Diurnal variation of CO O and evaporative site …eco.ibcas.ac.cn/group/lingh/siep/pdf/StaleIsotopesyueduw...neer, Piper aduncum,on two different days in Trinidad during February 1995

ABSTRACT

Concurrent measurements of gas exchange, instanta-neous isotope discrimination (∆) against 13CO2 andC18O16O, and extent of 18O enrichment in H2O at theevaporative sites, were followed in a tropical forest pio-neer, Piper aduncum, on two different days in Trinidadduring February 1995. ∆13CO2 differed from that pre-dicted from measurements of internal:external CO2 con-centration (Ci/Ca) and showed a wide range of valueswhich decreased throughout the course of the day.Derivation of Cc (the CO2 concentration at the carboxy-lation site) was not possible using carbon isotope dis-crimination under field conditions in situ and wasderived assuming a constant value of internal transferconductance (gw). Under low rates of assimilation thederived Cc/Ca, like Ci/Ca, remained relatively stable overthe course of both days and ∆C18O16O followed evapora-tive demand. Lower values of ∆C18O16O on day 2occurred in response to the indirect effect of increasedleaf-to-air vapour pressure deficits (VPD) and reducedstomatal conductance. For the first time, direct determi-nation of the δH2

18O of transpired water vapour (δt)allowed derivation of evaporative site enrichment with-out the prerequisite of isotopic steady state (ISS) definedin the Craig and Gordon model. Generally, δt was lessenriched than the source water (δs) in the morning andmore enriched in the afternoon, which would be pre-dicted from an increase and decrease in ambient VPD,respectively. On both days, leaves of P. aduncumapproached ISS (indicated where δt ≈ δs) between 1300and 1500 h. Evaporative site enrichment was maintainedinto the late afternoon, despite a decrease in ambientVPD. The data presented provide a greater insight intothe natural variation in isotopic discrimination underfield conditions, which may help to refine models of ter-restrial biome discrimination.

Key-words: Piper aduncum; 13C; instantaneous isotope discrim-ination; leaf gas exchange; 18O; Trinidad; water vapour.

INTRODUCTION

Plant processes serve a unique role as integrators and indica-tors of global environmental change (Yakir et al. 1993).Isotopic analysis of atmospheric 13CO2 (Mook et al. 1983;Broadmeadow et al. 1992; Ciais et al. 1995), C18O16O(Francey & Tans 1987; Farquhar et al. 1993; Ciais & Meijer1997; Ciais et al. 1997) and H2

18O (Salati et al. 1979; Jacob& Sonntag 1991) has suggested that the terrestrial biosphereexerts a substantial influence on global cycles of CO2 andwater. Attention has, therefore, focused on the mechanismscontrolling fractionation in biological systems. Intuitively,terrestrial photosynthetic gas exchange will provide a largebiospheric contribution to global biogeochemical cycles,along with soil respiration and aquatic photosynthesis. Inorder to assess any contribution by terrestrial photosynthesis,we must first understand the processes governing fractiona-tion and the natural variation in biological discrimination.

During photosynthetic gas exchange, a combination ofdiffusive and enzymatic discrimination against the heavier13C leaves plant material depleted in the heavier isotope rel-ative to atmospheric CO2 (Farquhar, Ehleringer & Hubick1989). Additionally, through rapid exchange between CO2

and water in the chloroplast, the 18O leaf water isotope sig-nal is transferred to atmospheric CO2 (Francey & Tans1987; Farquhar & Lloyd 1993; Yakir et al. 1994) and pro-vides a non-destructive probe to investigate terrestrial–atmospheric exchanges of CO2 and water vapour (Farquharet al. 1993; Yakir & Wang 1996). The magnitude of dis-crimination expressed by the leaf is a result of both leafwater enrichment and the diffusional resistance of CO2

from ambient air to the chloroplast (Farquhar & Lloyd1993; Flanagan et al. 1994). The latter is represented by theexpression Cc/Ca, the concentration of CO2 within thechloroplast of a leaf relative to that in ambient air, and is akey intracellular parameter which represents leaf photosyn-thetic gas exchange and is important for interpreting oxy-gen and carbon isotope discrimination of CO2 duringphotosynthesis. Estimation of Cc is possible by analysingthe difference between observed instantaneous carbon iso-tope discrimination and that predicted from concurrentmeasurements of gas exchange [∆i – ∆obs; Evans et al.(1986); von Caemmerer & Evans (1991)].

Plant, Cell and Environment (1998) 21, 269–283

© 1998 Blackwell Science Ltd

Diurnal variation of ∆13CO2, ∆C18O16O and evaporative site

enrichment of δH218O in Piper aduncum under field conditions

in Trinidad

K. G. HARWOOD,1 J. S. GILLON,1 H. GRIFFITHS1 & M. S. J. BROADMEADOW2

1Department of Agricultural and Environmental Science, Ridley Building, University of Newcastle-upon-Tyne, Newcastle-upon-Tyne, NE1 7RU, UK, 2Forestry Commission, Forest Research Agency, Alice Holt Lodge, Farnham, Surrey GU10 4LH, UK

ORIGINAL ARTICLE OA 220 EN

Correspondence: K. G. Harwood. Fax: (44) 191 2225229; e-mail:[email protected]

269

Measurements of photosynthetic response and isotopediscrimination under steady-state conditions are necessaryto gain an understanding of the interaction between physio-logical and biochemical processes that occur during photo-synthetic gas exchange. Because response times of gasexchange vary with light and water stress, the natural vari-ation in environmental conditions under some field situa-tions may mean that photosynthetic steady state is rarelyachieved. Stomatal conductances can take 20 min toachieve steady state following changes in irradiance(Barradas & Jones 1996) and have been reported to takebetween 20 and 40 min for Piper auritum and P. aequale(Tinoco-Ojanguren & Pearcy 1992). However, field datahave shown that despite large shifts in A and gs, Ci/Ca canremain relatively constant throughout the day [seeTenhunen et al. (1984), Wise et al. (1991) and referencestherein] from which simple discrimination models wouldpredict a constant ∆13CO2 (Farquhar et al. 1989). This sug-gests that steady-state discrimination may occur withoutthe prerequisite of steady-state photosynthesis.

However, initial observations of diurnal changes ininstantaneous discrimination for P. aduncum (a tropicalpioneering tree) under field conditions in Trinidad revealedlarge variability in the measured discrimination signalcompared with that predicted (M. S. J. Broadmeadow,unpublished field observations; Gillon et al. 1997). Recentlaboratory work has suggested that under low rates ofassimilation, changes in (photo)respiratory CO2 evolutioncan affect the on-line signal (Gillon & Griffiths 1997).Models predicting discrimination are essential for under-standing both net and gross exchanges of CO2 between thebiosphere and the atmosphere. However, whether theobserved oxygen and carbon discrimination in CO2 fol-lows that predicted, despite large changes in A and gs underfield conditions, has not been extensively investigated for13CO2 and only under laboratory conditions for C18O16O.

Additionally, estimation of 18O leaf water enrichment atthe sites of evaporation (δe) is important in analysing iso-topic leaf water budgets. Using the Craig & Gordon (1965)model of evaporative enrichment for hydrological systems,the enrichment of water at the evaporating surfaces of aleaf can be expressed as the proportion of H2

18O evapo-rated relative to H2

16O (White 1988; Flanagan, Comstock& Ehleringer 1991a; see Appendix A).

