Elevated CO2 enhances stomatal responses to osmotic stress and abscisic acid in Arabidopsis thaliana

ABSTRACT

Carbon dioxide and abscisic acid (ABA) are two major sig-nals triggering stomatal closure. Their putative interactionin stomatal regulation was investigated in well-watered air-grown or double CO2-grown Arabidopsis thalianaplants,using gas exchange and epidermal strip experiments. Withplants grown in normal air, a doubling of the CO2 concen-tration resulted in a rapid and transient drop in leaf con-ductance followed by recovery to the pre-treatment levelafter about two photoperiods. Despite the fact that plantsplaced in air or in double CO2 for 2 d exhibited similar lev-els of leaf conductance, their stomatal responses to anosmotic stress (0·16–0·24 MPa) were different. The decreasein leaf conductance in response to the osmotic stress wasstrongly enhanced at elevated CO2. Similarly, the drop inleaf conductance triggered by 1µM ABA applied at the rootlevel was stronger at double CO2. Identical experimentswere performed with plants fully grown at double CO2.Levels of leaf conductance and carbon assimilation ratemeasured at double CO2 were similar for air-grown andelevated CO2-grown plants. An enhanced response to ABAwas still observed at high CO2 in pre-conditioned plants. It isconcluded that: (i) in the absence of stress, elevated CO2

slightly affects leaf conductance in A. thaliana; (ii) there is astrong interaction in stomatal responses to CO2 and ABAwhich is not modified by growth at elevated CO2.

Key-words: Arabidopsis thaliana; abscisic acid; carbon diox-ide; elevated CO2; leaf conductance; stomata; water stress.

Abbreviations: A, carbon assimilation rate; ABA, abscisicacid; Ci, intercellular space CO2 concentration; g, leaf con-ductance; WUE, water use efficiency.

INTRODUCTION

Stomata regulate the transpiration flux according to environ-mental conditions. Among the parameters affecting stomatalaperture, abscisic acid (ABA) and CO2 are of major interest.

ABA plays a crucial role in plant adaptation to water stress(Giraudat 1995). Synthesized with some delay, during awater stress, ABA induces a decrease in guard cell turgorresulting from a concerted modulation of ion channel activi-ties (Blatt & Armstrong 1993; Pei et al. 1997), in order tolimit water losses. CO2 is a second signal resulting in areduction of stomatal aperture, but the mechanisms under-lying this response are still debated (Willmer & Fricker1996). It has been proposed that the apoplastic malate poolreflects the ambient CO2, and that a high malate concentra-tion in the apoplast could lead to stomatal closure throughthe activation of anion channels in the guard cell plasmamembrane (Hedrich et al. 1994). There are few studies andcontroversial results concerning a putative interactionbetween ABA and CO2 sensing in regulating stomatalmovements. Mansfield (1976) observed a total indepen-dence of the ABA and CO2 responses in Xanthium stru-marium. In contrast, the absence of CO2 has been reportedto inhibit the effect of externally applied ABA in the samespecies (Raschke 1975) and in Solanum melongena(Eamus & Narayan 1989). Raschke & Hedrich (1985) haveshown that the sensitization of guard cells to CO2 by ABAmay depend on species and on the degree of stomatal aper-ture. More recently, an ABA-enhanced response to CO2

has been observed in soybean (Bunce 1998).Previous studies have shown that stomatal response to

growth in high CO2 was highly variable, depending onspecies and experimental conditions (Drake, Gonzalèz-Meler & Long 1997). In the context of constant increase inatmospheric CO2, these interactions between ABA andCO2 sensing at the level of stomata must be better under-stood to accurately predict the effects of increasing CO2

concentrations on plant water use. Thus, the aim of thiswork was (i) to investigate the dependence of stomatalresponse upon osmotic stress and ABA in different CO2

concentrations in A. thaliana, a species of fundamentalgenetic interest (ii) to evaluate whether this relation wasaltered by the CO2 concentration prevailing during growth.To address these questions, changes in mean leaf conduc-tance triggered by osmotic stress or ABA were monitoredin A. thalianaby gas exchange techniques at different CO2

levels for plants grown at normal or double concentrationof CO2. Additionally, dose–response curves of stomatalresponse to ABA from plants grown in normal or elevatedCO2 were established in epidermal strip experiments.

