PhotosynthesisResearch 45:111-119, 1995. © 1995KluwerAcademicPublishers° Printedin theNetherlands. Regular paper

The interactive effects of elevated CO2 and 03 concentration on photosynthesis in spring wheat I.E McKee, P.K. Farage & S.R Long Department of Biology, University of Essex, Wivenhoe Park Colchester, Essex C04 3SQ, UK Received24 March 1995;acceptedin revisedform20 June 1995 Key words: carbon dioxide, gas exchange, ozone, RuBisCO, stomata, Triticum aestivum L.

Abstract

This study investigated the interacting effects of carbon dioxide and ozone on photosynthetic physiology in the flag leaves of spring wheat (Triticum aestivum L. cv. Wembley), at three stages of development. Plants were exposed throughout their development to reciprocal combinations of two carbon dioxide and two ozone treatments: [CO2] at 350 or 700/zmol mo1-1, [03] at < 5 or 60 nmol mo1-1. Gas exchange analysis, coupled spectrophotometric assay for RuBisCO activity, and SDS-PAGE, were used to examine the relative importance of pollutant effects on i) stomatal conductance, ii) quantum yield, and iii) RuBisCO activity, activation, and concentration. Independently, both elevated [CO2] and elevated [03] caused a loss of RuBisCO protein and Vcmax.In combination, elevated [CO2] partially protected against the deleterious effects of ozone. It did this partly by reducing stomatal conductance, and thereby reducing the effective ozone dose. Elevated [03] caused stomatal closure largely via its effect on photoassimilation. Introduction

Atmospheric carbon dioxide concentrations have fluctuated over many millennia (Bamola et al. 1987). In the industrial era, the global atmospheric CO2 concentration has risen significantly (Neftel et al. 1985), and further large rises are predicted for the next century and beyond (IPCC 1992). In addition to its contribution to the 'greenhouse effect', atmospheric [CO2] has a direct impact on plant physiology and crop production (Bowes 1993). In parallel with the recent trend in [CO2], tropospheric ozone concentrations have been rising (IPCC 1992). In industrially and agriculturally developed regions there is a rising frequency of high [03] episodes, and the projected impact on agricultural production is considerable (Chameides et al. 1994). Fundamental to the broad range of secondary effects resulting from growth under elevated [CO2] are the direct responses of stomatal conductance and photosynthetic metabolism. Elevated carbon dioxide concentrations generally result in reduced stomatal conductance (Morison 1985). In C3 plants, a reduction

in ribulose-l,5-bisphosphate carboxylase/oxygenase (RuBisCO) activity and concentration has often been reported (Sage et al. 1989; McKee and Woodward 1994a), despite which, a shift in photoassimilatory control away from RuBisCO limitation may occur (Socias et al. 1993; McKee and Woodward 1994a). Growth at elevated ozone levels affects crop physiology in superficially similar ways. There may be a reduction or an increase in stomatal conductance (Darrail 1989; Hassan et al. 1994), a reduction in RuBisCO activity and amount (Pell and Pearson 1983; Lehnherr et al. 1987; Dann and Pell 1989; Farage et al. 1991), and eventually a decline in quantum use efficiency (Nie et al. 1993). The coincident rise in concentration of these key atmospheric pollutants, their effects on crop physiology and yield, and the apparently similar loci of their respective actions make the study of the interaction of their effects important, both in order to understand the physiology and for predictive purposes. There is little published evidence of the combined effects of carbon dioxide and ozone on crops (Kramer et al. 1991;

112 Barnes and Pfirrmann 1992; Polle et al. 1993; Heagle et al. 1993). The mechanisms of interaction are unclear from these reports, but there is an indication that elevated [CO2] may protect against the deleterious effects of ozone. This study investigated the following possibilities. i) Carbon dioxide and ozone effects on RuBisCO activity may be realised by a decline in RuBisCO activation state, or by a decline in RuBisCO concentration. ii)Elevated [CO2] may enhance the deleterious effects of elevated [03] if the decline in RuBisCO activity caused by each pollutant separately is simply additive in combination. iii) Elevated [CO2] may negate the effect of elevated [03] on RuBisCO by shifting photoassimilatory control away from RuBisCO limitation. iv) Elevated [CO2] may protect against the deleterious effects of elevated [03] by reducing stomatal conductance, and thereby reducing the uptake of ozone. v) Ozone may have a direct effect on guard cell turgor, or may have an indirect effect on stomata/conductance via its action on the mesophyll; a reduction in photoassimilatory rate may cause a rise in intercellular [CO2] (Ci) and, thereby, a reduction in stomatal aperture. vi) The effects of elevated [CO2] and [03] on photoassimilatory metabolism may vary during leaf development. In order to examine these possibilities, spring wheat was chosen as a model crop; its photosynthetic physiology has been widely studied. In order to study photosynthetic responses during the grain-filling period, and so that examining changes in treatment interaction during leaf development should not be confused with shading effects, the flag leaf was chosen as the model leaf.

