%. At a RWCnear 60%, C. dactylon leaves still had a relatively high value of A(around 25 µmol min−1 g1 DW) and showed net photosynthesiseven below 40% RWC.

The stomatal conductance to water vapour (gs, Fig. 3d–f )showed a similar behaviour to A in response to stress. InP. dilatatum and Z. japonica gs decreased to a low value at60% RWC, but in C. dactylon gs decreased less steeply and wasmeasurable even below an RWC of 35%. In the range of RWC

208 Functional Plant Biology A. E. Carmo-Silva et al.

from 100 to 80%, gs decreased by 74% in P. dilatatum, 43% inZ. japonica and only 23% in C. dactylon. There was a linearrelationship between A and gs in C. dactylon and Z. japonica,whereas in P. dilatatum a curvilinear relationship was obtained(Fig. 4).

Instantaneous WUE decreased linearly with RWC inC. dactylon and Z. japonica (Fig. 3h, i) but more steeply inthe latter species. For P. dilatatum instantaneous WUE valuesappeared to form two groups, one from 100 to 85% and a secondfrom 80 to 75% RWC (Fig. 3g). The average instantaneous WUEvalue was higher in the first group (6.8 ± 1.98 µmol CO2 mmol−1

H2O) than in the second (2.8 ± 1.91 µmol CO2 mmol−1 H2O). Inall three species there was a decrease in WUE with the increasein water stress shown by the decrease in RWC.

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Fig. 4. Net photosynthetic rate (A) v. stomatal conductance to watervapour (gs) for the leaves of Paspalum dilatatum (a, R2 = 0.676), Cynodondactylon (b, R2 = 0.649) and Zoysia japonica (c, R2 = 0.672) submittedto different levels of water deficit. Measurements were performed at25 ± 2◦C, atmospheric concentrations of CO2 and O2 and a PPFD of∼500 µmol m−2 s−1. Each data point represents a different sample.

Rubisco and PEPC activities

The highest values for Rubisco initial activity (Vi, Fig. 5a–c)and PEPC physiological activity (Vphysiol, Fig. 5d–f ) wereobserved in fully hydrated and moderately stressed leaves ofC. dactylon.

Paspalum dilatatum Rubisco Vi was not affected by waterdeficit (Fig. 5a), whereas in C. dactylon and Z. japonica Vi

decreased linearly with drought (Fig. 5b, c). With a decreasein RWC from 100 to 80%, Rubisco Vi decreased 18% in bothC. dactylon and Z. japonica. PEPC Vphysiol was not affectedby water deficit in P. dilatatum and C. dactylon (Fig. 5d, e),but decreased in Z. japonica at RWC values lower than 70%(Fig. 5f ).

Chlorophyll a fluorescence

There was a slight decrease in the maximum photochemicalefficiency of PSII reaction centres of dark-adapted leaves(Fv/Fm) of P. dilatatum and C. dactylon (Fig. 6a, b), but notfor leaves of Z. japonica (Fig. 6c), as the RWC fell below 60%.In all three species a similar value for Fv/Fm (close to 0.8) wasobserved in fully hydrated leaves.

The effective quantum yield of PSII electron transport (�PSII,Fig. 7a–c) and the photochemical quenching coefficient (qP,Fig. 7d–f ) were little affected in the range of RWC from100 to 70%, but decreased at lower RWC. No significantvariation (P > 0.05) with leaf RWC was observed for the non-photochemical quenching coefficient (qN, Fig. 7g–i) in any ofthe three species. In fully hydrated leaves, qN was ∼0.7 inC. dactylon and ∼0.8 in P. dilatatum and Z. japonica.

Discussion

Water relations and photosynthetic CO2 assimilation

The relationship between the concentration of PEG4000 and thenutrient solution �w (Fig. 1) was described by a second-degreepolynomial regression, as previously reported by Lawlor (seeSlavık 1974) and Money (1989). Since the concentration ofPEG4000 was not linearly related to the water potential of thenutrient solutions used to cause water stress, it was not useddirectly as a measure of the imposed water deficit. Leaf RWC isa good indicator of plant water status (Chaves 1991; Lawlor andCornic 2002) and was used as a measure of the severity of waterstress.