Plant, Cell and Environment (1999) 22, 301–308

© 1999 Blackwell Science Ltd

Elevated CO 2 enhances stomatal responses to osmotic stress

and abscisic acid in Arabidopsis thaliana

J. LEYMARIE,* G. LASCÈVE & A. VAVASSEUR

CEA/Cadarache – DSV – DEVM – Laboratoire des Echanges Membranaires et Signalisation, F-13108, Saint Paul lez DuranceCedex, France

ORIGINAL ARTICLE OA 220 EN

Correspondence: Alain Vavasseur. Fax: (33) 4 42 25 46 56; e-mail:[email protected]

*Present address: Max Planck Institut für Züchtungforshung, MDL,Carl-von-Linne-Weg 10, 50829 Köln, Germany.

301

MATERIALS AND METHODS

Plant material

Arabidopsis thaliana(L.) Heynh ecotype Landsberg erectaplants were grown individually in pots (65 mm × 65 mm× 70 mm) of sand watered with half-strength Hoagland’ssolution (Epstein 1972) in a growth chamber (8 h lightperiod, 22 °C; 16 h dark period, 20 °C; 70% relativehumidity) at a CO2 concentration of 370µmol mol–1 (con-trol plants) or 740µmol mol–1 (elevated CO2 plants). Light(250 µmol m–2s–1) was supplied by halogen lamps (HQI-TS, 150 W/NDL; Osram, München, Germany).

Whole plant gas exchange measurements

Four-week-old plants (leaf area: 6–8 cm2) were placed at alow vapour pressure deficit (VPD < 1 kPa) into an experi-mental chamber composed of independent shoot and rootcompartments with control of air humidity and temperatureas previously described (Leymarie, Vavasseur & Lascève1998). Water vapour pressures at the inlet and the outlet ofthe shoot compartment were continuously measured withtwo dew point hygrometers (Hygro M4; General Eastern,Woburn, MA, USA). Normal air (atmospheric air) wasdetermined as containing 370 ±20µmol mol–1 CO2.Different concentrations of CO2 (185, 555, 740µmol mol–1)were obtained by adding pure CO2 to normal air or CO2-freeair. The CO2 concentrations at the inlet and outlet of theplant chamber were monitored with a differential infra-redgas analyser (225 MK 3; ADC, Hoddesdon, UK). Light (8 h,220µmol m–2 s–1) was supplied by halogen lamps (HQI-TS150 W/NDL; Osram, München, Germany). The leaf surfacearea was measured daily from enlarged photographs.

ABA (cis-trans, in methanol) and polyethylene glycol(PEG 3500) 6·7 or 10% w/w, resulting in a 0·16 and0·24 MPa reduction in osmotic pressure, respectively(Wescor 5500; Wescor, Logan, Utah, USA), were added tothe aerated nutrient solution 1 h after the onset of light. Themethanol concentration in the solution never exceeded0·001 v/v, and such a concentration in control experimentswas found to have no effect on leaf conductance. One-thirdof the nutrient solution (70 mL) was changed twice a day.

Experiments with paradermal sections

Leaves from 4–5-week old plants (two plants for eachexperiment) grown either in normal air or in CO2-enrichedair (740µmol mol–1) were harvested in darkness at the endof the night period. Sections of abaxial epidermis tangen-tial to the leaf surface, referred to as paradermal sections,were prepared using a razor blade and placed cuticle-up inPetri dishes containing a buffered solution [10 mM

Mes/KOH; 10 mM KCl; 7·5 mM potassium iminodiacetate(K2IDA); pH 6·15] that was thermoregulated at 20 °C. Theimpermeant anion IDA was used to diminish the chlorideconcentration in the bathing medium since it has beenshown that a high KCl concentration strongly reducesABA and CO2 sensitivity in different species including A.