ture was controlled with a 24/18 °C day/night regime, vapour pressure deficit (VPD) was controlled at 0.5 kPa, and the plants were grown in 0.65 dm 3 pots, in an inorganic medium (Silvaperl; William Sinclair Horticultural Ltd, Gainsborough, UK), watered to saturation on alternate days with deionised water or a modified Shive's solution (McKee and Woodward 1994b). Air entering each chamber was filtered of particulates and contained negligible ozone. Plants were subjected to two [CO2] treatments in combination with two [03] treatments. [CO2]and [03] were controlled using a computerised feedback-control algorithm. Pollutant concentrations were measured in each chamber sequentially using an infra-red [CO2] analyser (model WMA-2; PP-Systems, Hitchin, UK) and a UV [O3] analyser (model 1008-AH; Dasibi Environmental Corp., Glendale, USA; calibrated by Quantitech Ltd., Milton Keynes, UK). The algorithm compared the measured concentration with the pre-set concentration. Using solenoids and mass-flow controllers it then adjusted the dose rates of pure carbon dioxide (Certificated by Linde Gas UK Ltd, Stoke-on-Trent, UK) and ozone (generated from pure oxygen using an electrical discharge generator (model BA 023012; Wallace and Tiernan, Tonbridge, UK)) accordingly. [CO2] was controlled at a constant 350 #mol mo1-1 or 700 #mol mo1-1 with negligible fluctuation. [O3] was controlled in a sine wave form during the photoperiod peaking at either < 5 nmol mol- 1 or 60 nmol mo1-1. In order to segregate treatment effects from chamber effects, and to reduce the effect of environmental heterogeneity within the chambers, plants and associated treatments were randomised between chambers, and plants were randomised within chambers, on alternate days throughout the experiment. Thus the variance associated with slight chamber differences was homogenised across all treatments. Plants were spaced to avoid co-shading so that the response of one plant to a treatment should not affect the response of another.

Materials and methods

Sampling

Plant growth conditions

Flag leaves from main stems were analysed at three stages of plant development: at panicle emergence (I), at seventh leaf senescence (II), and at eighth (penultimate) leaf senescence (III). At the final analysis point, early signs of senescence were appearing at the tip of the flag leaves. At each stage, gas exchange analysis was conducted to measure maximum quantum efficiency of carboxylation (q~), maximum carboxyla-

Spring wheat plants (Triticum aestivum L. cv. Wembley) were grown in controlled environment chambers (model SGC660/C/HQI; Sanyo Gallenkamp, Loughborough, UK) illuminated at a mean photosynthetically active radiation (PAR) fluence rate of 645 #mol m -z s -1 over a 16/8 hour day/night cycle. Tempera-

113 tion velocity at the in vivo state of activation (Vcmax), and stomatal conductance (gs). At the same time, flag leaves were also harvested for analysis of RuBisCO activity and activation state in vitro, and for SDSPAGE analysis of RnBisCO protein concentration. For each analysis, measurements were independently replicated four times. Data were tested for normality and analysed using ANOVA and Student's t-test to compare unpaired sample sets.