Plants respond to a decrease of water availability eitherby avoiding tissue dehydration or by tolerating low tissuewater potentials (Mundree et al. 2002; Chaves et al. 2003). Themechanisms involved in the avoidance of tissue dehydration arethe closure of stomata and the reduction of light absorbance,for example, by rolling the leaves or increasing reflectance bythe presence of a dense trichome layer. Tolerance of low waterpotentials may involve osmotic adjustment, more rigid cell wallsor smaller cells (Chaves et al. 2003). The higher values of RWCin Z. japonica compared with P. dilatatum and C. dactylonas water potential decreased (Fig. 2) may be explained by thepresence of trichomes on the leaves (data not shown) and theobserved leaf curling. The RWC of C. dactylon decreased to amuch lower value than in the other two species and in responseto a small decrease in the water potential of the nutrient solution(Fig. 2). The relationship of stomatal conductance to water

Photosynthetic responses of C4 species to water deficit Functional Plant Biology 209

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Fig. 5. Rubisco initial activity (Vi) (a–c) and phosphoenolpyruvate carboxylase (PEPC) physiological activity (Vphysiol) (d–f) at different leaf relativewater contents (RWC) in the leaves of Paspalum dilatatum (a, d), Cynodon dactylon (b, R2 = 0.686; e) and Zoysia japonica (c, R2 = 0.453; f, R2 = 0.760).Each data point represents a different sample.

deficit measured by RWC (Fig. 3d–f ) shows that the stomata ofP. dilatatum are the most sensitive and those of C. dactylon leastsensitive to water deficit. The photosynthetic rates (Fig. 3a–c)show that C. dactylon retains active metabolism even at severewater deficit. The higher values of instantaneous WUE (Fig. 3h),together with higher values of gs, found in C. dactylon areconsistent with the lower sensitivity of A in this species to waterdeficit. It is concluded that P. dilatatum avoids tissue dehydrationby stomatal closure, Z. japonica avoids dehydration partly bystomatal closure and partly by protection against excessiveirradiance by trichomes and rolling its leaves, but C. dactylontolerates water deficit.

Instantaneous WUE decreased in C. dactylon and Z. japonica(Fig. 3h, i) with decreased RWC because the transpiration rate(and gs) was relatively less sensitive to stress than A. A similarresult was obtained by Marques da Silva and Arrabaca (2004a)for S. sphacelata under rapidly and slowly induced water stressbut the decrease was greater with rapidly induced stress. Marocoet al. (2000) found that instantaneous WUE was not changedby water stress in leaves of two Sahelian C4 grasses. Fig. 3gshows that the data for instantaneous WUE in P. dilatatum canbe best considered in two groups with respect to RWC from 100to 85% and from 80 to 75%. The decrease in the mean value ofthe first group to the second group was related to the very lowvalues of A found in severe water deficit conditions (Fig. 3a).C. dactylon always showed the highest values of instantaneousWUE. Ghannoum et al. (2002) found that nine NAD-MEAustralian C4 grasses had higher WUE than nine NADP-MEspecies.

Biochemical and photochemical limitationsto photosynthesisDecreased stomatal aperture has been recognised as one of theearliest responses to drought (e.g. Flexas and Medrano 2002;Chaves et al. 2003) and can occur even before changes in RWC(Majumdar et al. 1991; Zhang et al. 2001). Marques da Silvaand Arrabaca (2004a) concluded that with rapidly induced waterdeficit stomatal closure was mainly responsible for the decreaseof A in S. sphacelata (NADP-ME). The regression best fittingthe data for A v. gs in P. dilatatum (Fig. 4a) shows that stomatalclosure did not decrease photosynthesis in the early stage ofwater deficit down to a RWC of 93%. Below this threshold, therewas a progressive and similar decrease in gs and A with stressintensity. The linear decrease of A with decreased gs observedin C. dactylon and Z. japonica (Fig. 4b, c) is consistent witha close correlation between the two parameters. However, therelatively greater sensitivity of A than gs to RWC, especiallyin these two species, indicates that non-stomatal as well asstomatal limitations to photosynthesis may be involved. Similarconclusions for sugarcane (NADP-ME) were reached by Duet al. (1996).

The decrease of Rubisco Vi with drought in C. dactylon andZ. japonica (Fig. 5b, c) was consistent with the observations ofDu et al. (1996) and Marques da Silva and Arrabaca (2004b) fora rapidly imposed water deficit and may have contributed to thedecreased A with decrease in RWC. In P. dilatatum there wasno significant trend in Rubisco activity that could contribute tothe decrease in A. Decreased PEPC Vphysiol under severe waterdeficit could be a factor among the metabolic limitations to

210 Functional Plant Biology A. E. Carmo-Silva et al.

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y = 0.289 + 0.013 x – 0.0001 x2

y = 0.060 + 0.019 x – 0.0001 x2

Fv/F

m

RWC (%)

Fig. 6. Maximum photochemical efficiency of PSII reaction centres ofdark adapted leaves (Fv/Fm) at different leaf relative water contents (RWC)in the leaves of Paspalum dilatatum (a, R2 = 0.645), Cynodon dactylon(b, R2 = 0.765) and Zoysia japonica (c). Each data point represents adifferent sample.

photosynthesis in Z. japonica (Fig. 5f ). A decrease in PEPCactivity with water deficit was also found by Du et al. (1996)and Marques da Silva and Arrabaca (2004b). PEPC activity wasnot significantly affected by drought (P > 0.05) in P. dilatatumand C. dactylon (Fig. 5d, e). A similar conclusion for maize wasreached by Saccardy et al. (1996).

