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Blackwell Science, Ltd

Limitations to CO2 assimilation in ozone-exposed leaves of Plantago major

Y. Zheng1, H. Shimizu2 and J. D. Barnes1

1Air Pollution Laboratory, Department of Agricultural and Environmental Science, Ridley Building, Newcastle University, Newcastle upon Tyne, NE1 7RU,

UK; 2Centre for Global Environmental Research, National Institute for Environmental Studies, Onogawa 16–2, Tsukuba, Ibaraki, 305–0053, Japan

Summary

• The potential limitations on net leaf carbon assimilation imposed by stomatalconductance, carboxylation velocity, capacity for ribulose 1,5-bisphosphateregeneration and triose phosphate ultilization rate were derived from steady-stategas exchange measurements made over the life-span of two leaves on plants of an‘O3-sensitive’ population of Plantago major grown at contrasting atmospheric O3concentrations.• Parallel measurements of chlorophyll fluorescence were used to monitor changesin the quantum efficiency of PSII photochemistry, and in vitro measurements ofRubisco activity were made to corroborate modelled gas exchange data.• Data indicated that a loss of Rubisco was predominantly responsible for thedecline in CO2 assimilation observed in O3-treated leaves. The quantum efficiencyof PSII was unchanged by O3 exposure.• Stomatal aperture declined in parallel with CO2 assimilation in O3-treated plants,but this did not account for the observed decline in photosynthesis. Findingssuggested that O3-induced shifts in stomatal conductance result from ‘direct’ effectson the stomatal complex as well as ‘indirect effects’ mediated through changes inintercellular CO2 concentration. Leaves on the same plant exposed to equivalentlevels of O3 showed striking differences in their response to the pollutant.

Key words: ozone (O3), Plantago major, gas exchange, photosynthesis, stomatalconductance, relative stomatal limitation, chlorophyll fluorescence, Rubisco.

© New Phytologist (2002) 155: 67–78

Author for correspondence: Jeremy Barnes Tel: +191 2227374 Fax: +191 2225229 Email: J.D.Barnes@ncl.ac.uk

Received: 5 December 2002 Accepted: 8 March 2002

Introduction

Photosynthesis has long been known to be reduced by O3 andthe effects play a key role in determining the adverse effects ofthe pollutant on the productivity of sensitive taxa (Heath,1994; Pell et al., 1997; Farage & Long, 1999). However, manyaspects related to the depression of photosynthesis by O3remain poorly understood (Pell et al., 1994a).

It has been established that O3 commonly causes aconcomitant decline in stomatal conductance (gs) and the rateof CO2 assimilation (A). However, few studies conducted underenvironmentally relevant conditions have questioned whetherthe shifts in gs in plants exposed to O3 are the product ofeffects of the pollutant on the stomatal complex or effects onA (Heath, 1994). Even fewer studies have examined stomatalvs non-stomatal limitations of photosynthesis (Farage &

Long, 1995) and the timing of effects in relation to plant age/prior O3 history (Krupa & Manning, 1988). Efforts tounderstand the physiological basis of the decline in CO2assimilation induced by O3 have focused on changes in theamount and activity of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco EC 4.1.1.39) (Farage & Long, 1992; Pellet al., 1994b). Indeed, O3 exposure has been shown to decreaseboth the amount and activity of Rubisco, effects that areaccompanied by a decrease in mRNA transcript abundancefor both the large (LSU) and small (SSU) subunits of theenzyme (Pell et al., 1994b; Pell et al., 1997). Rather less attentionhas been paid to the impacts of O3 on photosynthetic limitationsimposed by (i) the capacity to regenerate Ribulose-1,5-bisphosphate (RuBP), dependent on the activity of theenzymes associated with the interconversion of 3-, 4-, 5- and6-carbon intermediates in the Calvin cycle and the potential

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of the thylakoid reactions to supply ATP and NADPH(Sharkey, 1985), and (ii) end-product feedback inhibitionwhich may arise as a consequence of the direct repression ofphotosynthetic genes by soluble carbohydrates (Sheen, 1994;Krapp & Stitt, 1995) and/or indirectly via limitations imposedby the utilization of triose phosphates, and subsequent regenera-tion of Pi, in the synthesis of starch and sucrose (Herold, 1980).

Ozone can directly affect stomatal aperture and impairstomatal performance (Robinson et al. 1998) – effects that maylead to reductions in the dose of the pollutant absorbed byplant tissues and afford a mechanism to restrict pollutantuptake. The extent to which O3-induced shifts in stomatalconductance contribute to the decline in CO2 assimilationremain uncertain. Some studies indicate that the shift in g sinduced by O3 is the cause of the reduction in CO2 assimilation(Moldau et al., 1990, 1993; Kull et al., 1996), while othershave shown that the shift in gs is a downstream consequenceof the increase in the intercellular CO2 concentration (ci)resulting from the effects of O3 on photosynthetic metabolism(Farage et al., 1991; McKee et al. 1995). The reasons for thesediscrepancies are unclear, and it remains to be establishedwhether there are genuine differences in responses betweentaxa or, whether these incongruities arise as a result of thediverse range of experimental methods and approaches thathave been employed.