Gas exchange analysis Plants were selected from each treatment at random for measurement between 3 and 7 hours into the photoperiod. Over this period there was no detectable trend in photosynthetic response. Gas exchange studies were conducted using a programmable gas exchange system (model MPH-1000; Campbell Scientific Inc., Logan, USA) incorporating an infra-red gas analyser (model LI-6262; LiCor, Lincoln, USA) and a Peltier temperature controlled leaf cuvette. The gas analyser was calibrated against standards every week, and the zero calibration was adjusted on every measurement day. The MPH-1000 was modified by the addition of a semi-automatic zero calibration device; this allowed easy recalibration of zero-shift during use. PAR was controlled to within 10 #mol m -2 s -1 of growth value, using the same type of lamp as in the growth chambers (model HQI-E400W/DV; Urban Enviroscape Ltd., UK). Leaf temperature and dewpoint temperature were controlled at 24 °C and 18 °C, respectively. Leaves were equilibrated in the leaf chamber at growth Ci prior to measurement of gs, Vcmax and ~b. Vcmax was calculated as in McKee and Woodward (1994a); A/Ci curves were plotted close to the Ci compensation point. ~bwas calculated from the initial slope of A/intercepted PAR fluence rate, measured in saturating [CO2] rather than reduced [O2], according to Ehleringer and Bjorkman (1977). In order to test whether the effect of [03] on stomatal conductance was attributable to a rise in Ci, caused by a decline in the rate of photoassimilation, gs of control and ozone treated flag leaves was compared. For the ozone treated leaves, the carbon dioxide concentration in the leaf cuvette was then manipulated to lower Ci to the mean level found in control leaves. After half an hour, gs was remeasured to determine whether it had acclimated to control levels. At stage II, full A/Ci curves were plotted, and growth Ci was estimated as in McKee and Woodward (1994a), so as to determine whether

photoassimilatory control shifted away from RuBisCO limitation in the elevated [CO2] treatments.

Assay of RuBisCO activity and activation Flag leaves were equilibrated in the leaf chamber as for gas exchange measurements in order both to allow a direct comparison between in vivo and in vitro measurements and to standardise conditions, reducing systematic variance. The portion of leaf within the chamber was removed, rapidly frozen and stored in liquid nitrogen (McKee and Woodward 1994a). The procedures for the extraction and enzymatic assay of RuBisCO activity and activation were modified from those outlined in Ward and Keys (1989). Chemicals were obtained from the Sigma Chemical Company UK Ltd., (Poole, UK). The extraction buffer contained 100 mol m -3 Bicine adjusted to pH 8, 20 mol m -3 MgC12, 50 mol m -3 DTT, 1% Bovine Serum Albumin and 0.2% PVPP by mass. Leaf portions (approximately 350 mm 2) were extracted in 3000 mm 3 of buffer, in liquid nitrogen, in a mortar. Each homogenate was rapidly thawed by agitation and immediately centrifuged at 12000 g and 0 °C. A sample of the supernatant sol was transported on ice for immediate photometric assay of RuBisCO activity and activation state. The assay buffer contained 100 mol m -3 Bicine adjusted to pH 8.2, 50 mol m -3 DTT, 10 mol m -3 NaHCO3, 20 mol m -3 MgC1 2, 20 mol m -3 NaC1, 0.66 mol m -3 RuBP, 42 tool m -3 NADH, 5 mol m -3 ATP, 5 mol m -3 phosphocreatine, 100 #kat mm -3 PGK, 100 #kat mm -3 GAPdH, 150/zkat mm -3 creatine phosphokinase. For each extract two assays were conducted; one to assay the initial activity of RuBisCO (i.e. the maximum activity measured at (as near as possible) the in vivo state of activation), the other to assay the activated activity (i.e. the maximum activity measured after the maximum achievable activation of the enzyme with Mg 2+ and CO2). The activation state could then be estimated by expressing the initial activity as a percentage of the activated activity. The two assays were conducted at 25 °C, in stirred quartz cuvettes. For initial activity assay, 40 mm 3 of extract was added to 960 mm 3 of buffer. For activated activity assay the extract was preincubated with the Bicine, DTT, NaI-ICO3, and MgC12 components for seven minutes at 25 °C (lower temperatures were found to give incomplete activation) prior to starting the reaction with the other constituents. In each case, the activ-

114 ity of RuBisCO was measured using the absorbance change at 340 nm, i.e. the rate of NADH depletion which, in this coupled reaction, is double the rate of carboxylation.

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Assay of RuBisCO concentration Leaf portions (approx. 350 1ran3) were extracted in 3000 mm 3 of a buffer containing 60 mol m -3 Tris, pH 6.8, 50 mol m -3 DTT, and 7% activated charcoal powder by mass, in liquid nitrogen, in a mortar. Each homogenate was rapidly thawed by agitation and immediately centrifuged at 15 000 g and 0 °C for 30 minutes. This extraction procedure yielded the highest recovery of RuBisCO of any we tried. The protein concentration of the supernatant was estimated using the bicinchonic acid assay system (Pierce, Rockford, IL, USA) according to the manufacturer's protocol. The proportion of protein attributable to RuBisCO was determined by SDS-PAGE separation on 10-17.5% gradient polyacrylamide gels using an electrophoresis apparatus (model AE-6450; Atto Corporation, Tokyo, Japan). The gels were stained with Fast Green stain and were scanned using a laser-scanning densitometer (Model 300A, Molecular Dynamics, Sunnyvale, CA, USA). The linearity of the densitometer was calibrated against protein standards, and the gels were loaded so as to keep RuBisCO densities within the calibrated range.