Whilst the model is useful for estimating the degree ofenrichment at the evaporation sites (δe), it is, by derivation,dependent on the leaf being at isotopic steady state (ISS),the point at which transpired water vapour has the sameoxygen isotope composition as the stem water. The timefor leaf water to attain ISS is proportional to leaf waterturnover rate [defined as the ratio of leaf water volume totranspiration rate, V/E: Farris & Strain (1978); Flanagan,Bain & Ehleringer (1991b); Wang & Yakir (1995)] and themagnitude of the change in environmental conditions thatthe leaf is exposed to (Flanagan et al. 1991b; Yakir et al.1994). Under transient environmental conditions, we can-not always assume that a leaf is at ISS (Wang & Yakir1995). Direct measurement of the δ18O of transpired water

vapour during photosynthetic gas exchange would, there-fore, allow: (i) the identification of proximity to ISS; (ii) anestimation of evaporative site enrichment irrespective ofISS; and (iii) provide a greater insight into the range ofδ18O water vapour signal transferred to the surroundingenvironment, which may have important implications forscaling up of water transfer from leaf to canopy to atmo-sphere.

The objectives of this investigation were to follow pho-tosynthetic gas exchange, discrimination against 13CO2

and C18O16O along with the extent of evaporative siteenrichment in H2

18O, under field conditions for a decidu-ous rainforest in Trinidad. In particular, the natural fluctua-tions throughout the day were examined, irrespective ofwhether steady-state photosynthetic gas exchange or iso-tope discrimination were attained. From this we hoped toascertain whether steady-state photosynthesis was a pre-requisite for steady-state isotopic discrimination, as well asevaluating whether discrimination models reliably predictdiscrimination, and therefore Cc, under natural conditionsin the field.

MATERIALS AND METHODS

Experiments were conducted in the field, at the AsaWright, Simla Research Station, Trinidad [see Borlandet al. (1993) for a full site description] on 2 d in February1995. An elevated bamboo platform was constructed sothat the upper leaves of the P. aduncum canopy (thoseexposed to full sunlight) could be reached. Leaves selectedwere fully expanded and located at the same height andposition within the canopy. To gain a representative sampleof gas exchange and discrimination characteristics, randomleaves were used on day 1 (8 February 1995). On day 2 (16February 1995), the same three leaves were measuredthrough the day to account for any effects of leaf-to-leafvariation.

Additionally, three leaves were collected for δ18O leafwater determination, at 0930 and 1230 h on the first dayand placed into Exetainers (Europa Scientific, Crewe,UK). Spring water was collected from a ground waterspring within 0·5 km of the field site. Rain water was col-lected in a rain gauge, positioned in a clearing, fitted with afunnel to minimize evaporation and the oxygen isotopecomposition was analysed for each major precipitationevent, throughout the field campaign. Volumes of 1 cm3 ofcollected rain or spring water were injected into pre-evacu-ated Exetainers (Europa Scientific, Crewe, UK).

Photon flux and temperature

Instantaneous photon flux density (PFD) was followedthroughout the day using the sensor attached to theportable infra red gas analysis system (IRGA) used for gasexchange measurements (see below). Measurements oftotal daily PFD were also recorded using an integratinglight meter (Delta T, Cambridge, UK). Shade air tempera-ture and relative humidity measurements were taken with

270 K. G. Harwood et al.

© 1998 Blackwell Science Ltd, Plant, Cell and Environment, 21, 269–283

the dew point humidity sensor (Protimeter, Marlow, UK)throughout the course of both days.

Leaf water potential

Xylem sap pressure was measured using a pressure cham-ber to indicate leaf water potential (Soil MoistureEquipment Corporation, Santa Barbara, CA, USA) for day1 only (because of the limited nitrogen supply in theremote field location). Leaves were taken from the sameheight and position in the canopy over the day (n = 3).

Gas exchange

Gas exchange measurements were conducted by enclosing10–20 cm2 sections of leaf within a leaf cuvette attached toa CIRAS-1 portable IRGA (PP Systems, Hitchin, UK) onleaves throughout the day, under ambient light, CO2 con-centration and temperature. The leaf temperature was cal-culated using an energy balance calculation (Parkinson1983) and gas exchange parameters were recalculated forthe respective leaf areas. On day 1, a conifer PLC 3 head(ADC, Hoddeston, U.K.) was used, whilst in the hotterconditions on day 2 this was replaced with a broad-leafPLC (PP Systems, Hitchin, UK) attached with an externalcooling fan. The air supply was taken from a 5 dm3 buffer-ing volume ≈ 15 m upwind of the platform to provide a ref-erence supply with a stable CO2 concentration and isotopicsignature. The flow of air passed through the cuvetteranged between 350 and 430 cm3 min–1. Gas exchangemeasurements were taken immediately before and after the15 min on-line collection periods, and were averaged forthe collection time. The P. aduncum leaves were hypos-tomatous with an average total leaf area of 85 cm2. Meanstomatal density taken from five stomatal epidermalimpressions of three leaves was 193 mm–2. Leaf conduc-tance to water vapour was corrected for hypostomy(CIRAS manual, PP systems 1994). Differences betweenthe two types of cuvettes used were accounted for by cor-recting for boundary layer resistances of 0·45 and0·28 m2 s–1 mol–1 for days 1 and 2, respectively.

Collection of CO2 and water vapour for isotopeanalysis

Atmospheric CO2 and water vapour were collected using amodified glass collection line (Griffiths et al. 1990;Broadmeadow et al. 1992) attached to a rotary vacuumpump. The standard collection procedure was modified toenable separation of CO2 from water vapour for C18O16Oand H2

18O analysis. Air from either upstream (reference)or downstream (analysis) of the leaf cuvette was passed atpositive pressure to a metering valve (Swagelock, Ohio,USA). The atmospheric CO2 and water vapour weretrapped in a 6 mm internal diameter double spiral cold trap,immersed in liquid nitrogen, under a partial vacuum.Reference air was typically collected twice at the begin-ning and end of the day and on average after every third

analysis collection, to account for diurnal variations in theambient isotopic composition of canopy air, δa [Fig. 1, seealso Borland et al. (1993); Harwood (1997)].Cryogenically trapped CO2 and water vapour were isolatedand evacuated down to 10–2 mbar (10–3 kPa).

The CO2 was then liberated using acetone cooled to–70 °C (by addition of liquid nitrogen), for at least 2 minand condensed into a Pyrex side arm and sealed using abutane blow torch. As a precautionary measure, the sealedPyrex tube was immediately stored in a liquid nitrogen dryshipper (Jencons, Leighton Buzzard, UK). Should com-plete separation of the CO2 and H2O not have been

Carbon and oxygen isotope discrimination in Piper aduncum 271


Figure 1. Diurnal variation in (a) δa13CO2, (b) δaC

18O16O and (c)δaH2

18O of cuvette air supply on day 1 (l) and day 2 (●). Best fitpolynomial regressions were applied for both days from which theisotopic composition at the time of the analysis (δin) was derived.The fitted trend lines are represented by fine and thick lines for day1 and day 2, respectively. The CO2 reference at 1014 h on day 1(arrowed) was excluded from the regression as the internal massspectrometer precision of the sample was four times greater thanthat observed for all other samples.

achieved during sampling, cold storage prevented equili-bration (Harwood 1997) allowing the C18O16O signal tobe preserved.