The present study was undertaken on an ‘O3-sensitive’population of Plantago major. Previous work has revealedconsiderable variation in the O3 resistance of discretepopulations of this species across Europe (Davison & Barnes,1998) – a phenomenon related to the extent of the reductionin stomatal conductance induced by O3 (Reiling & Davison,1995) and which is influenced by plant age/prior O3 history(Lyons & Barnes, 1998). It is not clear at this stage whetherthe observed differences in stomatal response contribute to thedifferences in O3 sensitivity between populations or whetherthe stomata respond to an increase in ci as a result of a declinein the capacity for CO2 fixation. The objective of the presentstudy was therefore to: probe the relative limitations on Aimposed by changes in stomatal conductance, Rubiscoactivity, RuBP regeneration capacity and triose phosphateutilization; test Reiling and Davison’s (Reiling & Davison,1995) theory that the decrease in gs induced by O3 in Plantagomajor is caused by direct effects on the stomatal complex,rather than a down-stream consequence of the changes in cithat accompanies the inhibition of photosynthetic metabolism;and examine the effects of O3 on leaf gas exchange on plantsat different developmental stages with contrasting O3 histories.

Materials and Methods

Plant culture and fumigation

Seed of Plantago major L. ‘Valsain’ was germinated in apropagator containing a standard potting compost (John

Innes no. 2) in a controlled environment chamber ventilatedwith charcoal/Purafil®-filtered air (‘clean air’ < 5 nmolO3 mol−1 dry air). The propagator lid was removed followinggermination, and 7-d-old-seedlings (i.e. at the three-leafstage) transplanted individually into 2.5 cm2 plugs of 10 × 7modules containing the same compost. Seedlings wereallowed 24 h to recover after transplantation, then transferredto duplicate controlled environment chambers ventilatedwith either ‘clean air’ (CFA) or ‘ozone’ (O3) (CFA plus15 nmol mol−1 O3 overnight rising to a maximum between12:00–16:00 hours of 75 nmol mol−1) (Zheng et al., 1998).Plants were transplanted into progressively larger pots (0.45and 1.74 dm3) containing the same standard compost after 14and 28 d, respectively, watered daily and fertilized 7, 21 and35 d after transplantation with a medium-strength com-mercial nutrient solution (Phostrogen, Corwen, Clwyd, UK).

Leaf gas exchange

After 19 and 33 d exposure to CFA or O3, the newly emergingleaf borne on 2–3 plants per chamber was labelled (leaves 7and 10, respectively) and leaf gas exchange determined atintervals following the attainment of full expansion (9 d fromleaf emergence). Measurements were made using a sphericalleaf cuvette (manufactured by PP Systems, Hitchin, UK),incorporated within an open gas exchange system, housed inan additional controlled environment cabinet constructedfrom 3 mm perspex, ventilated with ‘clean air’ (charcoal/Purafil®-filtered air) from the same air handling systemsupplying the fumigation chambers and illuminated by metalhalide lamps (Connect Lighting Systems 250 W unit fittedwith Sylvania HQI-TD 250 W/NDL lamp) providing aPPFD of 300 µmol m−2 s−1 at plant canopy height.Temperature, photoperiod, relative humidity and air velocityin the cabinet housing the leaf cuvette were maintained thesame as that in the fumigation chambers. The leaf cuvetteused was similar to that developed by Ireland & Long (1989),and described in detail by Long & Drake (1991).Atmospheric composition was modulated by mixingpredetermined levels of compressed gases (CO2, N2 and dryCO2-free air) with the aid of mass flow controllers (BrooksInstruments B. V., Veenendaal, The Netherlands, model5850TR MFCs/5878 controller). The gas flow to and fromthe cuvette was monitored with the aid of flow meters (PlatonFlow Control Ltd, Basingstoke, UK). Before entering the leafcuvette, the air was purified by passing through a charcoal/Purafil®/potassium permanganate-filter and humidified overdistilled water maintained in a temperature controlled waterbath to achieve a leaf-atmosphere vapour pressure deficit(VPD) of 1.3 ± 0.04 kPa. The air leaving the leaf cuvette wasdried over anhydrous magnesium perchlorate before enteringan infrared gas analyzer (IRGA). Absolute CO2 concentrationwas continuously monitored by an ADC IRGA (ADC Ltd,Hoddesdon, UK, model LCA-2), with the change in CO2

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across the cuvette measured by a twin-channel LICOR IRGA(Li-Cor Inc., Lincoln, NB, USA, model 6252). Both IRGAswere calibrated against compressed mixtures of CO2 in air,cross-calibrated against gravimetrically prepared CO2standards (Distillers MG, Reigate, Surrey; accuracy ± 2 µmolCO2 mol−1 dry air). The dewpoint of the air entering andleaving the cuvette was measured with the aid of cooled-mirrors (Protimeter Plc, Marlow, UK, model DP989M). Airand leaf surface temperatures were measured with the aid ofcross-calibrated thermocouples. Measurements were made in‘clean air’ at a leaf temperature of 24 ± 1°C, under a PPFDof 1000 µmol m−2 s−1 supplied by an integral fan-cooled15 V/150 W Xenon lamp (Osram Xenophot HLX 64634,St Helens, Merseyside, UK). Prior experimentation revealed(i) no significant recovery in the light-saturated rate of CO2assimilation (Asat) and stomatal conductance (g s) within 6 hof transferring plants from O3 to CFA and (ii) the level ofirradiance employed was high enough to achieve Asat in thesechamber-grown plants of Plantago major.