thaliana (Wardle & Short 1981; Leymarie et al. 1998).Normal air or CO2-enriched air was continuously bubbledthrough the bathing medium (10 mM Mes/KOH; 10 mM

KCl; 7·5 mM potassium iminodiacetate; pH 6·15). After30 min in darkness, the paradermal sections were incubated2 h under light (450µmol m–2s–1) at 20 °C. Then, ABA wasadded to the solution and measurements of stomatal aper-tures were performed after 3 h. The ABA was dissolved inmethanol and the final methanol concentration in the solu-tion never exceeded 0·001 v/v. The same amount ofmethanol was added to the controls. Stomata withoutunderlying mesophyll contamination were observed at theedges of paradermal sections (Fig. 1) and used for measure-ments. Only ‘mature stomata’, whose ostiole length washigher than one-third of the length of stoma were taken intoaccount. For each treatment at least 60 stomatal apertures(10 each from six different paradermal sections) were mea-sured (Lascève, Leymarie & Vavasseur 1997). All experi-ments were replicated four times with different plants.

Statistical analyses

The significance of each effect was tested by one-way analy-sis of variance (ANOVA), or, when populations were notnormally distributed or exhibited unequal variances, by one-way ANOVA on ranks. To test significance of interactions, datawere subjected to a two-way ANOVA. Pairwise comparisonswere carried out using Tukey’s test. Analyses were performedusing Sigmastat 2·0 (Jandel Corporation, Chicago, IL, USA).

RESULTS

Changes in CO 2 concentration and leafconductance in A. thaliana

Figure 2 presents the response of leaf conductance to a tran-sition from normal air to CO2 concentrations ranging fromhalf to twice the normal level of CO2. Decreased CO2resulted in a substantial increase in leaf conductance

302 J. Leymarie et al.

© 1999 Blackwell Science Ltd, Plant, Cell and Environment,22, 301–308

Figure 1. Open stomata from Arabidopsis thaliana; (m), ‘mature’stomata; (*), ‘immature’ stomata.

(88 ± 17 mmol m–2 s–1) lasting for at least the three follow-ing photoperiods. In contrast, a doubling of CO2 transientlyreduced the leaf conductance (–47 ± 7 mmol m–2 s–1 after2 h), and leaf conductance returned, during the followingphotoperiod, to the level measured in normal air (controlvalues in Table 1). At elevated CO2, assimilation rate wasincreased at an almost constant level of transpiration, and ahigher WUE was found: 3·4 ± 0·2µmol CO2 mmol–1 H2O,compared with 2·4 ± 0·2 in normal air.

The response of leaf conductance to an osmoticstress is enhanced at elevated CO 2

The leaf conductance of plants grown in normal air orplaced for 2 d in elevated CO2 were not significantly

different from each other (P = 0·70). However, theirresponse to an osmotic stress of 0·16 or 0·24 MPa, result-ing from the application of 6·7 or 10% PEG 3500 in thenutrient solution, was markedly different, with the plantsplaced in high CO2 showing the strongest decrease in leafconductance (Table 1). Figure 3 illustrates the modificationof leaf conductance of a plant submitted to a decrease of0·24 MPa in the osmotic pressure in the nutrient solution.The osmotic stress resulted in a decrease in the basal levelof leaf conductance in both CO2 concentrations but thedaily change in leaf conductance was affected more signif-icantly at elevated CO2 (Figs 3 & 4). A moderate osmoticstress (0·16 MPa), which has no detectable effect on thelight-induced shift in leaf conductance in normal air, trig-gered a significant inhibition under elevated CO2 (Fig. 4).When all data were considered, a two-way analysis of vari-ance (ANOVA) indicated a significant interaction betweenthe 2-d PEG treatment and CO2 concentration (P = 0·031).Even for a mild stress (0·16 MPa), this interaction withCO2 was significant (P = 0·039). The carbon assimilationrate was not significantly affected by the 0·24 MPa osmoticstress in normal air (P = 0·45) but a significant reductionoccurred at elevated CO2 (P = 0·019) resulting in almostsimilar levels of carbon fixation in both atmospheric CO2

conditions (Table 1). It is interesting to note that WUE dur-ing the water stress was higher at high CO2 (7·0 ± 0·8) thanin normal air (3·2 ± 0·2).