A new Pyrex side arm was then fitted to the collectionline and evacuated. The water was liberated by heating thetrap using a butane blow torch and cryodistilled into thefreshly evacuated side arm until no more water wasobserved to condense in the side arm (this was usually inthe order of 2–3 min). An aliquot of CO2 was then intro-duced into the collection line, so that the δH2

18O could bemeasured through equilibration with CO2. This wasachieved by backfilling a gas tight syringe with CO2, ofknown isotopic signature from a modified commercialdrink carbonation cylinder and injecting 1 cm3, via a sep-tum fitting, into the collection line. The CO2 was thenpassed into the side arm, frozen above the water sampleand sealed in the vial using the blow torch.

Protocol for determination of C18O16O

Samples of CO2 were stored in liquid nitrogen untilpurification. Each sample was then taken individually,warmed to room temperature, wiped to remove any exter-nal moisture and placed in a bottle breaker attached to asample purification line. The transfer time from liquidnitrogen to breaking the sample tube under vacuum wastypically less than 2 min, so as not to allow equilibrationwith any residual water.

Technique development showed that small volumes ofCO2 inferred incomplete separation, or collection, of theCO2 and water vapour which lead to large outliers. Suchsamples were, therefore, rejected. This was validated inlaboratory trials leading to precision for separated CO2 of± 0·08‰ for δ13CO2 and ± 0·07‰ for δC18O16O (standarddeviations, n = 8) where isotope compositions areexpressed as δ using the per mille (‰) notation given by δ(‰) = [(Rsample/Rstandard) – 1] ×1000. For 13CO2, R = 13C/12Cand the standard is Pee Dee Belemnite (PDB) and forC18O16O, R = 18O/16O and the standard is CO2 derived fromPDB (PDB–CO2).

Protocol for determination of δH218O of water

vapour

Samples containing water and equilibration CO2 were leftfor a minimum of 3 d to equilibrate, after which the sam-pled was purified in accordance with the standard proce-dure. Corrections were made for equilibration temperatureand respective volume of gas and liquid according toScrimgeour (1995).

The volume of water vapour present in the side arm(involved in equilibration) was calculated from the flowrate of air passing through the collection line (the rate setby the needle valve), the duration of the collection, and thewater vapour concentration of the air supply as measuredby the CIRAS water vapour IRGAs attached to the collec-tion line. The average volume of water collected over15 min generally varied between 0·05 and 0·07 cm3,

depending on the ambient vapour pressure. Repro-ducibility of water vapour samples equilibrated with CO2

were typically ± 0·09‰ (n = 8) and were expressed in the δnotation relative to the water standard, standard meanocean water (SMOW).

Purification and mass spectrometric analysis ofon-line samples

All atmospheric CO2 and water equilibrated CO2 sampleswere purified by cryodistillation under vacuum on a purifi-cation line by passing the gas through two acetone traps atdry ice temperature (– 70 °C, as described in Griffiths et al.(1990) to remove any water and non-condensable gases.Samples were analysed using a dual inlet mass spectrome-ter modified to a VG 903 triple collector specification byProvac Services (Crewe, UK) and run using the EuropaIRMS (Crewe, UK) data collection software, against aworking standard of 99·995% CO2 (BDH High PurityGases, Poole, UK), with a δ13CO2 of – 43·18‰ and aδC18O16O of – 28·72‰ versus PDB–CO2.

Samples of CO2 were corrected for the presence ofN2O (as N2O has the same molecular mass, 44, as CO2).Mass spectrometer sensitivity to N2O was determinedusing the method described in Freidli & Siegenthaler(1988), whereby pure CO2 and N2O are mixed using massflow controllers to produce different CO2–N2O gas mix-tures (where ρ represents the ratio of the volume of N2Oto CO2) and admitted into the mass spectrometer. Thecorrections derived for the ratio of N2O:CO2 representingambient air (ρ = 0·00088, 310 p.p.b.:350 p.p.m., respec-tively) of + 0·24‰ for δ13C and + 0·31‰ for δ18O were inagreement with those observed in other investigations(Freidli & Siegenthaler 1988; Flanagan & Varney 1995).Diurnal variation in ambient N2O was considered as neg-ligible and, thus, corrections to CO2 were made using anN2O concentration of 310 p.p.b. and the respective CO2

concentration using the equations of Freidli &Siegenthaler (1988).

Source and rain water

Rain and source water were analysed using a direct equi-libration method (Scrimgeour 1995), whereby the wateris equilibrated with CO2 of known volume and isotopiccomposition. Samples were left for at least 3 d to equili-brate, after which they were run on the ANCA CF-IRMS(Europa Scientific, Crewe, UK). Working standards alsounderwent the same equilibration process as the samples,to account for any variation in sample preparation. Aftermass spectrometer analysis, samples were corrected fortheir respective volumes, δ18O of the equilibration CO2

and equilibration temperature. Reproducibility ofrepeated runs of the same sample generally yielded astandard deviation of ± 0·07‰ (n = 8). The oxygen iso-tope composition of all source, rain and leaf water (seebelow) samples were expressed in the δ notation relativeto SMOW.



δH218O Leaf water

The δH218O of the leaf water samples were kindly

analysed by Prof. D. Yakir and colleagues at the WeizmannInstitute, Rehovot, Israel. Leaf water was extracted viavacuum distillation for analysis as described in Wang &Yakir (1995). External precision for the water samples wasapproximately ± 0·2‰.

On-line discrimination of 13CO2 and C18O16O

Measured (observed) discrimination of 13CO2 (∆obs) wascalculated as described by Evans et al. (1986) below,whereby:

ξ (δout – δin) × 1000∆obs = –––––––––––––––––– , (1)

1000 + δo – ξ (δout – δin)

where ξ = ce/(ce – co) and δin, ce and δout, co are the isotopiccomposition and CO2 concentration entering and leavingthe cuvette, respectively. Values of co were corrected to thevapour pressure of ce to account for any dilution of CO2

leaving the cuvette from increased water vapour concen-tration. Background ambient CO2 typically varied between–9·5 and –8·5‰ δ13CO2 (Fig. 1a) whilst δC18O16O rangedfrom –0·5 to 0·5‰ (Fig. 1b). To ensure consistency in theselection of δin for measurements of concurrent discrimi-nation of 13CO2, C18O16O and H2

18O, values were interpo-lated from temporal polynomial regressions of thereference air isotope composition (δa) for both days at thetime of any given analysis (Fig. 1). The more conventionalmethod, expressing the isotope composition of ambient airas a function of the inverse of the concentration, could notbe applied as no relationship held with either δaC

18O16Oand 1/[CO2] or δaH2

18O and 1/[H2O], consistent with otherobservations within this canopy (Harwood 1997).

Instantaneous ‘on-line’ discrimination of C18O16O wasderived using the same equation as for ∆13CO2 (Farquhar& Lloyd 1993) by simply substituting the oxygen isotopecomposition of CO2 for δin and δout in Eqn 1.

Modelled discrimination of ∆13CO2 and ∆C18O16O

Modelled discrimination against 13CO2 (∆i) was derivedfrom the following equation (Evans et al. 1986):

(Ca – Ci) b'Ci fΓ*∆i = a ––––––– + –––– – –––– , (2)

Ca Ca Ca

where Ca and Ci refer to the concentration of CO2 in the airand intercellular leaf spaces, respectively (where Ca repre-sents the CO2 concentration exposed to the leaf and isrepresented by the CO2 concentration leaving the cuvette),b′ is an assumed value of net discrimination of Rubisco(here taken as 29‰), a is the fractionation due to diffusionthrough the stomata (4·4‰); Γ* is the CO2 compensationpoint in the absence of dark respiration (calculated fromthe regression equations listed by Brooks & Farquhar(1985) and f represents the fractionations associated with

photorespiration [here taken as 7‰; Rooney (1988);Gillon & Griffiths (1997)].