The attached leaf was sealed in the cuvette and allowed toequilibrate to the cuvette environment (30–40 min). Then,the CO2 concentration and dewpoint of the air entering andleaving the cuvette were measured at a reference CO2 concen-tration (Ca) of 350 µmol mol−1. Subsequent measurementswere made on the same leaf following step-wise changes in thereference CO2 concentration, allowing 30–40 min for leavesto attain steady-state rates of gas exchange at each CO2 level.Reference CO2 concentrations used for the construction ofA/ci response curves were: 80, 130, 165, 200, 250, 350, 700,1000 µmol mol−1. The projected area of the leaf enclosedwithin the cuvette was traced and subsequently determinedusing a Delta-T Devices area meter (Cambridge, UK). Light-saturated rates of net CO2 assimilation (Asat), stomatalconductance (gs) and intercellular space CO2 concentration (ci)were calculated according to Von Caemmerer & Farquhar, 1981).

The light- and CO2-saturated rate of CO2 assimilation(Amax) was derived from asymptotic curves (y = a + bekx) fittedto A /ci response data according to Delgado et al. (1993);where y equals the rate of net CO2 assimilation; x equals theintercellular CO2 concentration and a represents Amax.

The relative stomatal limitation of photosynthesis (RSL)(i.e. the proportionate decrease in Asat attributable to stomata)was calculated from A /ci curves using the method of Farquhar& Sharkey (1982):

Where A0 represents the rate of CO2 assimilation when ciis 350 µmol mol−1 (i.e. the potential rate of CO2 assimilationin the absence of stomatal limitation at 350 µmol CO2 mol−1

dry air), and A represents the rate of CO2 assimilation at anambient CO2 concentration of 350 µmol mol−1 (i.e. theactual rate of CO2 assimilation in the presence of stomatallimitation at 350 µmol CO2 mol−1 dry air).

In vivo estimates of the maximum rate of Rubiscocarboxylation (Vc,max), maximum RuBP regeneration capacitymediated by light harvesting and electron transport (Jmax,RuBP)and the potential capacity of starch and sucrose synthesis toutilise triose phosphates and subsequently regenerate inorganicphosphate (Pi) for photophosphorylation (triose phosphateutilization (TPU)) were calculated by iteratively fitting curves(using nonlinear least square regression methods) to A /ciresponse data according to Harley & Sharkey (1991); underthe assumption that (i) the transport of 4 electrons generatessufficient energy (ATP) and reductant (NADPH) for theregeneration of RuBP in the Calvin cycle (Farquhar & VonCaemmerer, 1982) (ii) CO2 assimilation is limited solely bythe amount, activity and kinetic properties of Rubisco ata ci below 200 µmol mol−1 under light-saturating conditions(iii) inorganic phosphate (Pi) limits carboxylation at high ci,and (iv) the Michaelis–Menten constants for carboxylationand oxygenation (Kc and Ko, respectively), the specificity fac-tor of Rubisco (τ) are similar in all C3 species, and unaffectedby exposure to O3. Jordan & Ogren’s (1984) measured valuesfor Kc, Ko and τ were employed in calculations, correcting fortemperature- and pressure-dependencies according to Harleyet al. (1992). The efficiency of light energy conversion (α) wasassumed to be constant at 0.24 mol electrons mol−1 photons– based on an average quantum use efficiency of 0.073 molCO2 mol−1 photons absorbed (Ehleringer & Bjorkman,1977) and a leaf absorptance of 83% (Ehleringer & Pearcy,1983). The rate of nonphotorespiratory CO2 evolution in thelight (Rd) and the CO2 compensation point in the absence ofRd (Γ*) were derived according to Brooks & Farquhar (1985),from measurements made on three independent CFA andO3-treated plants of Plantago major ‘Valsain’ after 32 dexposure in duplicate controlled environment chambers.Measurements were made employing the same gas exchangesystem used to undertake A/ci analyses. Rates of CO2assimilation were measured over a range of ci’s close to thecompensation point (15, 30, 60, 80 µmol mol−1) at contrastingPPFDs (150, 400 and 600 µmol quanta m−2 s−1) at a leaftemperature of 24°C. The response of A was found to belinear over this range of ci and regressions were fitted to dataobtained at each level of irradiance. The co-ordinates at whichthe regression lines intersected each other were used to estimateboth Rd and Γ*. This procedure provided values for CFA andO3-exposed plants of −0.033 and −0.17 µmol m−2 s−1 for Rdand 42.6 and 44.6 µmol mol−1 for Γ*, respectively (seeFig. 1). Further details are given by Zheng (1998) regardingthe simplifying assumptions that were necessary, the estimationof Rd and Γ* and the manner in which the latter values wereemployed in the analysis of A /ci response curves.