The response of leaf conductance to ABA isenhanced at elevated CO 2

Application of 1µM ABA in the nutrient solution of plantspreviously placed for 2 d in different CO2 concentrationsresulted in a drop in leaf conductance after a time lag ofabout 45 min. The amplitude of this drop was stronglycorrelated to the ambient CO2 concentration (Fig. 5,Table 2). While the response was only transient in normalair, with a rapid recovery to the initial level of leaf conduc-tance, at high CO2 the application of ABA induced a per-manent reduction in leaf conductance for at least the threefollowing photoperiods. As observed during an osmoticstress, the elevated CO2 levels also resulted in a higherABA-induced drop in leaf conductance (Fig. 6). Indeed,there was a significant interaction between a 2-d ABAtreatment and CO2 concentration in leaf conductance

Elevated CO2 and stomatal response to ABA 303


Figure 2. Effect of a change in CO2 concentration on leafconductance of A. thaliana. Typical responses to a change fromnormal air to different CO2 concentrations ranging from 185 to740µmol mol–1. The numbers in front of dashed lines refer to thevalues of leaf conductance at the onset of light. Black and whitebars correspond to night and day periods, respectively.

Table 1. Effect of an osmotic stress on leaf conductance, g (mmol m–2 s–1) and assimilation rate, A (µmol m–2 s–1) according to CO2concentrations. Plants were placed for 2 d in normal or elevated CO2 (370 and 740µmol mol–1, respectively) before being subjected toosmotic stress. Measurements were taken after 6 h exposure to light 1 d before (control) and 24 and 48 h after addition of PEG. Each value isthe mean of at least three independent experiments ± SE

PEG 6·7% PEG 10%CO2 µmol mol–1 370 740 370 740

g A g A g A g A

Control 387 ± 13 7·6 ± 0·1 361 ± 11 9·7 ± 0·2 365 ± 29 7·5 ± 0·6 343 ± 26 9·6 ± 0·4PEG 24 h 330 ± 33 7·5 ± 0·3 310 ± 14 9·0 ± 0·5 209 ± 9 6·6 ± 0·8 136 ± 31 8·5 ± 0·7PEG 48 h 318 ± 28 7·4 ± 0·2 258 ± 25 7·9 ± 0·5 230 ± 26 7·0 ± 0·5 113 ± 33 7·2 ± 1·0

regulation (P = 0·018). In contrast, the assimilation rateswere not significantly affected by 1µM ABA treatment(P = 0·123).

Growth at elevated CO 2 does not modify stomatalresponse to ABA

Additional experiments with plants fully grown from ger-mination at 740µmol mol–1 CO2 were conducted at ele-vated CO2 levels (Figs 5 & 6). In the absence of stress,these plants exhibited a leaf conductance in the range ofthose found for control plants in normal air (P = 0·352,Table 2). At elevated CO2, the assimilation rates (A), inter-cellular space CO2 concentrations (Ci) and the A/Ci ratiofor plants grown in normal and elevated CO2 concentra-tions were not significantly different (P > 0·05, Table 3),indicating no evidence of reduction of photosynthetic effi-ciency in response to CO2 growth conditions. Moreover,the response to an application of ABA was not significantlydifferent for plants grown either in air or double CO2

(ABA–culture interaction P = 0·687). Thus, the CO2 con-centration prevailing during the growth period apparentlydid not induce any adaptation of the response of leaf con-ductance to ABA. Stomatal responses to ABA and CO2

according to CO2 growth conditions were also investigatedin epidermal strips bioassays. The CO2 concentration dur-ing growth did not significantly affect stomatal density(Table 4). Whatever the growth conditions, in the absenceof ABA, the stomatal apertures measured under light innormal air or elevated CO2 were not significantly different(P > 0·6, Fig. 7). For both growth conditions, the responseto low concentrations of ABA was higher in elevated CO2.The only noticeable difference was that plants grown indouble CO2 were slightly less sensitive to ABA at highCO2 (Fig. 7, inset) but there was no significant interactionculture× measure (P > 0·05).