Chlorophyll a fluorescence measurements showed that thephotochemical reactions may be involved in the limitations ofA, especially in P. dilatatum and C. dactylon, under severewater deficit. The maximum efficiency of energy capture byopen PSII reaction centres (estimated by the Fv/Fm ratio) inP. dilatatum and C. dactylon was not affected at RWC valuesdown to 70% (Fig. 6a, b). Similar conclusions have been reached

for C3 (for review see Lawlor 2002; Lawlor and Cornic 2002and Chaves et al. 2003) and for C4 plants (Saccardy et al. 1998;Ghannoum et al. 2003; Marques da Silva and Arrabaca 2004a).There was no effect on Fv/Fm in Z. japonica even at RWCvalues down to 55% (Fig. 6c), indicating greater resistance ofthe photochemical apparatus to drought than in the other twospecies. Jagtap et al. (1998) found that Fv/Fm ratio in C4 specieswas strongly affected by water stress. Chaves (1991) has pointedout that the discrepancies in the literature for chlorophyll afluorescence results might be explained by secondary effectsof other stresses, such as the light intensity.

�PSII (Fig. 7a–c) decreased only for RWC values below70–80%. Similar results were reported by Lal and Edwards(1996) for other C4 plants, whereas Ghannoum et al. (2003)and Marques da Silva and Arrabaca (2004a) observed a declineof �PSII even with small decreases in RWC. Unlike theimmediate decrease of A with decrease in RWC (Fig. 3a–c),�PSII (Fig. 7a–c) remained almost constant, suggesting thatunder moderate stress alternative electron sinks must beavailable to dissipate the excess of electrons not used in carbonassimilation. This was the conclusion also for other C4 plantsunder water deficit conditions (Loreto et al. 1995; Lal andEdwards 1996; Ghannoum et al. 2003). Electron transfer to O2

through photorespiration and the Mehler-ascorbate reaction (e.g.Cornic and Briantais 1991; Havaux 1992; Biehler and Fock1996) have been proposed as possible mechanisms by whichexcess of photochemical energy can be dissipated to maintainthe high quantum yield of PSII electron transport. However,photorespiration is slow in unstressed C4 plants.

The value of �PSII depends on the efficiency of the primaryphotochemical reaction in open PSII reaction centres and onqP (Genty et al. 1989). The similar response to water stress ofqP and �PSII in the three species (Fig. 7), suggests that �PSII

decreased mainly as a result of the closure of PSII reactioncentres. The decrease in qP with RWC values below 70–80%(Fig. 7d–f ), indicates an increase in the reduction state of the firstelectron acceptor of the PSII reaction centre, QA, under severedrought conditions. The results for qP (Fig. 7) are similar to thoseby Lawlor and Cornic (2002) for C3 plants but in the NADP-MEC4 plants Sorghum bicolor (Loreto et al. 1995) and S. sphacelata(Marques da Silva and Arrabaca 2004a) qP decreased even withmoderate water deficit.

An increase of qN with water deficit is frequently referred toin the literature for C3 plants (e.g. Lu and Zhang 1999; Tezaraet al. 1999). With the C4 plant S. sphacelata, either an increasedor decreased qN with moderate water deficit was observeddepending on the way stress was imposed (Marques da Silvaand Arrabaca 2004a). Conversely, water stress did not affect qNin P. dilatatum, C. dactylon or Z. japonica (Fig. 7g–i) despitethe decrease of A with RWC (Fig. 3a–c) and the decrease of qPand �PSII for RWC values lower than 70–80% (Fig. 7). Thus,alternative electron sinks must dissipate the excess excitationenergy in the three species during water deficit to compensatefor decreases in A and �PSII.