Measurement of in vitro Rubisco activity

Measurements of in vitro Rubisco activity were undertaken onfully expanded leaves over the course of a single day, 23 d after

RSLA A

A

=

−

×0

0

100

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the emergence of leaves 7 and 10. Measurements were performedusing an NADH-linked coupled spectrophotometric assay(Ward & Keys, 1989), based on the original methoddeveloped by Lilley & Walker (1974). Optimized assays ofthis type have been shown to yield data comparable toradioisotopic measurements of Rubisco activity (Keys &Parry, 1990; Reid et al., 1997). Assays were performed onleaves detached from four independent plants per treatment(two plants per chamber). Leaves were harvested individuallyat regular periods during the day and their area/fresh weightrecorded. Crude extracts were rapidly prepared byhomogenising c. 200 mg fresh leaf tissue in 6 ml of ice-cold extraction buffer containing 100 mM HEPES-KOH[pH 7.5], 15 mM MgCl2, 5 mM EGTA, 15% (w/w)polyethylene glycol [PEG 20 000] and 14 mMmercaptoethanol; adding 100% (w/w) insolublepolyvinylpolypyrrolidone [PVPP] immediately beforegrinding. The extracts were centrifuged at 12 000 g for 1 minat 4°C, then the supernatant immediately decanted into fresh1.5 ml Eppendorf tubes.

Initial Rubisco activity (i.e. the maximum activitymeasured as near as possible to the in vivo state of enzymeactivation) was assayed at 25°C immediately following thepreparation of crude extracts in a reaction mixture containing150 mM Bicine (pH 8.0), 25 mM NaHCO3, 20 mM MgCl2,3.5 mM ATP, 0.25 mM NADH, 5 mM phosphocreatine,80 nkat1 glyceraldehyde-3-phosphate dehydrogenase (EC1.2.1.12), 80 nkat 3-phosphoglyceric phosphokinase (EC2.7.2.3), 80 nkat creatine phosphokinase (EC 2.7.3.2),plus 50 µl extract. NADH oxidation was initiated in thespectrophotometer by the addition of RuBP (to provide afinal RuBP concentration of 0.5 mM). Total Rubisco activity(i.e. the maximum activity measured following the optimalachievable activation of the enzyme with Mg2+ and CO2) wasassayed following 15 min incubation of the reaction mixture

minus the three enzymes and RuBP at 25°C. Absorbancechanges were recorded at 340 nm using an automatedUV/Vis spectrophotometer (Unicam-Philips SP8700, Pye-Unicam Ltd, Cambridge, UK). Each assay was performed induplicate and was corrected for independent controls run forevery sample, to which RuBP was not added. The reaction waslinear over 5 min and enzyme rates were found to be directlyproportional to the amount of extract added to the reactionmixture. Rubisco activity was calculated from the changein absorbance over the first minute, based on a reactionstoichiometry of 2 : 1 (NADH : CO2). The activation state ofRubisco was estimated by expressing the initial activity as apercentage of the total activity. Data were expressed on a leaf areabasis to enable comparisons with in vivo estimates of Vc,max.

Chlorophyll fluorescence

Modulated chlorophyll fluorescence measurements weremade 8 h into the photoperiod at weekly intervals overthe first five weeks of the life-span of leaves 7 and 10.Measurements were made using a PAM-2000 fluorimeter(Walz, Effeltrich, Germany) with the minimum level offluorescence (Fo) obtained under modulated red light (2 µmolm−2 s−1, frequency 20 kHz) and maximal fluorescence yields(Fm and Fm′ ) recorded following exposure to a saturating lightpulse (0.8 s) of 8000 µmol m−2 s−1, provided by an 8-V/20 Whalogen lamp (Bellaphot, Osram). Fluorescence signals wereanalysed according to Genty et al. (1989): the relativequantum efficiency of PSII photochemistry (ΦPSII = [Fm′ – Fs]/Fm′ ) was measured in vivo under growth chamber conditions,while the maximum (or potential) quantum efficiency of PSIIphotochemistry (Fv/Fm = [Fm − Fo]/Fm) was measured after a40-min period of dark-adaptation.

Leaf surface morphology

Cured and rapid-set dental wash material (Wright HealthGroup Ltd, Dundee, Scotland) was applied to the abaxial and

1 One katal represents the conversion of one mole of substrate into one mole of product per second.

Fig. 1 Impact of O3 on non-photorespiratory CO2 evolution rate in the light (Rd) and the CO2 compensation point in the absence of Rd (Γ*). Values represent the mean (± SE) of 3 measurements made on the youngest fully expanded leaf of Plantago major ‘Valsain’ after 32 d exposure to either Charcoal/Purafil®-filtered air (CFA; < 5 nmol mol−1 O3) or O3 (< 15 nmol mol−1 overnight rising to a maximum between 12:00–16:00 hours of 75 nmol mol−1 O3). Squares, triangles and circles represent measurements made at PPFDs of 150, 400 and 600 µmol m−2 s−1, respectively. All measurements were made at a leaf temperature of 24 ± 1°C. Data in brackets represent the mean values of intercepts.

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adaxial surfaces of leaves 7 and 10 (9 d after leaf emergence)borne on 8 independent plants per treatment (4 plants perchamber). When set (c. 2 min), the wash material was peeledfrom the leaf surface, and cellulose acetate (CA) impressionsmade from the template. Randomly selected areas(7 × 0.1 mm2) were examined on each CA ‘film’ using a10 × 40 light microscope. The number of stomata andepidermal cells and the aperture of three stomata in each fieldof view were recorded. This enabled the estimation of stomataland epidermal cell densities (numbers per mm2 leaf surface);stomatal index (number of stomates expressed in terms of thenumber of epidermal cells per unit leaf area); stomatalaperture (pore size), and percentage pore area (the percentageof total leaf surface area occupied by stomatal aperture).