DISCUSSION

Stomatal response to CO 2 in Arabidopsis

CO2 and ABA sensing in stomata is certainly located inthe guard cell since guard cell protoplasts retain theirability to shrink in response to both signals (Gotow,Kondo & Syono 1982; Fitzsimons & Weyers 1986). Inthe integrated plant system, the stomata respond to theintercellular space concentration of CO2 determined byatmospheric concentration and by the assimilation rate ofCO2 in the mesophyll (Mott 1988). On the basis ofnumerous experiments with various species, it is gener-ally accepted that a doubling of atmospheric CO2 willresult in an approximately 20–40% reduction in leaf con-ductance (Morison 1987; Beerling et al. 1996; Morison1998). However, large variations in response to high CO2

have been observed according to the duration of exposi-tion to CO2, the plant species (Bunce 1992; Beerlinget al. 1996), and the growth conditions (Talbott,Srivastava & Zeiger 1996). In this study, while air-grownplants of A. thalianawere maintained in hydroponic con-ditions, we observed that lowering the CO2 concentrationto half the normal concentration induced a permanentenhancement of leaf conductance. Conversely, a doublingof CO2 only provoked a transient 15% reduction in con-ductance followed by a rapid restoration of the initiallevel of leaf conductance. Similarly, high CO2 was poorlyeffective in inducing stomatal closure in bioassays con-ducted with epidermal strips from well-watered plantswhile in similar conditions the removal of CO2 induced asignificant stomatal opening (Leymarie et al. 1998).Thus, in well-watered conditions, the stomatal behaviourwas only slightly affected by a doubling of CO2 concen-tration.



Figure 3. Typical responses of leaf conductance to an osmoticstress (0·24 MPa), resulting from the application of PEG 3500(10% w/w) in the nutrient solution, plants being previously placedfor 2 d in normal air (dotted line) or in 740µmol mol–1 CO2 (solidline). Black and white bars correspond to night and day periods,respectively.

Figure 4. Daily change in leaf conductance observed before andafter a 2-d osmotic stress (PEG 3500: 6·7%, 0·16 MPa or 10%,0·24 MPa), plants being already placed for 48 h in normal air or inelevated CO2. The daily change in leaf conductance is taken as themaximal conductance minus the minimal conductance recordedduring a photoperiod (mean of at least three replicates ± SE).

Elevated CO 2 enhances stomatal responses toosmotic stress and ABA

In contrast, the impact of high CO2 on stomatal conduc-tance was more obvious after application of an osmoticstress. With plants transferred to elevated CO2, the short-term response to an osmotic stress was considerablyenhanced compared with the response observed in normalair, revealing a strong interaction between CO2 sensing andwater stress in stomatal regulation of gas exchanges in A.thaliana. These results underline the importance of theplant water status on stomatal sensitivity to CO2 and couldat least partly explain the large variability reported on theeffect of elevated CO2 on stomatal conductance. During

drought, the sensitization of stomata to ABA by CO2 couldenable the leaf conductance to follow more accuratelychanges in assimilation while saving water (Raschke1987). It is interesting to note that when a short-term waterstress was applied at high CO2, the leaf conductance wasdiminished by up to 67% while assimilation was onlyreduced by 25%. Such a limitation in leaf conductance atelevated CO2 allows a doubling of WUE.

