Does engineering abscisic acid biosynthesis in Nicotiana plumbaginifolia modify stomatal response to drought?

© 2001 Blackwell Science Ltd 477

Plant, Cell and Environment (2001) 24, 477–489

leaf water potential; Ypre-dawn, leaf water potential mea-sured at pre-dawn; gs, stomatal conductance.

INTRODUCTION

Stomatal control of transpiration is a major process deter-mining short-term response of the whole plant to drought.It directly influences soil water depletion rates, plant waterpotentials and fluxes of solutes in the xylem stream (Tardieu& Simonneau 1998). Stomatal response to drought is medi-ated by root-born abscisic acid (ABA) (Zhang & Davies1991) and it has been suggested that plant response to waterstress could be improved by manipulating rates of ABAbiosynthesis. However, the consequences of such manipula-tions remain difficult to predict for several reasons.

The role of ABA in plant response to soil drying can bealtered or overcome by other mediators. Hydraulic controlof stomatal conductance by leaf water potential or xylemembolism predominates in some adult trees (Fuchs & Livingston 1996; Triboulot et al. 1996; Salleo et al. 2000).In herbaceous isohydric species, such as maize, stomatalresponse to ABA concentration in the xylem sap([ABA]xyl) can also be altered by changes in leaf waterpotential (Tardieu & Davies 1992; Tardieu & Simonneau1998). In addition to increases in [ABA]xyl, other drought-induced changes in the composition of the xylem sap cancontrol stomatal response to soil drying. This includeschanges in the concentration of ABA-glucose-ester as apotential source of free ABA (Cornish & Zeevaart 1984;Lehmann & Glund 1986; Dietz et al. 2000), decreased con-centrations of hormones such as cytokinins (Blackman &Davies 1985; Incoll, Ray & Jewer 1990; Fubeder et al. 1992;Badenoch-Jones et al. 1996) or auxins (Dunleavy & Ladley1995), and possibly changes in ion concentrations (Schurr,Gollan & Schulze 1992; Ruiz, Atkinson & Mansfield 1993;Ridolfi et al. 1996). Apoplastic pH has also been shown toincrease with the depletion of soil water (Wilkinson et al.1998) and change in apoplastic pH was suggested to controlstomatal response to ABA in the early development of adrought stress when there is no detectable change in[ABA]xyl. Such a role of increased apoplastic pH on stom-atal closure was clearly demonstrated in vitro (Wilkinson &Davies 1997) and in planta by comparing wild-type tomatoand the ABA-deficient mutant flacca (Wilkinson et al. 1998)but remains to be evaluated in planta for other species.

ABSTRACT

The consequences of manipulating abscisic acid (ABA)biosynthesis rates on stomatal response to drought wereanalysed in wild-type, a full-deficient mutant and fourunder-producing transgenic lines of N. plumbaginifolia.The roles of ABA, xylem sap pH and leaf water potentialwere investigated under four experimental conditions:feeding detached leaves with varying ABA concentration;injecting exogenous ABA into well-watered plants; andwithholding irrigation on pot-grown plants, either intact orgrafted onto tobacco. Changes in ABA synthesis abilitiesamong lines did not affect stomatal sensitivity to ABA con-centration in the leaf xylem sap ([ABA]xyl), as evidencedwith exogenous ABA supplies and natural increases of[ABA]xyl in grafted plants subjected to drought. The ABA-deficient mutant, which is uncultivable under normal evaporative demand, was grafted onto tobacco stock andthen presented the same stomatal response to [ABA]xyl aswild-type and other lines. This reinforces the dominant roleof ABA in controlling stomatal response to drought in N.plumbaginifolia whereas roles of leaf water potential andxylem sap pH were excluded under all studied conditions.However, when plants were submitted to soil drying ontotheir own roots, stomatal response to [ABA]xyl slightly differed among lines. It is suggested, consistently with allthe results, that an additional root signal of soil drying modulates stomatal response to [ABA]xyl.

Key-words: Nicotiana plumbaginifolia; abscisic acid;drought; stomatal conductance; xylem sap pH.