Statistical analyses

Statistical analyses were performed using SPSS (SPSS Inc.,Chicago, Il, USA). Data were first checked for normaldistribution and homogeneity of variation, then the influenceof chamber on measured variables determined by multivariateanalysis of variance (MANOVA). No significant chamber-to-chamber variation was found within treatments, so data werereanalysed using a reduced ANOVA model – under theassumption that plants in replicate chambers were as likely tobe as similar, or as different from, plants within an individualchamber. Time-course data were subject to RM-ANOVA,then data at each of the individual measurement dates werereanalysed and significant differences between treatmentswere determined using one-way ANOVA.

Results

Visible injury

Plants exposed to 15 nmol mol−1 O3 overnight rising toa maximum between 12 : 00 and 16 : 00 hours of75 nmol mol−1 over a period of 88 d (cumulative AOT40 =14960 nmol mol−1 h) developed no visible symptoms offoliar injury and no signs of O3-induced senescence duringthe measurement period.

Leaf gas exchange

Gas exchange measurements made 9, 23 and 45 d after leafemergence at a c. of 350 µmol CO2 mol−1 dry air (i.e. theCO2 concentration at which plants were grown) revealedmarkedly different effects on leaves of the same plant exposedto equivalent O3 concentrations over their life-span (Fig. 2).Ozone reduced Amax (P < 0.001) and Asat,350 (P < 0.0001) inleaf 7, but there were no significant effects on leaf 10.Although the magnitude of the O3 effect on leaf 7 appearedto increase with leaf age, statistical analysis indicated that theO3 × leaf age interaction was not significant at the 5% level.

Both leaves showed a significant (P < 0.0001) decline inAsat,350 with leaf age. Stomatal conductance, on the otherhand, was found to decline significantly (P < 0.05) followingexposure to O3 in both leaves; reductions in g s at growth CO2concentrations averaging 15% and 10% for leaves 7 and 10,respectively. Effects of O3 on A and g s were associated with asignificant (P < 0.004) increase in ci (+8 µmol mol−1 averagedacross measurement dates) in leaf 7, but there was nosignificant change in ci in leaf 10.

Figure 3 shows A /ci response curves constructed 9, 23 and45 d after the emergence of leaves 7 and 10 on plants exposedto CFA. Asat decreased in parallel with gs (Asat = 0.07gs – 4.2,r2 = 0.3607, P < 0.0001), but no significant shifts in RSLwere observed in O3-treated plants over the first 5 wks inthe life-span of leaves 7 and 10 (Fig. 4). These results implytherefore that the inhibition of CO2 assimilation by O3 wasprimarily caused by increased mesophyll limitations tophotosynthesis. The relationship between A and ci was usedto analyse those steps in the photosynthetic processes whichare affected by O3 under light-saturating conditions. Vc,max(P < 0.0001), Jmax,RuBP (P < 0.001) and TPU (P < 0.001)were reduced significantly by O3 in leaf 7 (Fig. 4) andalthough there was some suggestion that the effects of O3increased with leaf age, statistical analyses revealed nosignificant O3 × leaf age interaction (O3 × leaf age P > 0.05).No significant changes in Vc,max, Jmax,RuBP and TPU wereobserved in leaf 10 exposed to equivalent O3 concentrationsat the same stages of leaf development (see Fig. 4). Regressionof Vc,max against Jmax,RuBP revealed a strong linear relationshipbetween these parameters across the entire dataset (Fig. 5).

Measurement of in vitro Rubisco activity

Figure 6 shows the effects of O3 on Rubisco activitydetermined immediately following extraction (initial) andafter maximum achievable activation of the enzyme by Mg2+

and CO2 (total), 23 d after the emergence of leaves 7 and 10.No significant effects of O3 on initial and total Rubiscoactivity were evident 6 h into the photoperiod, followingexposure to 15 nmol mol−1 O3 overnight. However, 3 h intothe daily exposure to elevated O3 (a maximum between12 : 00 and 16 : 00 hours of 75 nmol mol−1) initial Rubiscoactivity was reduced by 27% (P < 0.05) in leaf 7 and thiseffect persisted throughout the rest of the day, with nosignificant recovery in Rubisco activity when theconcentration in the chambers returned to night-time levelslate in the day (16 : 00–20 : 00). The reduction in Rubiscoactivity could not be overcome by fully activating the enzymeand changes in total activity mirrored the effects on initialactivity (see Fig. 6). No significant effects of O3 on initial andtotal Rubisco activity were observed over the course of the dayin leaf 10. Assays indicated an activation state of 49 ± 1.9%for Rubisco, and O3 exposure resulted in no significantchange in the degree of enzyme activation.

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In vitro measurements of initial activity (Fig. 6) showedrelatively good agreement with in vivo estimates of Vc,maxderived from A/ci response curves (Fig. 4) given the fact thatdata were recorded on different plants at the same stage ofleaf development. However, in vitro estimates of initialRubisco activity were consistently lower than estimates ofVc,max (20–30% lower on average). The reason for this isunclear. Light microscopy and low temperature scanningelectron microscopy revealed no evidence to support anincrease in stomatal heterogeneity or stomatal patchinessfollowing the O3 treatment (Zheng, 1998). Thus, we believethe discrepancy between these datasets, which essentiallymeasure the same parameter, to arise from differences in leafdevelopment rates between experiments conducted undersimilar conditions, inhibition of enzyme activity by phenolicsubstances during the preparation of crude enzyme extractsand/or incomplete extraction of Rubisco during thehomogenisation of material for in vitro assay.