The offset between the modelled 13CO2 discrimination,∆i (Eqn 2) and that measured, ∆obs (Eqn 1), expressed as∆i – ∆obs has traditionally been attributed to additionaldrawdown imposed by the internal resistance to CO2 diffu-sion from the intercellular air space CO2 concentration (Ci)to that at the sites of carboxylation in the chloroplast [Cc;Evans et al. (1986); von Caemmerer & Evans (1991)].

The difference between measured and modelled discrim-ination (∆i – ∆obs) has, therefore, been used to estimate Cc

by rearranging the following equation (von Caemmerer &Evans 1991):

∆i – ∆obs = (b′ – a)(Ci – Cc)/Ca + (fΓ*)/Ca. (3)

Additionally, if values of gw are known then from Ficks’law of diffusion, estimates of Cc can also be derived usingthe following expression (von Caemmerer & Evans 1991;Harley et al. 1992):

A = gw(Ci – Cc)/P, (4)

where A is assimilation in µmol m–2 s–1, P is atmosphericpressure and gw is the internal transfer conductance forCO2 (in mol m–2 s–1 bar –1). Whilst conductance for CO2

(gs) between Ca and Ci can vary, the internal transfer con-ductance (gw) from Ci to Cc within the same leaf can beconsidered as constant.

Assuming there is complete equilibration between CO2

and water within the chloroplast, the on-line discriminationof C18O16O can be predicted using the following equation(Farquhar & Lloyd 1993):

Cc∆C18O16O = a + ––––––– (δc – δa) , (5)Ca – Cc

where δ is the oxygen isotope composition of CO2, C is theconcentration of CO2 and subscripts c and a refer to thechloroplast and ambient air, respectively, and a is theweighted mean of discrimination occurring during the dif-fusion from ambient air to the sites of carboxylation withinthe chloroplast, here taken as 7·4‰ (Farquhar et al. 1993).

Determination of the δH218O of transpired water

vapour (δt)

The δH218O of transpired water vapour was derived by

mass balance from measuring the water vapour concentra-tion and isotope composition before and after a leafcuvette, as follows (see Appendix A):

δt = ξL(δout – δin) + δin, (6)

where ξL = eout/(eout – ein) and δin, ein and δout, eout are theoxygen isotope composition and vapour pressure (in mbar)of the water vapour entering and leaving the cuvette,respectively. This was substituted for Rs in Eqn A3 andsolved for Re, to provide an estimate for enrichment at theevaporative sites of the P. aduncum leaves, without theprerequisite of ISS. The oxygen isotope composition of the



reference water vapour, δa H218O, varied between –7·5 and

–10·0‰ on day 1 and between –9·0 and –10·9‰ on day 2(Fig. 1c). Variation in the flux and isotope composition oftranspired water vapour under the high and variable leaf-to-air VPDs may have accounted for the differences inδaH2

18O between days 1 and 2. As with CO2, the value ofH2O δin at the time of the analysis was derived from atemporal polynomial regression of the δ18O of watervapour entering the cuvette (Fig. 1c).

RESULTS

Photon flux, temperature and leaf water status

The average PFD on day 1 was lower and more intermit-tent than on day 2, due to greater cloud cover (Fig. 2a & b).The accumulated PFD recorded reached 26·7 and37·2 mol m–2 d–1, for days 1 and 2, respectively. In general,day 1 experienced substantially lower temperatures andhigher relative humidities throughout the photoperiod thanday 2 (data not shown). These brighter, drier conditions onday 2 resulted in higher average leaf temperatures of37·3 °C between 1000 and 1400 h on day 2, compared witha mean of 31·6 °C for the same period on day 1 (Fig. 2c).

The leaf water potential (Ψ, measured on day 1 only)decreased from early morning, reaching a minimum of–1·1 MPa at 1300 h and then increased in the afternoon(Fig. 2d). The leaf water potential had fully recovered tothe predawn value of –0·21 MPa, by 1800 h. In general, thediurnal transition in leaf water potential was gradual,reflecting the overall diel progression in evaporative

demand, and contrasted with the variability seen in leaf gasexchange which was influenced by individual leaf stomatalresponses (Fig. 3a & b).

Gas exchange

On day 1, gas exchange measurements were made continu-ously throughout the day on a range of leaves. Assimilationwas greatest in the morning around 0800 h and declined asthe day progressed (open symbols, Fig. 3a), whilst the gen-eral diurnal trend in stomatal conductance tended to followleaf-to-air VPD (Fig. 3b & d). Under cloud cover at mid-day (Fig. 2a), a decrease in leaf-to-air VPD (Fig. 3d) gaverise to a transient increase in average A and gs from 4·2 to5·8 µmol m–2 s–1 (Fig. 3a) and 120–185 mmol m–2 s–1

(Fig. 3b), respectively. The concentration of substomatalCO2, expressed as Ci/Ca, remained relatively stablethroughout the day (Fig. 3c), with a mean value of 0·78(standard error = 0·02, n = 12), with the exception of theearly morning, between 0800 and 0900 h, where high ratesof assimilation increased the drawdown from Ca to Ci

reducing Ci/Ca to 0·65.For day 2, the same three leaves were followed through-

out the day (closed symbols, Fig. 3). Higher PFD and tem-peratures (Fig. 2) induced greater leaf-to-air VPDs(Fig. 3d) reaching a maximum of 42 mbar (4·2 kPa) at mid-day. Assimilation and stomatal conductance were greatestin the morning before 1000 h and declined over the courseof the day. Both A and gs on day 2 were slightly reduced inthe drier conditions, compared with day 1. For all leaves,



Figure 2. Diurnal variation in (a, b) above canopy photon flux density (PFD) and (c) leaf temperature for day 1 (l) and day 2 (●). (d) Variationin leaf water potential (expressed by xylem sap pressure) for the average of three Piper aduncum leaves over day 1 (n = 3, error bars showstandard error).

Ci/Ca slightly increased over the day (Fig. 3c), due to thedrop in assimilation, but on average was significantlylower (P = 0·00026, students t-test) than day 1 at 0·68(standard error = 0·02, n = 11).

Instantaneous isotope discrimination

On day 1, measured ∆13CO2 declined gradually over theday, from above 30‰ in the morning to around 10‰ in thelate afternoon (open symbols, Fig. 4a). Whilst the samplingprocedure showed that individual leaves can have distinctresponses, as demonstrated by the regular decline over thecourse of day 2 in all three leaves (closed symbols,

Fig. 4a), variation in measured discrimination was alsosystematically related to changing environmental condi-tions. The first measurements of both days, taken as soonas rates of photosynthesis for isotopic discrimination weremeasurable (the time at which solar elevation exceeded thesurrounding canopy) produced unusually high discrimina-tion signals (circled points, Fig. 4a) compared with thatmodelled (Fig. 4b). This ‘postdawn’ response wasobserved not only on both days in 1995, but also on thesame P. aduncum canopy 3 years earlier (M. S. J.Broadmeadow, unpublished field observations 1992;Gillon et al. 1997).