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All results are presented relative to growth stages I, II and III. This developmental chronology differs slightly from calendar time because the ozone treatment causes a slight acceleration of development and senescence (data not shown). At each developmental stage, all treatments had completed an approximately equal proportion of flag leaf duration (to within 18 h). The results of the in vivo gas exchange analyses are presented in Fig. 1. Maximum intercepted quantum yield declined with leaf age (Fig. 1a). At no stage did the elevated [CO2] treatment affect ~band only in early senescence (stage III) did the ozone treatment cause a marginally significant decline. In combination, elevated [CO2] appeared to protect against the late effect of elevated [03] on qS. Since ~bwas measured in saturating [CO2], changes in this parameter indicate changes in the efficiency of thylakoid related processes.

III

Growth Stage Fig. 1. The effects of elevated [CO2] and [03] on: (a) quantum yield of photoassimilafion (b) maximum carboxylation velocity measured in vivo (c) stomatal conductance Each parameter was measured using the flag leaves of spring wheat (T. aestivum L. cv. Wembley), at three stages of development: I panicle emergence, II seventh leaf senescence, III eighth leaf senescence. The plants were grown throughout their development under one of four treatment combinations: ~ control conditions (350 Izmol mo1-1 [CO2], < 5 nmol tool -1 [O3]), ~ with elevated [CO2] (700 #mol mo1-1 [CO2]), illii!I!~liwith elevated [03] (peak 60 nmol mo1-1 [O3]), or ~ with elevated [CO2] and [03] (700 izmol mo1-1 [CO2], peak 60 nmol mol- l [03]). Each mean represents measurements on four replicate leaves and the statistical significance of Student t-tests, comparing each treatment with the control, is shown using three logistic levels of null hypothesis probability: *p < 0.08, **p < 0.04, ***p < 0.02. Three way ANOVA showed that the interaction between carbon dioxide and ozone effects on maximum carboxylation velocity varied significantly with the developmental stage (***).

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Maximum carboxylation velocity, determined in vivo, declined with leaf age (Fig. lb). At panicle emergence (stage I) elevated [CO2] caused a decrease in Vemax. Relative to the control, this decrease became less pronounced with leaf age. Initially [03] had little effect o n Vcmax; the ozone induced decline became more pronounced with leaf age. In the young leaf (stage I), the effects of [CO2] and [03] on Vcmax were roughly additive. By stage II, elevated [CO2] appeared to p r o t e c t a g a i n s t t h e d e t r i m e n t a l effect o f o z o n e o n Vcm~x; this p r o t e c t i v e e f f e c t c o n t i n u e d into e a r l y senescence.

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G r o w t h Stage Fig. 3. The effects of elevated [CO2] and [03] on: (a) maximum carboxylation velocity measured in vitro, at the in vivo activation state, b) maximum carboxylation velocity measured in vitro, following maximally achievable activation with CO2 and Mg2+ (c) activation state, i.e. expressing data from (a) as a percentage of (b). All other information is as for Fig. 1.

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Stomatal conductance remained stable, declining slightly in early senescence (Fig. lc). At all stages, elevated [CO2] caused a similar reduction in gs. Elevated [03 ] induced a decline in gs and the effect became more pronounced with leaf age. The effects of carbon dioxide and ozone in combination were not simply additive; in the presence of elevated [CO2], elevated [03] did not induce a statistically significant decline (relative to elevated [CO2] alone) until stage III (p < O.O4). Despite the decline in Vcmax (Fig. lb), elevated [CO2] caused a shift away from the RuBisCO limited region of the A / Ci response curve (Fig. 2). Figure 3 shows data from the in vitro analysis of RuBisCO activity and activation state. The initial activity (Fig. 3a), which relates directly to the Vcmax measured in vivo (Fig. lb), declined with leafage. The effects of [CO2] and [03] paralleled the pattern seen in vivo. Changes in Vcmax and the initial activity in vitro appeared to be driven largely by changes in the activated RuBisCO activity (Fig. 3b). This parameter relates directly to the concentration of RuBisCO protein (Fig. 5). At panicle emergence, both elevated [CO2] and [03] caused a marginally significant decline in activation state (Fig. 3c), though, in combination, the effect was not significant. By stage III, the RuBisCO activation state of the ozone treated flag leaves