ABA is certainly the major intermediate coupling sensingof water stress to stomatal closure, as demonstrated by thewilty phenotype of ABA-deficient mutant plants(Koornneef et al. 1982). In the present study, in whole plantexperiments or in epidermal strip bioassays, high CO2 wasable to enhance stomatal sensitivity to ABA. These obser-vations strongly argue for a direct interaction between ABAand CO2 sensing at the guard cell level and fit well with theearlier observation of a CO2-enhanced ABA sensitivity inX. strumarium (Raschke 1975). The molecular basis of thisinteraction between responses to CO2 and ABA in guardcells remains to be elucidated, but calcium signalling inguard cells could well be a point of integration of these twosignals. Supporting this hypothesis, exogenous ABA has



Figure 5. Typical response of leaf conductance to an applicationof 1 µM ABA in the nutrient solution, plants being previouslyplaced for 2 d in different CO2 concentrations (185, 370, 555,740µmol mol–1 CO2) except 740* plants which were grown fromgermination at 740µmol mol–1 CO2. Numbers in front of dashedlines refer to leaf conductance at the onset of light. Black and whitebars correspond to night and day periods, respectively.

CO2 µmol mol–1 185 370 555 740 740 *

Control 420 ± 35 364 ± 21 344 ± 7 329 ± 4 367 ± 14ABA 1 µM 24 h 364 ± 17 261 ± 29 215 ± 36 206 ± 5 234 ± 28ABA 1 µM 48 h 362 ± 24 288 ± 29 202 ± 38 185 ± 14 194 ± 22

Table 2. Effect of ABA on leaf conductance(mmol m–2 s–1) according to CO2concentration. Plants were placed for 2 d inthe indicated CO2 concentrations beforeapplication of 1µM ABA. Measurementswere taken after 6 h exposure to light 1 dbefore (control) and 24 and 48 h afteraddition of ABA. Each value is the mean ofat least three independent experiments ± SE.740*, plants were grown from germination at740µmol mol–1 CO2

Figure 6. Daily change in leaf conductance before and 48 h afterapplication of 1µM ABA according to CO2 concentration. Plantswere placed in the various CO2 conditions 2 d before ABAapplication except 740* plants which were grown fromgermination at 740µmol mol–1 CO2 (mean of at least threereplicates ± SE).

been shown to trigger Ca2+ influx in guard cells (McAinsh,Brownlee & Hetherington 1990; Schroeder & Hagiwara1990; Gilroy et al. 1991; McAinsh, Brownlee &Hetherington 1992; Lemtiri-Chlieh & MacRobbie 1994)and Ca2+ is also involved as a second messenger in the CO2

signal transduction pathway (Webb et al. 1996).

Growth at elevated CO 2 and stomatal responses

In general, growth at elevated CO2 affects stomatal density(Poole et al. 1996). A survey has shown that among 100species examined 74% exhibited a reduction in stomataldensity in response to growth at elevated CO2 (Woodward& Kelly 1995). In the present study, there was no signifi-cant effect of a doubling of CO2 on abaxial stomatal den-sity in A. thaliana. Even though this observation does notsupport a direct effect of high CO2 on stomatal patterning,a long-term effect based on a selective advantage cannot beexcluded. Furthermore, there was no evidence of a modifi-cation of stomatal sensitivity to CO2 resulting from growthconditions since stomatal conductance and A/Ci at740µmol mol–1 CO2 were similar for both air-grown andadapted plants. Such absence of stomatal acclimation tohigh CO2 has been observed in Phaseolus vulgaris(Radoglou, Aphalo & Jarvis 1992). In contrast, changes instomatal sensitivity to CO2 resulting from growth at highCO2 have been described (Morison 1998) leading to adecreased sensitivity in Eucalyptus tetrodontaandChenopodium album(Berryman, Eamus & Duff 1994;Santrucek & Sage 1996) and conversely to an enhancedsensitivity in Triticum aestivum(Tuba, Szente & Koch1994). Moreover, the interaction of ABA and CO2 sensingwas not affected by growth conditions since stomatalresponses of air-grown or elevated CO2-grown plants to an

application of ABA were almost similar. These results sug-gest that CO2 enrichment could have a large impact inenhancing stomatal response to drought stress.