Chlorophyll Fluorescence

Chlorophyll fluorescence measurements tracked over thefirst 5 wk in the life-span of leaves 7 and 10 revealed nosignificant effects of O3 on the relative quantum efficiency ofphotosystem II (PSII) photochemistry (ΦPSII) measuredunder growth conditions or the maximum quantumefficiency of PSII photochemistry (Fv/Fm) (see Fig. 7). Bothparameters were observed to decline significantly (P < 0.001)as leaves aged, ΦPSII declining from 0.78 to 0.71 and Fv/Fmfrom 0.84 to 0.76, over a 5-wk period.

Leaf surface morphology

Measurements derived from epidermal impressions 9 d afterthe emergence of leaves 7 and 10 are shown in Table 1.Statistical analysis revealed that O3 significantly (P < 0.001)increased stomatal and epidermal cell densities on the adaxial

Fig. 2 Impact of O3 on the light- and CO2-saturated rate of CO2 assimilation (Amax), light-saturated CO2 assimilation rate at an ambient CO2 concentration of 350 µmol mol−1 (Asat,350), stomatal conductance to water vapour (gH2O) and intercellular CO2 concentration (ci) in Plantago major ‘Valsain’. Values represent the mean of measurements made on leaf 7 and 10, at 9, 23 and 45 days after leaf emergence (DALE). Plants were exposed to either Charcoal/Purafil®-filtered air (CFA; < 5 nmol mol−1 O3, open symbols) or O3 (< 15 nmol mol−1 overnight rising to a maximum between 12:00–16:00 hours of 75 nmol mol−1, closed symbols). Measurements were made on 4–6 independent plants per treatment at a PPFD of 1000 µmol m−2 s−1 at a leaf temperature of 24 ± 1°C. Significant differences from CFA are denoted: *, P < 0.05; **, P < 0.01.

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and abaxial surface of leaf 7, though the effect was dominatedby changes on the adaxial surface (O3* surface P < 0.01).Furthermore, O3-induced changes in stomatal densityexceeded those in epidermal cell density resulting in asignificant (P < 0.001) increase in the stomatal index onthe adaxial surface of leaf 7. There was again evidence ofcontrasting effects dependent upon leaf surface (O3 × surfaceP < 0.05) and stage of plant development (O3 × plant age P< 0.001). Ozone decreased (P < 0.001) average stomatalaperture on the adaxial (29–51%) and abaxial (9–68%) leafsurfaces and reduced (P < 0.001) percentage pore area onboth leaf surfaces.

Discussion

This study confirmed previous observations (Reiling &Davison, 1995; Zheng, Lyons & Barnes, 2000) thatenvironmentally-relevant O3 concentrations inhibitphotosynthesis and reduce stomatal conductance in sensitivelines of Plantago major in the absence of visible symptoms offoliar injury. Furthermore, substantial differences in responseto similar levels of O3 were found between leaves on the sameplant.

The O3-induced decline in Asat observed in leaf 7 (Figs 2and 3) was accompanied by a parallel reduction in Rubiscoactivity (measured both in vivo (Fig. 4) and in vitro (Fig. 6)),

Jmax,RuBP and TPU (Fig. 4). In comparison, only minorchanges in RSL were observed (Fig. 4). The O3-induceddecline in Rubisco activity appeared to be due to a reductionin the amount of Rubisco protein (see Western blots inZheng, 1998) and not simply to a deactivation of the existingenzyme (Fig. 6). This finding was supported by a decline inthe soluble protein content of leaf 7 (Zheng, 1998). Similareffects of O3 on Rubisco activity and content have beenreported in Triticum aestivum (Lehnherr et al., 1987; Farageet al., 1991; McKee et al., 1995), Pisum sativum (Farage &Long, 1995), Raphanus sativus (Atkinson et al., 1988), Populusmaximowizii × trichocarpa (Pell et al., 1992; Landry & Pell,1993), Solanum tuberosum (Enyedi et al., 1992; Eckardt &Pell, 1994), Medicago sativa (Pell & Pearson, 1983) andOryza sativa (Nakamura & Zaka, 1978) under a variety ofexperimental conditions. In some cases, the effects of O3 havebeen shown to be associated with changes in mRNA levelscoding for the LSU, but more often the SSU, of Rubiscobefore discernible effects on protein content are evident –suggesting that O3 may affect the biosynthesis of the enzyme(Pell et al., 1994b) as well as possibly enhancing the rate ofits degradation (Pell et al., 1997). However, the mechanismstriggering these processes remain unclear.

The maximum capacity for RuBP regeneration (Jmax,RuBP),modelled from A/ci response curves, was found to decline inparallel with the change in Vc,max in leaf 7 following exposure

Fig. 3 Impact of O3 on A/ci relationships of Plantago major ‘Valsain’. A/ci curves were constructed for 4–6 independent plants per treatment with measurements made on leaves 7 and 10, at 9, 23 and 45 d after leaf emergence (DALE). Plants were exposed to either Charcoal/Purafil®-filtered air (CFA; < 5 nmol mol−1, open symbols) or O3 (< 15 nmol mol−1 overnight rising to a maximum between 12:00–16:00 of 75 nmol mol−1, closed symbols). A/ci relationships were monitored under a saturating PPFD (1000 µmol m−2 s−1) at a leaf temperature of 24 ±1°C.