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The effectsof elevated [CO2] and [03] on: (a) the concentration of RuBisCO protein (b) the activatabilityof RuBisCO, i.e. the activatedRuBisCO activity(Fig. 2b) expressedper unit of protein (Fig. 4a). All otherinformationis as for Fig. 1. Fig. 5.

had risen significantly. Otherwise, changes in activity were attributable to changes in the maximally activated RuBisCO activity. The relationship between the initial activities (Fig. 3a) and Vcmaxvalues (Fig. lb) is shown in Fig. 4. The in vitro values were generally lower than, and a fairly constant proportion of, the in vivo values. However, in the elevated [O3] treatment, the ratio rose above equality at stage III (see discussion below). Changes in the amount of RuBisCO protein (Fig. 5a) followed, and confirm, the pattern seen in the activated RuBisCO activity (Fig. 3b). The relationship between the amount of RuBisCO protein and the activated RuBisCO activity is shown in Fig. 5b where activated RuBisCO activity is expressed per unit of protein rather than per unit of area. This parameter dif-

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Growth Stage Fig. 6. The mechanism of the effect of ozone on stomatal conductance. Plants from the control treatment ~ were compared with plants from the elevated [03] treatment ~ For the ozone treated leaves, the carbon dioxide concentration in the leaf cuvette was then manipulated to lower C~ to the mean level found in control leaves ~i!~!iiii~iiAll other information is as for Fig. 1.

fers from the specific activity of RuBisCO because it uses the activated RuBisCO activity, not Vcmax.Rather, it relates to the 'activatability' of RuBisCO. The activatability was stable except that, in the elevated ozone treatment, it declined in early senescence. At stages I and II the effect of ozone on stomatal conductance could be removed by adjusting Ci to control levels (Fig. 6). At stage III, the effect of elevated [03] was not completely removed by this adjustment.

Discussion The late effect of [03] on ~b (Fig. la, stage III) is consistent with earlier investigations on wheat in which the related parameter ~bpsu declined only in the oldest portions of leaf (Nie et al. 1993) or following high, acute exposure (Farage et al. 1991). The same proportion of flag leaf duration had been completed in all treatments at stage III so that, even taking account of the ozone induced acceleration of senescence (Ojanpera et al. 1992), the decline in q5 appeared ahead of the general senescent trend. The most complex interaction between [03] and [CO2] responses was seen in their combined effect on RuBisCO activity (Fig. lb, Fig. 3). The interaction

changed with leaf maturity. From the in vivo data (Fig. lb) it was found that the carbon dioxide induced reduction in Vcmax appeared earlier than the ozone effect (stage I). As the ozone effect became more pronounced (stages II and III), elevated [CO2] protected against the decline in activity. Many studies on wheat and other crops have shown a decline in RuBisCO activity caused by elevated [03] (Lehnherr et al. 1987; Dann and Pell 1989; Farage et al. 1991; Pell et al. 1992) or by elevated [CO2] (Porter and Grodzinski 1984; Vu et al 1987; Sage et al. 1989; McKee and Woodward 1994a). Some studies have shown no decline or an increase in RuBisCO activity in response to elevated [CO2] (Arp and Drake 1991). This study leads to the important finding that in the presence of moderately elevated [03] (the peak concentrations used in this study are already frequently encountered in the field (PORG 1993) and are in line with predicted values for the next century), elevated [CO2] may lead to an apparent increase in RuBisCO activity attributable to its protective role against ozone damage. In addition to protecting against the ozone induced loss of RuBisCO activity, elevated [CO2] also converts the residual loss of activity from a loss of usable activity to a loss of excess activity by shifting control of photoassimilation away from the RuBisCO limited region of the A/Ci response (Fig. 2). The in vivo Vcmax data (Fig. lb) were supported by the in vitro measurement of initial RuBisCO activity (Fig. 3a). There is a slight, but revealing, discrepancy between these data sets which both aim to measure essentially the same parameter (Fig. 4). As the leaf ages, the ratio of the in vitro/in vivo values rises in the elevated [03] treatment. This may have been due to patchy assimilation, leading to an underestimation of Vcmax in vivo (Terashima et al. 1989; Cardon et al. 1994), which emphasises the need for in vivo estimation of Vcmax to be corroborated by in vitro assay. The question of whether the pattern of Vemaxvariation was attributable to changes in activation state or to variation in the concentration of RuBisCO was investigated (Fig. 3b,c and Fig. 5). Apart from a marginally significant indication that, at least initially, a decline in percentage activation may have been involved (Fig. 3c, stage I), later reductions in Vcr~x were not driven by reductions in activation state but correlated well with the pattern of activated RuBisCO activity (Fig. 3b) for all treatment combinations. A more detailed study would be required to determine whether a decline in activation state precedes the decline in activated RuBisCO activity seen in elevated [03] and in elevat-