Results in relation to the reported occurrence of C4subtypes in natural habitats

The different responses of C4 species to drought reported inthe literature can be associated with interspecific variation and

Photosynthetic responses of C4 species to water deficit Functional Plant Biology 211

0.0

0.2

0.4

0.6

0.8

1.0

0.0

0.2

0.4

0.6

0.8

1.0

0.0

0.2

0.4

0.6

0.8

1.0

30 40 50 60 70 80 90 100 30 40 50 60 70 80 90 100 30 40 50 60 70 80 90 100

y = –0.422 + 0.024 x – 0.0001 x2y = –0.701 + 0.036 x – 0.0002 x2y = –1.309 + 0.047 x – 0.0003 x2

y = –0.786 + 0.027 x – 0.0002 x2y = –0.988 + 0.035 x – 0.0002 x2y = –0.897 + 0.031 x – 0.0002 x2

Paspalum dilatatum Cynodon dactylon Zoysia japonicaΦ

PS

IIq

Nq

P

RWC (%) RWC (%) RWC (%)

ba c

d e f

g h i

Fig. 7. Effective quantum yield of PSII electron transport (�PSII) (a–c), photochemical quenching coefficient (qP) (d–f) and non-photochemicalquenching coefficient (qN) (g–i) at different leaf relative water contents (RWC) in the leaves of Paspalum dilatatum (a, R2 = 0.660; d, R2 = 0.780;g), Cynodon dactylon (b, R2 = 0.471; e, R2 = 0.423; h) and Zoysia japonica (c, R2 = 0.211; f, R2 = 0.237; i). Measurements were obtained underan actinic light intensity of 590 µmol m−2 s−1. Each data point represents a different sample.

also with the way experiments are undertaken. We believethat these differences may also be related to the C4 subtypesand their distribution. Hattersley (1992) showed that plantsbelonging to the metabolic subtypes NADP-ME and PEPCKpredominate in areas of Australia with good rainfall, whereasNAD-ME species are more prevalent in drier sites. A study ofC4 grasses in the USA (Taub 2000) revealed that the distributionof the Panicoideae family (e.g. P. dilatatum) was positivelycorrelated with the precipitation gradient, whereas plants fromthe Chlorideae family (e.g. C. dactylon and Z. japonica) werenegatively correlated with the precipitation gradient and existedin more arid areas. Photosynthetic CO2 assimilation (Fig. 3) byC. dactylon (NAD-ME) was much less sensitive to decreasedRWC than by P. dilatatum (NADP-ME) or Z. japonica (PEPCK)when water stress was rapidly imposed. Furthermore, A inZ. japonica was less sensitive than in P. dilatatum to droughtstress. Thus, the lower photosynthetic sensitivity of C. dactylonto water deficit could be related to the distribution of NAD-ME species in arid environments and the higher photosynthetic

sensitivity of P. dilatatum may be related to the presence ofNADP-ME species in areas with better precipitation levels. Theresponse of Z. japonica to water stress was intermediate betweenthat of C. dactylon and P. dilatatum. To understand the relativeimportance of family as opposed to C4 metabolic subtype indetermining resistance to water stress would require a moreextensive survey.

Conclusions

Paspalum dilatatum and Z. japonica responded to water deficitby closing their stomata at an early stage to maintain leafhydration whereas C. dactylon lost water rapidly (decreasein RWC) but was able to maintain higher values of A,gs and instantaneous WUE at low RWC. Photosynthesis byP. dilatatum was the most affected by rapidly imposed waterstress and was associated with decreased stomatal conductance.Decrease in Rubisco activity may contribute to the decrease ofphotosynthesis by C. dactylon and Z. japonica with decreased

212 Functional Plant Biology A. E. Carmo-Silva et al.

RWC but PSII photochemistry could only contribute in severedrought. C. dactylon is the most resistant of the threespecies to rapidly imposed water stress and P. dilatatum isleast resistant.

Acknowledgements

This work was partially supported by ‘Programa de DesenvolvimentoEducativo para Portugal’ (PRODEP III) and by ‘Federacao Portuguesa deGolfe’. The authors thank Dr GL Lockett, Margot Forde Forage GermplasmCentre, New Zealand, for providing the seeds of P. dilatatum and Dr DanielRibeiro, Geodesenho, Portugal, for providing the seeds of C. dactylonand Z. japonica. The authors thank Ms Manuela Lucas of the Centro deEngenharia Biologica, Faculdade de Ciencias da Universidade de Lisboa,Campo Grande, 1749–016 Lisbon, Portugal, for technical assistance; andDr Stephen J Powers of the Biomathematics and Bioinformatics Division,Rothamsted Research, Harpenden, Herts., AL5 2JQ, UK, for advice onnon-linear modelling. Note: Ana E. Carmo-Silva and Ana S. Soares havecontributed equally to the work presented.

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Manuscript received 31 October 2006, accepted 29 January 2007

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