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to O3 (Fig. 4). Moreover, there was a linear relationshipbetween Vc,max and Jmax,RuBP (Fig. 5) across treatmentssuggesting that plants preserve a close functional balance inthe allocation of resources between these processes. Similarobservations of a tight coupling between Vc,max and Jmax,RuBPhave been made in a range of species across a range ofexperimental conditions and have been interpreted to reflectadjustments within the photosynthetic apparatus facilitatingthe optimal utilization of available resources, especially

Fig. 4 Impact of O3 on relative stomatal limitation to photosynthesis (RSL), in vivo maximum rate of Rubisco carboxylation (Vc,max), maximum rate of electron transport contributing to RuBP regeneration (Jmax,RuBP) and rate of triose phosphate utilization (TPU) of Plantago major ‘Valsian’. Data were derived from modeled A/ci curves. Values represent the mean (± SE) of 4–6 independently constructed curves for different plants. Measurements were made on leaf 7 and 10, at 9, 23 and 45 d after leaf emergence (DALE)). Plants were exposed to either Charcoal/Purafil®-filtered air (CFA; < 5 nmol mol−1 O3, open symbols) or O3 (< 15 nmol mol−1 overnight rising to a maximum between 12:00–16:00 hours of 75 nmol mol−1, closed symbols). Measurements were made at a PPFD of 1000 µmol m−2 s−1 at a leaf temperature of 24 ± 1°C. Significant differences from CFA are denoted: *, P < 0.05; **, P < 0.01.

Fig. 5 Relationship (Vc,max = 0.35 + 0.31 Jmax,RuBP; r2 = 0.872, P < 0.0001) between in vivo maximum rate of Rubisco carboxylation (Vc,max) and maximum rate of electron transport contributing to RuBP regeneration (Jmax,RuBP) of Plantago major ‘Valsain’. Values were derived from A/ci response curves and each point represents the mean of 4–6 independent measurements per treatment. Plants were exposed to either Charcoal/Purafil®-filtered air (CFA; < 5 nmol mol−1 O3, open symbols) or O3 (< 15 nmol mol−1 overnight rising to a maximum between 12:00–16:00 hours of 75 nmol mol−1, closed symbols). A/ci relationships were measured at saturating PPFD (1000 µmol m−2 s−1) at a leaf temperature of 24 ± 1°C.

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nitrogen (Wullschleger, 1993). Since O3-induced reductionsin Jmax,RuBP, were found in the absence of equivalent changesin ΦPSII and Fv/Fm (Fig. 7), it was concluded that the maximumrate of RuBP regeneration was not limited by effects of O3 onPSII photochemistry. This finding corroborates the conclusionsdrawn by previous authors who have investigated the effectsof O3 on PSII activity (Farage et al., 1991; Baker et al. 1994;Farage & Long, 1995). Effects on Jmax,RuBP possibly reflectedan inability to regenerate RuBP via the Calvin cycle followingO3-treatment, as a result of the reduced activities of regenerative

Calvin cycle enzymes. These enzymes are believed to be undera high degree of self-regulation (Geiger & Servaites, 1994)and a reduction in RuBP regeneration capacity might beexpected in situations where there is a marked decline inVc,max (Long & Drake, 1992). Effects on Jmax,RuBP similarto those induced by O3 in the present study have beenreported following the exposure of leaves to damaging levels ofultraviolet-B radiation (Allen et al., 1997; Baker et al., 1997).

Modelled data suggested that O3 may also reduce triosephosphate utilization resulting in enhanced limitation of

Fig. 6 Impact of O3 on initial (squares) and final (circles) Rubisco activity on leaves of Plantago major ‘Valsain’. In vitro measurements of Rubisco activity were made over the course of a single day, on the youngest fully expanded leaf after 28 (leaf 7) and 42 d (leaf 10) exposure to either Charcoal/Purafil®-filtered air (CFA; < 5 nmol mol−1 O3, open symbols) or O3 (< 15 nmol mol−1 overnight rising to a maximum between 12:00–16:00 hours of 75 nmol mol−1, closed symbols). Each value represents the mean (± SE) of four independent assays performed on leaves from different plants. Significant differences from the CFA are indicated: *, P< 0.05.

Fig. 7 Impact of O3 on the relative quantum efficiency of PSII photochemistry (ΦPSII) and the maximum quantum efficiency of PSII photochemistry (Fv/Fm) in leaves of Plantago major ‘Valsain’. Measurements were made on leaf 7 and 10, at 7, 16, 23, 30 and 37 d after leaf emergence (DALE). Plants were exposed to either Charcoal/Purafil®-filtered air (CFA; < 5 nmol mol−1, open symbols) or O3 (< 15 nmol mol−1 overnight rising to a maximum between 12:00–16:00 hours of 75 nmol mol−1, closed symbols). Values (± SE) represent measurements made on 5 independent plants per treatment. Ozone effects were not significant at the 5% level.