118 ed [CO2]. Dann and Pell (1989), working on ozone effects on Solanum tuberosum, also found no change in RuBisCO activation state, and Enyedi et al. (1992) found no difference in the sulphydryl content of RuBisCO between ozone sensitive and insensitive cultivars. By stage III the activation state in the elevated [03] treatment had actually risen. This may indicate that ozone induced RuBisCO loss was driven by a process independent of mechanisms involved in optimising the activity of RuBisCO. The activated RuBisCO activity (Fig. 3b) corresponds to the maximally activatable RuBisCO activity under the conditions of assay. Changes in this parameter could relate to changes in the concentration of enzyme or to changes in the activatability of RuBisCO, e.g. due to inhibition, dissociation or modification. Assay of RuBisCO concentration (Fig. 5a) showed that concentration changes were largely responsible for the pattern of activated RuBisCO activity variation. At stage III, there is a slight decline in activatability in the elevated [03] treatment (Fig. 5b). If this indicates dissociation or modification prior to proteolysis, then, at least at this late stage, a greater rate of loss of RuBisCO may be involved, in addition to (or rather than) a reduced rate of synthesis. Studies on other species have shown that the protective role of elevated [CO2] against ozone damage could not be attributed to an increase in either antioxidant status (Polle et al. 1993) or polyamine levels (Kramer et al. 1991). The results of this study are consistent with the hypothesis that elevated [CO2] protects against ozone damage via a reduction in stomatal conductance, reducing the flux density and total dose of ozone to the mesophyll (Fig. lc). Other changes in sensitivity may occur (Badiani et al. 1993; Heath 1994; Kangasjarvi et al. 1994), but the stomatal effect seems to be dominant in this species. Other studies have shown that whilst stomatal closure in elevated [CO2] is an almost universal phenomenon (Mott 1990), stomatal conductance may rise or fall in response to ozone damage, dependent on the species and the sensitivity of guard cells relative to the surrounding epidermal cells (Darrall 1989; Hassan et al. 1994). The question of whether ozone has a direct effect on conductance or whether it acts via its effect on assimilation (causing a rise in Ci and thereby reduced stomatal conductance) was investigated (Fig. 6). The reversal of the ozone induced decline in gs, achieved by manipulating Ci for the treated leaves down to control levels, indicates that the ozone effect on conductance was attributable to Ci feedback from the reduction in photoassimilation. At

the latest stage of measurement this mechanism could not entirely account for the ozone effect. An additional mechanism may either involve a direct action on guard cell membranes, or damage to mesophyll cells may elicit a 'plant growth regulator' response. In conclusion, both elevated [CO2] and elevated [03] caused a loss of RuBisCO protein leading to a decline in Vcmax. In the case of elevated [CO2] this constitutes a loss of excess activity (Long 1991; McKee and Woodward 1994a), whereas, in the case of elevated [O3], this constitutes a loss of usable activity, leading to a reduction in photoassimilation rate (Reich and Amundson 1985; Lehnherr et al. 1987; Farage et al. 1991). The effects of both pollutants were not simply additive in combination. The effects of both pollutants are likely to be less pronounced at the whole canopy level than at the individual flag leaf level (Grandjean Grimm and Fuhrer 1992; Amthor et al. 1994), though, as the treatment levels in this study are not excessively high, the mechanisms discussed here should apply in the field. Elevated [ C Q ] protected against the deleterious effect of ozone by a) reducing stomatal conductance (i.e. reducing the effective ozone dose), and b) shifting photoassimilatory control away from RuBisCO limitation. Thus, in the presence of moderately elevated ozone levels, elevated [ C Q ] may appear to cause an increase in Vcm~x. In T. aestivum, moderately elevated [03] causes stomatal closure largely via its effect on photoassimilation. The interaction between carbon dioxide and ozone effects varies with leaf development.

Acknowledgements We would like to thank Mrs J.E Bullimore and Mr D.J. Allen for technical assistance. This work was funded by the Biotechnology and Biological Sciences Research Council under grant BAGEC 084/320.

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