With Ci/Ca relatively constant over both days (Fig. 3c),modelled ∆13CO2 (∆i) also remained constant (Fig. 4b), sothat the observed discrimination (∆obs) could be eithergreater or lower than that modelled (∆i) from Ci/Ca

(Fig. 4c). The offset between observed and modelled dis-crimination (∆i – ∆obs), when compared with the quotientof assimilation and CO2 concentration, A/Ca, can usuallybe used to derive the internal resistance to CO2, from ambi-ent air to the sites of carboxylation in the chloroplast, Cc

(Evans et al. 1986; von Caemmerer & Evans 1991; Evans& von Caemmerer 1996). However, a general decrease in∆i – ∆obs with A/Ca was observed (Fig. 4c) and contrastedwith the increase expected in order to calculate gw.

The systematic shift in ∆13CO2 was not a function of thegas exchange system: calculation of ∆obs is partly depen-dent on ξ, the fractional uptake of CO2 (Eqn 1), wherelarge values of ξ (low CO2 drawdown) may reduce theaccuracy of ∆obs (von Caemmerer & Evans 1991).However, the relationship between ξ and ∆i – ∆obs for P.aduncum revealed that scatter occurred over the full rangeof ξ (8–45), not just at large values (Fig. 4d). In addition,the majority of the data points fell outside the range of∆i – ∆obs associated with a mass spectrometric precision of± 0·1‰ (shaded triangle, Fig. 4d).

On day 1, measured ∆C18O16O generally increased from10‰ in the morning, to 37‰ at 1330 h, and declined againin the afternoon (open symbols, Fig. 5a). As with ∆13CO2,variations from this trend were related to both distinct leafresponses (demonstrated by the three individually mea-sured leaves on day 2, closed symbols, Fig. 5a) andchanges in environmental conditions induced by intermit-tent cloud cover (Fig. 2a & b). For day 2, leaves 2 and 3followed a similar diurnal pattern, but had maximum mid-day enrichment of 21 and 23‰, respectively, somewhatlower than that measured on day 1. In contrast, ∆C18O16Oof leaf 1 remained fairly constant between 8 and 12‰, overthe three time intervals measured (also Fig. 5a).

Because of the large variation in ∆obs relative to ∆i for13CO2 discrimination (Fig. 4c), the concentration of CO2 inthe chloroplast (Cc) for P. aduncum was estimated usingEqn 4. Internal CO2 transfer conductance (gw) was derivedfrom taking the assimilation of 16 µmol m–2 s–1 attainedunder 1000 µmol m–2 s–1 PFD for the pioneering species P.auritum (Tinoco-Ojanguren & Pearcy 1992) and the rela-tionship between assimilation under high light and gw inthe literature (von Caemmerer & Evans 1991; Lloyd et al.



Figure 3. Diurnal variation in (a) assimilation (A), (b) stomatalconductance (gs), (c) Ci/Ca and (d) leaf-to-air vapour pressuredeficits (VPD), for different Piper aduncum leaves on day 1 (l)and three separate leaves on day 2 (▲ leaf 1, ● leaf 2, ■ leaf 3).Points are the average of two readings.

1992; Loreto et al. 1992; Evans et al. 1994; Epron et al.1995). Assuming a constant Rubisco sink strength, theinternal CO2 transfer conductance (gw) for P. aduncum (ahypostomatous tree) was estimated as 0·25 mol m–2 s–1

bar–1.Assuming constant gw and under low rates of assimila-

tion, by definition, the offset between Ci and Cc, and, hence,Cc/Ca, in P. aduncum will have remained relatively stableover the course of the day. A reduced chloroplast CO2 con-centration on day 2 (closed symbols, Fig. 5a) is, therefore,suggested to partially account for the lower observed valuesof ∆C18O16O. Furthermore, the low C18O16O discriminationobserved for leaf 1 on the second day corresponded withlower values of Cc/Ca (closed triangles Fig. 5a & b).Measured values of ∆C18O16O generally lay between thatpredicted (Eqn 5, dashed lines, Fig. 5b) for chloroplast waterin equilibration with that at the evaporative sites of between2 and 10‰. This agreed well with the evaporative enrich-ment derived from the δ18O of transpired water vapour (seeFig. 6b) for day 1, but did not reflect the high evaporativeenrichment (20‰) in the late afternoon of day 2.

On both days, the δH218O signal of transpired water was

generally more depleted than stem water in the morning

and more enriched in the afternoon (Fig. 6a). The soleexception to this trend was at 0740 h on day 1, whichapproximated that of source water and may have occurreddue to low and stable VPD conditions early in the morning.An increase in the δH2

18O of transpired water vapour overthe day, would be expected with an approach to steadystate, under increasing and decreasing VPD, respectively.The transpired water vapour had, on average, approxi-mately the same signal as the stem water (–3·2‰) between1300 and 1500 h on both days indicating that the leaveswere probably close to or at ISS.

Leaf water enrichment at the evaporative sites (δe)derived from the measurements of transpired watervapour, was tightly related for all leaves over both sam-pling days, but consistently higher on day 2 (Fig. 6b).Again, there was no systematic relationship with the frac-tional proportion of water lost (ξL), as for both days 17 ofthe 23 data points ranged between 3 and 7 (data notshown). The largest remaining values of ξL recordedoccurred shortly after dawn and approaching dusk andranged from 8 to 15.

Evaporative site enrichment (δe) generally increased inthe morning, peaked between 1300 and 1400 h, and then



Figure 4. Diurnal variation in (a) instantaneous observed ∆13CO2 for different Piper aduncum leaves on day 1 (l) and three separate leaveson day 2 (▲ leaf 1, ● leaf 2, ■ leaf 3), (b) discrimination from predicted Ci/Ca for days 1 and 2 (symbols as above). Relationship between theoffset in predicted and measured ∆13CO2 (∆i – ∆obs) with (c) the quotient A/Ca and (d) the expression of the amount of CO2 fixed relative to theconcentration in the atmosphere ξ. The shaded triangle in (d) represents the range of values associated with a mass spectrometric precision of± 0·1‰. High values of ∆13CO2 observed at the beginning of both days are circled in (a), (b) and (d) to ease interpretation.

became slightly reduced in the afternoon, on both days. Onday 1, δe reached a maximum enrichment of 12·7‰ at1334 h and remained high in the afternoon (Fig. 6b). Forday 2, the maximum enrichment was higher, reaching20·9‰ at 1423 h, for both leaves 1 and 2. The transientdecrease in δe at 1300 h on day 1 was likely to have beendue to a decrease in air temperature and leaf-to-air VPD, asa result of cloud cover. The dashed lines in Fig. 6(b) repre-sent values of δe assuming that ISS held throughout theentire day. Such an assumption would have over-estimatedevaporative site enrichment under periods of increasingleaf-to-air VPD and under-estimated δe at times of decreas-ing leaf-to-air VPD, with an increased effect under highVPDs (up to 4‰, in Fig. 6b).

Bulk δH218O leaf water values for P. aduncum (taken

on day 1) were 2·56‰ (± 0·2, n = 3) and 7·01‰ (± 0·3,n = 3) for 0930 and 1230 h, respectively (crosses,Fig. 6b). Both measurements were in close agreementwith that calculated for the evaporation sites (δe),including the transient decrease in enrichment around1300 h on day 1 due to cloud cover.