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photosynthesis via the reduced availability of inorganicphosphate (Pi). A/ci measurements performed on leaf 7 in21% and 2% oxygen revealed no significant increase in Asat onswitching between O2 concentrations (data not shown) lendingsupport to this conclusion (Sharkey et al., 1986). Moreover,O3 exposure resulted in the enhanced accumulation of non-structural carbohydrates (mainly starch) in leaf 7, but not inleaf 10 (Zheng et al., 2000) – a situation that could result intop-down or bottom-up repression of Rubisco activity and otherphotosynthetic components in order to retain the functionalbalance between rates of CO2 fixation and carbohydrateutilization (Goldschmidt & Huber, 1992).

The O3-induced reduction in Asat in leaf 7 was accompa-nied by a parallel decline in conductance (15% lower onaverage). However, only minor changes in RSL were observed(Fig. 4). This indicates that the decline in gs was not respon-sible for the decline of Asat, an argument strongly supportedby the absence of any change in Asat in leaf 10 despitesignificantly lower stomatal conductance in O3-treated plants(Fig. 2). The extent of the decrease in g s induced by O3 wasgreater in leaf 7 than leaf 10, consistent with the view thatchanges in g s were, at least in part, a response to the increasein ci induced via the negative effects of O3 on photosyntheticmetabolism. However, similar effects could not explain theO3-induced decline in gs observed in leaf 10. Lightmicroscopy revealed that the reduction in gs induced by O3in both leaves was due to a decline in stomatal aperture, andnot to a reduction in stomatal density (Table 1), so the datafor leaf 10 suggest that ‘direct’ effects of O3 on stomatalaperture may have contributed to the O3-induced decline ings observed in the present study. This conclusion is supportedby other reports of O3-induced changes in gs independent ofeffects on the capacity for CO2 assimilation (Kleier et al.1998; Torsethaugen et al. 1999). Indeed, recent studies indicatethat gs may rise or fall in response to O3 damage, dependent ongenotype, O3 concentration, and the sensitivity of stomatalguard cells to O3 relative to that of surrounding epidermalcells (Robinson et al., 1998). Improved understanding of thetiming and mechanisms underlying stomatal closure inresponse to O3 is vital if the role that changes in stomatalaperture play in the avoidance of pollutant uptake is to beelucidated. Stomatal closure may afford little or no usefulprotection if, as it appears, the stomata close predominantly asa consequence of, rather than before, detrimental effects onleaf metabolism.

Marked differences were observed in the effects of O3 onphotosynthetic metabolism between leaves on the same plant,despite the fact that leaves were exposed to equivalent O3exposures at similar stages in their development. This findingis consistent with growth studies on Plantago major whichhave revealed a marked reduction in the impacts of O3 withplant age (Lyons & Barnes, 1998; Zheng, 1998), with similarshifts in O3 resistance reported for other species (Soja et al.,2000). The mechanisms underlying these shifts in resistance, Ta

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© New Phytologist (2002) 155: 67–78 www.newphytologist.com

Research 77

and whether they are triggered by the prior exposure to O3(i.e. ‘acclimation’) or are associated with plant developmentalstatus, remain to be elucidated. Measurements of stomatalconductance (see Fig. 2) suggested that the contrasting effectsof O3 on leaf 7 and 10 were not mediated by differences in O3uptake – a finding consistent with the view that shifts in O3resistance triggered by prior exposure to O3 and/or plantontogeny are linked to changes in the tolerance of tissuesfollowing uptake (Schraudner et al., 1998). Indeed, the reducedimpacts of O3 on leaf 10 vs leaf 7 are consistent with thesystemic induction of cellular oxidative defence and repairsystems under O3 stress (see Rao, Koch & Davis, 2000).

It is concluded that a loss of Rubisco protein constitutes theprimary cause of the O3-induced decline in CO2 assimilationin Plantago major. The effect would appear to be accompaniedby the down-regulation of the activity of other Calvin cycleenzymes and a possible increase in Pi limitation. Changes inthe quantum efficiency of PSII were not involved with theO3-induced inhibition of CO2 assimilation. Findings relatingto the effects of O3 on the photosynthetic physiology of otherspecies show the same basic pattern of effects (Farage & Long,1995). It remains to be established whether such shifts inphotosynthetic metabolism are driven by ‘direct’ effects of O3(or it’s reactive dissolution products) on Rubisco or whether theyarise as a consequence of the enhanced accumulation ofnonstructural carbohydrates in foliage. Although stomatalconductance declined in parallel with CO2 assimilation in O3-treated plants this did not account for the observed decline inphotosynthesis. The change in stomatal conductance wasfound to be due to a reduction in stomatal aperture, ratherthan an effect on the number of stomates per unit leafarea, with data suggesting evidence of ‘direct’ effects of thepollutant on the stomatal complex as well as effects mediatedthrough shifts in ci. Leaves on the same plant exposed toequivalent levels of O3 showed striking differences in theimpacts of the pollutant on photosynthetic capacity withthe pattern of effects consistent with the induction ofdefence and repair systems following O3-induced oxidativechallenge.

Acknowledgements

We express our sincere thanks to Tom Lyons (NewcastleUniversity), Kate Maxwell (Cambridge University), Ian McKee(Essex University), Chris Jeffrey (Edinburgh University) andFred Last (retired) for assistance during the course of thisstudy. The authors are indebted to The Royal Society, TheSwales Trust and the British Overseas DevelopmentAdministration for financing the study.

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