Elevated CO 2 and photosynthesis

Many studies have shown that photosynthesis acclimatesto long-term exposure to elevated CO2 (Drake et al. 1997),although this response is not universal (Curtis & Wang1998). There are at present few data concerning CO2 accli-mation in Arabidopsisand only recent studies have shownthat prolonged exposure to elevated CO2 altered the levelsof expression of sucrose phosphate synthase (Signora et al.1998) and Rubisco (Cheng, Moore & Seemann 1998).However, even after a 10-week-culture period at elevatedCO2, the maximal photosynthetic activity was only slightlyaffected (Signora et al. 1998). In the present work, assimi-lation rate, stomatal conductance, and intercellular CO2

concentration measured at double CO2 were very similarfor air-grown or double CO2-grown plants. Additionally,the increase in assimilation rate resulting from a transition



Control ABA 24 h ABA 48 h

A A/Ci A A/Ci A A/Ci

Air-grown 9·8 ± 0·7 13·5 ± 1·0 9·3 ± 0·6 13·3 ± 0·7 8·8 ± 0·4 12·5 ± 0·5Double 10·1 ± 0·3 13·9 ± 0·4 9·7 ± 0·8 13·5 ± 0·1 9·4 ± 0·5 13·4 ± 0·6

CO2-grown

Table 3. Assimilation rate (A, µmol m–2

s–1) and A versus Ci (Ci in mmol mol–1) ofplants grown in normal air or double CO2

before and after application of 1µM ABA.All measurements were performed at740µmol mol–1 CO2

Table 4. Stomatal density (stomata mm–2) of plants grown ateither 370 or 740µmol mol–1 CO2. NS, not significant effect ofgrowth condition on stomatal density. Values were obtained fromthe observation of 2200 stomata on 32 leaves from eight plants ineach condition

Double Air-grown plants CO2-grown plants

‘Mature’ stomata 159 ± 11 154 ± 7 (NS)‘Immature’ stomata 54 ± 7 56 ± 8 (NS)Total 213 ± 15 210 ± 14 (NS)

Figure 7. Stomatal response to ABA in epidermal strip bioassaysperformed either in normal air (triangle) or in elevated CO2

(740µmol mol–1, circle) with plants grown in normal air (opensymbols) or CO2-enriched air (filled symbols). Inset, effect ofelevated CO2 on stomatal response to ABA. ‘Delta high CO2’means difference between stomatal aperture measured in normaland CO2-enriched air (dotted line, air grown plants; solid line,elevated CO2 grown plants). Data are the means of 240measurements of stomatal apertures in four independentreplicates. Error bar represent standard error of the mean with aconfidence interval of 95%.

from normal air to double CO2 was maintained for at leasta week (data not shown). Thus, there was no evidence for areduction of photosynthesis under elevated CO2 in ourstudy but a complete analysis of the A/Ci relationshipwould be required to fully address this point. Such anabsence of acclimation of photosynthesis to elevated CO2

has been observed in cotton plants during the exponentialgrowth phase (Wong 1993), in young Phaseolus vulgarisplants (Radoglou et al. 1992) or in spring wheat (Garciaet al. 1998).

The present rise in global atmospheric CO2 level isexpected to have important consequences on plantbehaviour through modifications of the climate, changes inphotosynthetic activity and modification of stomatal con-ductance (Field, Jackson & Mooney 1995) and patterning(Woodward 1989). It is now evident that plant acclimationto elevated CO2 is highly varied, depending on species andenvironmental conditions. For A. thaliana, in well-wateredor stress conditions, the major effect of a doubling of ambi-ent CO2 that we noticed was a higher WUE. However,long-term studies should be conducted to integrate adap-tive processes and adverse effects such as a diminishedheat loss from transpiration (Eamus & Jarvis 1989; Eamus1991; Jarvis 1995).

ACKNOWLEDGMENTS

The authors wish to thank Dr Daniel Plante for criticalreading of the manuscript.

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Received 29 August 1998; received in revised form 9 October 1998;accepted for publication 9 October 1998



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Elevated CO2 enhances stomatal responses to osmotic stress and abscisic acid in Arabidopsis thaliana