DISCUSSION

Gas exchange and leaf water status

On both days, maximum assimilation of P. aduncumoccurred in the morning, around 0800 h and graduallydeclined over the day as leaf-to-air VPD increased andwater potential decreased (Figs 2d & 3a). This is similar tothat found for Jacaranda copaia, in French Guiana (Huc,Fehri & Guehl 1994) and in other field observations ofdiurnal gas exchange (Wise et al. 1990; Wise et al. 1991;Epron, Dreyer & Breda 1992). The minimum leaf waterpotential (Ψ) at midday of –1·1 MPa for P. aduncum wassimilar to that of –1·5 MPa for the pioneering tree species



Figure 5. Diurnal variation in (a) instantaneous ∆C18O16O fordifferent Piper aduncum leaves on day 1 (l) and three separateleaves on day 2 (▲ leaf 1, ● leaf 2, ■ leaf 3). (b) Relationshipbetween observed ∆C18O16O and Cc/Ca (derived from Eqn 4) withthat predicted (Eqn 5) from Cc/Ca with δ18O chloroplast water inequilibration with source water (–3·2‰) and evaporative site waterat 2, 10 and 20‰ (from the observed range of δe, Fig. 6b).

Figure 6. Diurnal variation of (a) the δ18O of transpired watervapour for different Piper aduncum leaves on day 1 (l) and threeseparate leaves on day 2 (▲ leaf 1, ● leaf 2, ■ leaf 3) with respectto source water at –3·2‰ (dashed line), indicating the vapourpressure deficit (VPD) status and general trend over the day (solidline). The rain water signal generally varied between + 1·0 and–1·0‰ throughout the field campaign, whilst the ground water hada δ18O of –3·2‰. With high evaporative demand and infrequentand low intensity rain showers (data not shown) it was likely thatthe water supply for the Piper originated from ground water, ratherthan precipitation-fed soil water. Isotopic composition of groundwater has been shown to remain constant despite input fromprecipitation with variable δ18O (Neal & Rosier 1990), so the δ18Oof spring water collected on 20 February 1995 was assumed to bethe source water signal for the 2 d investigated. (b) Evaporative siteenrichment (δe) for different leaves on day 1 and three separateleaves on day 2 (symbols as above). Solid lines represent diurnaltrends in δe for day 1 and day 2 calculated using δH2

18O oftranspired water vapour. Dashed lines represent the trendline fittedto the same data points derived assuming isotopic steady state(ISS) held throughout the day. Crosses indicate two averagemeasurements of bulk leaf water δH2

18O (n = 3) taken on day 1.

J. copaia in the field in French Guiana (Huc et al. 1994). Inthe afternoon, photosynthesis and stomatal conductanceremained depressed (Fig. 3a & b), despite a recovery in Ψ[Fig. 2d, cf. Quick et al. (1992)].

Higher leaf-to-air VPDs in the brighter conditions of day2 are likely to have accounted for the reduction in stomatalconductance observed in P. aduncum. Reduced stomatalconductance under high VPDs has been reported foranother pioneering Piper species (P. auritum, Tinoco-Ojanguren & Pearcy 1993), maintaining instantaneouswater use efficiency, as a decline in gs reduced assimilationand transpiration in tandem. The variability in stomatalconductance on day 1 was due to leaf-to-leaf variation andintermittent light resulting from cloud cover. Concurrentvariation in assimilation was minimal and may have beendue to the more rapid response of assimilation to changesin PFD (Tinoco-Ojanguren & Pearcy 1992; Barradas &Jones 1996).

Despite variation in A and gs, Ci/Ca remained relativelystable over the day. Observations of relatively stable Ci/Ca

have also been reported, despite variation in gs during lightflecking in a related species, P. auritum (Tinoco-Ojanguren& Pearcy 1992). Furthermore, due to the low rates ofassimilation, the theoretical drawdown between Ci and Cc

never exceeded 30 µmol mol–1 (corresponding with a max-imum assimilation of 7 µmol m–2 s–1), thus, the diurnalvariation in Cc/Ca approximated that of Ci/Ca.

On-line discrimination of 13CO2

Measured ∆13CO2 (∆obs) in P. aduncum was similar to thatfound for other C3 mesophytes (Evans et al. 1986).However, the variation in ∆obs could not be accounted forby Ci/Ca alone (Fig. 4). The observation of a wide range in∆obs compared with that modelled from gas exchange wasobserved on the same P. aduncum canopy 3 years earlier(M. S. J. Broadmeadow, unpublished field observations1992; Gillon et al. 1997).

Differences between modelled and observed on-line∆13C have previously been accounted for by gw, the CO2

transfer conductance from the leaf intercellular spaces tothe chloroplast (Evans et al. 1986; von Caemmerer &Evans 1991). The maximum drawdown of 30 µmol mol–1

from Ci to Cc likely to be encountered in P. aduncum, onlyreduces modelled ∆13C by 2‰ and does not explain thefull extent of the shift between modelled and measured∆13CO2 (∆i – ∆obs). Any effect of drawdown to Cc on ∆obs

should be reflected by a positive correlation between∆i – ∆obs and A/Ca (Evans et al. 1986). In contrast, on bothdays, P. aduncum appeared to exhibit a negative correla-tion, whereby the greatest values of ∆i – ∆obs coincidedwith low assimilation rates, when the drawdown from Ci toCc should have been minimal. Thus, the data are notexplained by conventional theory, as neither Ci or Cc couldfully account for the observed range of measured ∆13CO2.

Further analysis also revealed that large variations in∆obs could not be explained by the influence of mass spec-trometric precision at high ξ (Fig. 4d) or diurnal variation

in δa13CO2 (Fig. 1a). Thus, similarities between data on

both days and that recorded 3 years previously on the sameP. aduncum stand suggest the range of ∆13C observed heremay be a function of plant physiology rather than any tech-nical problems.

Increased stomatal patchiness has been associated withwater stress (see Eckstein et al. (1996) for a recent review)and also with concurrent changes in relative humidity andirradiance (Cardon, Mott & Berry 1994). Any existence ofpatchy stomatal closure may lead to an over-estimation ofCi calculated from measurements of gas exchange(Farquhar 1989; Mott 1995). In this case, actual leaf sub-stomatal CO2 concentration would be lower, and in turnreduce modelled discrimination (∆i). Any shift in ∆i wouldstill leave values of ∆obs both above and below that mod-elled and, thus, stomatal patchiness seems unlikely to bewholly attributable for the full range of discrimination val-ues observed. However, investigation of asymmetric stom-atal closure was beyond the scope of the field work andmore experimental work is required to assess any interac-tion with discrimination.

The range of A/Ca encountered with P. aduncum in thefield, 0·005–0·02 (Fig. 3c), was generally lower than val-ues found in controlled conditions [e.g. 0·04–0·12; Evanset al. (1986)]. Values of ∆i – ∆obs, increasing at lowerassimilation rates, suggest that measured ∆13CO2 is lowerthan modelled from Ci/Ca. This phenomenon was alsoobserved in Phaseolus vulgaris, where the δ13C of darkrespired CO2 was very negative at c. –50‰ when plantswere grown air depleted in 13C (Gillon & Griffiths 1997),as well as other gas exchange studies under field condi-tions [M. S. J Broadmeadow, unpublished field data 1992;K. G. Harwood, unpublished field data on Quercus 1996; J.S. Gillon, unpublished field data on Prosopis 1997; seealso Gillon et al. (1997)]. At such low assimilation rates, itwas suggested that CO2 from dark respiration in the lightcan make a significant contribution to net CO2 exchange,whereby respiratory CO2 is more depleted in 13C than theCO2 being assimilated via photosynthesis. Any back diffu-sion or re-fixation of this depleted CO2 from the leafreduces the enrichment in 13CO2 observed in air passingover the leaf (i.e. a smaller difference between δin and δout,Eqn 1). Thus, the net discrimination expressed by the leafis lower than predicted (i.e. reduced ∆obs). The data wouldalso suggest that respired CO2 is relatively more depletedin 13C than the initial Rubisco product. Although no frac-tionation during respiration has been reported for cells invitro (Lin & Ehleringer 1997), such a situation may arisefrom short-term differences in the isotopic composition ofthe respiratory substrates and CO2 being assimilated, dueto the slow turnover time of carbohydrate pools, c. 4 h(Parnik, Keerberg & Viil 1972). Similarly, a transientenrichment in the 13C of respired CO2 (relative to thatfixed by photosynthesis), possibly as a function of a noc-turnal metabolic modification of the carbohydrate pools’isotope composition, may account for the occurrence ofvery high ∆obs, above that predicted, evident in the fewhours after dawn on both days.



Any effects of slow carbohydrate pool turnover on ∆obs

due to increased rates of dark respiration or re-fixationoccurring at high temperatures, may be exacerbated in thetropics, and are unlikely to be encountered in crop speciesusually studied, with characteristically higher values ofA/Ca, under controlled laboratory conditions.

On-line discrimination in C18O16O

Discrimination in C18O16O reflects both the enrichment of18O in leaf water, with which it equilibrates before diffus-ing back out of the chloroplast, and also the extent of backdiffusion of CO2, which allows the leaf water enrichmentto be expressed (Farquhar et al. 1993). With a relativelysmall variation in Cc/Ca (derived from Ci/Ca) over thecourse of both days, ∆C18O16O generally reflected leafwater enrichment, increasing in the morning, until justafter midday and slightly decreasing in the afternoon(Fig. 5a). Discrimination of C18O16O is strongly influ-enced by the non-linear relationship with Cc/Ca (Farquharet al. 1993; Flanagan et al. 1994). This was demonstratedby a general reduction in values of ∆C18O16O on day 2compared with day 1, when stomatal conductance andCc/Ca were reduced under high VPDs (Fig. 5b). Using theestimated gw of 0·25 mol m–2 s–1 bar–1, the observed∆C18O16O generally lay between that predicted for chloro-plast enrichment of 2 and 10‰ (assuming complete equili-bration between δe and δc, Fig. 4c), lower than concurrentmeasurements of δe which ranged from 2 to 20‰ (Fig. 5b).Despite the codependence of ∆13CO2 and ∆C18O16O onthe extent of back diffusion of CO2 from the chloroplast(Evans et al. 1986; Farquhar et al. 1993; Williams &Flanagan 1996) the two processes were not necessarilycoupled in field conditions (Figs 4a & 5a), largely becauseCc/Ca imposes a linear control on ∆13CO2, whereas∆C18O16O is dependent on the non-linear relationship withCc/Ca and the interaction with VPD-induced evaporativeenrichment (Williams, Flanagan & Coleman 1996).Additionally, any contribution from dark respiratory CO2

may alter the signal of on-line ∆13CO2, yet not affect the∆C18O16O, as the δ18O signal of respiratory CO2 will belost in the equilibration with leaf water in the chloroplast.

Enrichment of leaf water at the evaporative sites(δH2

18O/δe)

In an approach to ISS, transpired water vapour was gener-ally less enriched in 18O than source water during periodsof increasing VPD (e.g. morning) and more enriched thansource water during times of decreasing VPD (e.g. after-noon). A relative stability in VPD combined with a slightlag in leaf water turnover rate, indicated that leaf waterenrichment was close to ISS between 1300 and 1500 h(Fig. 6a). This observation of proximity to ISS in the fieldhas important implications in modelling the contribution oftranspired water vapour to terrestrial water budgets usingthe isotopic composition of atmospheric water vapour(Bariac et al. 1989, 1994; Brunel et al. 1992). This

supports the assumption of ISS in modelling ∆C18O16Odiscrimination (Farquhar et al. 1993), because the timewhen ISS occurs coincides with the most photosyntheti-cally active time of the day, when the greatest fluxes ofCO2 and H2O should take place, for many terrestrial plants.However, both this and other field observations havedemonstrated that under some conditions, optimumexchange of CO2 and water vapour can occur in the earlypart of the day, both at the leaf level (Roberts et al. 1980;Leverenz et al. 1982; Roberts, Wallace & Pitman 1984;Briggs, Jurik & Gates 1986; Roberts, Cobral & Ferreira deAguiar 1990; Wise et al. 1990; Epron et al. 1992; Hinckleyet al. 1994) and at the whole canopy level (Grace et al.1995). In these cases, the associated changes in environ-mental conditions may yield non-steady-state photosynthe-sis and transpiration, for which steady-state estimations ofboth ∆13CO2 and ∆C18O16O, derived largely from labora-tory experiments, may be in error.

Analysis of transpired water vapour allowed the directcalculation of 18O enrichment of water at the evaporationsites (δe) over the full diurnal period, without the prerequi-site of ISS. For the conditions experienced, a conventionalderivation of δe assuming ISS throughout the day wouldlead to an over-estimation of evaporative site enrichmentearly in the morning and an under-estimation later in theafternoon. The extent of evaporative site enrichment waslargely maintained in the afternoon, despite the decrease inleaf-to-air VPD (Fig. 6b) suggesting that 18O enrichment atthe evaporative sites may also be affected by species-dependent rates of leaf water turnover (Wang & Yakir1995), in addition to evaporative control imposed by ambi-ent VPD. Maintenance of evaporative enrichment of leafwater late into the afternoon has also been noted for cotton(Yakir, DeNiro & Gat 1990), alfalfa (Bariac et al. 1989)and maize (Bariac et al. 1994) and also to some extent inmature oak and beech leaves (Forstel 1978) and barley(Walker & Lance 1991), but contrasts with the relativelystable leaf water enrichment found in sclerophyllousjuniper and mistletoe by Flanagan, Marshall & Ehleringer(1993). Two direct measurements of bulk leaf water 18Ocomposition were close to that derived for the evaporativesites and suggest that the evaporative enrichment modelprovided approximate estimates of bulk leaf water δH2

18Oat the two times investigated (cf. Farris & Strain 1978;Forstel 1978; Walker et al. 1989; Yakir et al. 1990;Flanagan et al. 1991a, b; Flanagan et al. 1993). The diurnaltrend in measured ∆C18O16O agreed qualitatively with themodelled evaporative enrichment (δH2

18O at the evapora-tion sites, or δe). However, quantitatively, ∆C18O16O wasgreater on day 1 and this chloroplast signal was tightlycoupled to changes in Cc/Ca, associated with VPD andconductance effects. Meanwhile, there was a much moregradual transition in leaf water enrichment at the evapora-tion sites (δe, Fig. 6b), similar to overall leaf water status(Fig. 2d) and contrasted with the more variable stomatalinfluence on ∆C18O16O (Fig. 5a). Values of measured∆C18O16O were low, particularly for day 2, whencompared with that predicted from the Farquhar and Lloyd



model using measured values of δe (Eqn 5, Fig. 5b).By rearranging the Farquhar and Lloyd C18O16O dis-

crimination model the measured ∆C18O16O can be used toyield an estimate of isotopic composition of water withinthe chloroplast (δc), which can then be compared withδH2

18O at the evaporative sites (δe). Preliminary analysisfor P. aduncum reveals that in most cases, δe is greater thanderived δc (below the 1:1 line of unity, Fig. 7). The modelused to derive δc is partially dependent on the estimatedvalue of gw used for deriving Cc (here taken as0·25 mol m–2 s–1 bar–1). However, estimation of δc usingboth double and half of the internal conductance valueselected of 0·125 and 0·5 mol m–2 s–1 bar–1, respectively[representing the natural range of gw reported in the litera-ture; Loreto et al. (1992)], only results in an averagerespective shift of 1·5 and 0·7‰ (error bars, Fig. 7). Thus,uncertainty in Cc can still not account for the full offsetobserved between δc and δe for P. aduncum.

Observations of chloroplast water less enriched than thatat the evaporative sites agrees with the conventional theoryof a continuum of enrichment from the unfractionatedsource water, to the most fractionated water at the sites ofevaporation (Farquhar & Lloyd 1993). Chloroplast watercan be depleted by up to 10‰ in δH2

18O compared withthat at the evaporative sites (Yakir et al. 1994) and couldpartially account for the low values of δc observed in thisstudy. However, since the bulk leaf water values were closeto that derived for the evaporative sites (Fig. 6), the lowervalues of ∆C18O16O (Fig. 5) and, hence, δc (Fig. 7) mayhave been a result of incomplete isotopic equilibrationbetween CO2 and water vapour within the chloroplast

(Farquhar & Lloyd 1993; Flanagan et al. 1994), althoughthis may also be questionable under such low rates of gasexchange. Understanding variations in δc and δe areimportant for discerning the physiological controls of leafwater enrichment and provides a greater insight into therelationship between leaf water enrichment and the δ18Osignal transferred to the leaf cellulose. These observationssuggest that further investigation into the variation in δe

with derived δc is required under field conditions and inparticular for reliable estimates of Cc.

Under high temperatures in Trinidad, measurement of∆13CO2 in the field deviated from that modelled from gasexchange measurements of Ci/Ca. Simple models appliedto calculating discrimination and deriving single pointvalues of gw, under tropical field conditions, should,therefore, be used with caution. This study also suggestsdiscrepancies may still exist between C18O16O and H2

18Oestimation of 18O leaf water enrichment under field con-ditions. Additionally, the assumption of ISS may not holdunder changeable VPDs and a more faithful representa-tion of δe can be derived from measuring the oxygen iso-tope composition of transpired water vapour. Thus,observed isotopic discrimination under natural field con-ditions can differ from that predicted under steady-statephotosynthesis in controlled laboratory experiments.Whilst the models provide us with an insight into themechanistics of discriminatory processes, investigationof discrimination under field conditions is necessary toensure accurate prediction of biome discrimination forglobal models (Farquhar et al. 1993; Ciais et al. 1995,1997) and to provide a greater understanding of thefactors controlling discrimination under natural, tran-sient, environmental conditions.

ACKNOWLEDGMENTS

Thanks must go to Charles McDavid, Department of PlantScience and Biochemistry and Joss Knight, Department ofPhysics both at the University of the West Indies, Trinidadfor the loan of equipment. Also to Dan Yakir and Xue FengWang for useful discussion and the laboratory at theWeizmann Institute, Israel for 18O leaf water analysis. Thecomments of three anonymous reviewers are alsoacknowledged for substantially improving this manuscript,one of whom provided the simplified expression for EqnA7. This work was supported by the National EnvironmentResearch Council (NERC) under small grant no.GR9/1767 and studentship no. GT4/92/27 to K.G.H.

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Received 2 September 1997; received in revised form 1 December1997; accepted for publication1 December 1997

APPENDIX A: ESTIMATION OF EVAPORATIVESITE ENRICHMENT IN 18O

Flanagan et al. (1991a) modified the Craig & Gordon(1965) equation, whereby the evaporative enrichmentwithin a leaf is derived from the proportion of 18O evapo-rated relative to 16O (E18/E16). The difference in evapora-tion of the heavy and light isotope can be seen in thetranspired water, and expressed as a function of the isotopecomposition of water at the evaporation sites, the kineticand equilibrium isotope effects, the leaf-to-air VPD experi-enced and the atmospheric water vapour signal, as follows:

Re(liquid)–––––––––( α*

ei – Raea)E18 1––– = Rt (vapour) = ––– –––––––––––––––––, (A1)E16 αk ei – ea

where R is the ratio of heavy to light isotopes, and sub-scripts t and e represent transpired water vapour and(liquid) water at the evaporation sites, respectively, α* isthe liquid–vapour equilibration isotope effect corrected toleaf temperature (Majoube 1971); αk is the kinetic isotopediffusion fractionation factor, through the stomatal pore,which is derived from the relative rates of diffusion of thelight to heavy isotope, which is 1·0285 (Merlivat 1978).When using a leaf cuvette to measure photosynthetic gasexchange, ea represents the H2O concentration the leaf isexposed to and is reflected by the H2O concentrationleaving the cuvette.

Because at ISS the transpired water vapour has the samesignal as source water, Rt can be substituted by Rs, the iso-topic composition for stem water, and then solved for Re asfollows (White 1988; Flanagan et al. 1991a):

ei – ea eaRe = α* αk Rs ––––– + Ra –– . (A2)[ ( ei

) ( ei)]

Since fractionation is different through turbulent andlaminar flow, the model was further adapted to take into

account leaf boundary layer effects as follows (Flanaganet al. 1991a):

ei – es es – ea eaRe = α* αk Rs ––––– +αkb Rs ––––– + Ra ––[ ( ei

) ( ei) ( ei

)],(A3)

where es is the vapour pressure at the leaf surface and αkb

is the ratio of the diffusion of light to heavy isotopemolecules in a boundary layer (1·0189). Vapour pressure atthe leaf surface is calculated from the evaporation andboundary layer resistance measured during leaf gasexchange by rearranging the following:

1–– (es – ea)rb

E = ––––––––– , (A4)P

where E is the evaporation, rb the boundary layer resis-tance and P atmospheric pressure.

Re can then be expressed in the delta notation withrespect to a set standard by δ (‰) = [(Rsample/Rstandard) – 1]×103 where Rstandard for 18O/16O is SMOW. Correctderivation of δe using the above, therefore requires the leafto be at ISS.

By consideration of mass balance, the vapour pressure ofH2

18O in air as it passes through a leaf chamber can bedescribed by

einRin + ediffRt = eoutRout , (A5)

where e is the vapour pressure, R is the ratio 18O/16O andthe subscripts in, out and diff refer to the water vapourentering the cuvette, leaving the cuvette and that added byevaporation from the leaf. Rt is the oxygen isotope ratio oftranspired water vapour. The above expression holdsassuming that there is no change in temperature, no con-densation within the cuvette, that all the water vapour iscollected and the ediff remains constant over the measure-ment period (the authors acknowledge X. F. Wang for help-ful discussion).

Solving for Rt, and expressing isotopic composition inthe small delta notation (‰) gives

eout einδt = –––– δout – –––– δin · (A6)ediff ediff

Using the term ξL to represent the proportion of H2O tran-spired relative to that in the ambient air allows simplifica-tion of the above expression to one analogous to measured∆13CO2, as follows:

δt = ξL(δout – δin) + δin , (A7)

where ξL = eout/(eout – ein) and δin, ein and δout, eout are theoxygen isotope composition and vapour pressure (in mbar)of the water vapour entering and leaving the cuvette,respectively.



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Diurnal variation of CO O and evaporative site …eco.ibcas.ac.cn/group/lingh/siep/pdf/StaleIsotopesyueduw...neer, Piper aduncum,on two different days in Trinidad during February 1995