Abbreviations: ZEP, zeaxanthin epoxidase; WT, wild-typeN. plumbaginifolia; zep, the ABA-deficient N.p. mutant,previously named aba2 (Marin et al. 1996); MS-3 and MS-8, two transgenic lines obtained by independent comple-mentations of N. p. zep with a full-length ZEP cDNA in thesense orientation; AS-7 and AS-15, two transgenic linesobtained by independent transformations of WT N.p. withan antisense ZEP fragment; [ABA]xyl, concentration ofABA in the xylem sap extruded from pressurized leaf; YL,

Correspondence: Thierry Simonneau. Fax: + 33 04 67 52 21 16;e-mail: [email protected]

Does engineering abscisic acid biosynthesis in Nicotianaplumbaginifolia modify stomatal response to drought?

C. BOREL,1 A. FREY,2 A. MARION-POLL,2 F. TARDIEU1 & T. SIMONNEAU1

1Laboratoire d’Ecophysiologie des Plantes sous Stress Environnementaux (LEPSE), UMR 759 INRA-ENSAM, 2 PlaceViala, 34060 Montpellier Cedex 1, France and 2INRA, Laboratoire de Biologie Cellulaire, 78026 Versailles Cedex, France

Manipulation of the ABA biosynthesis pathway mayindirectly influence stomatal aperture and transpirationrate via pleiotropic effects on leaf growth or photosynthe-sis rate. The biosynthetic pathway of ABA has been amplyclarified (Cutler & Krochko 1999) and involves importantphysiologically active intermediates such as carotenoids(Demmig-Adams & Adams 1996) that can induce notice-able pleiotropic effects, notably in plants engineered in thevery early steps of ABA synthesis. As an example, trans-genic plants with modified carotenoids composition can beaffected in their tolerance to high light (Niyogi, Grossman& Bjorkman 1998) and in their stomatal response to blue light (Zeiger & Zhu 1998). Other enzymes, such asthose altering the synthesis of a molybdenum cofactor(Leydecker et al. 1995), influence the terminal steps of ABAformation but are not specific to the ABA synthesispathway. Abscisic acid itself acts on leaf growth (Bacon,Wilkinson & Davies 1998) which can in turn control tran-spiration rate, modify leaf water potential, and influencestomatal response to ABA (Tardieu & Simonneau 1998).

The following contribution evaluates the consequencesof manipulating ABA biosynthesis rates on stomatal re-sponse to drought. A series of transgenic lines presenting arange of mild reductions in ABA synthesis rate has beenobtained in Nicotiana plumbaginifolia by manipulatingexpression of the zeaxanthin epoxydase (ZEP) gene(Audran et al. 1998). ZEP catalyses two of the final com-mitted steps of ABA synthesis (Marin et al. 1996). ReducedZEP expression in roots (Borel et al. 2001) and leaves (Freyet al. 1999) resulted in three- to five-fold lower [ABA]xyl intransgenic N. plumbaginifolia than in wild-type (Borel et al.2001). Despite these reductions, major pleiotropic effectsusually induced by manipulating ABA synthesis wereavoided. Notably, direct consequences of genetic manipu-lations on stomatal response to drought were isolated frompleiotropic effects on leaf growth or photosynthesis rate(Marin et al. 1996; Borel 1999).

A subset of N. plumbaginifolia transgenic lines togetherwith wild-type and the full-deficient mutant zep wereselected to examine in planta (i) if the rise in [ABA]xyl

induced by soil drying accounted for changes in stomatalconductance in all lines, and (ii) whether altering ABAaccumulation rates modified stomatal sensitivity to ABA.The relative contribution of ABA compared with othersignals of drought on the reduction of stomatal con-ductance was tested by uncoupling changes in [ABA]xyl

from that of soil water deficit. This was achieved by feedingexogenous ABA to whole plants or detached leaves. Thesame coupling between changes in xylem sap compositionand soil drying was restored for all lines by grafting pairsof plants of different lines on the same N. tabacum stock.Grafting zep onto tobacco stock also enabled the analysisof its response to drought conditions, whereas ABA-deficient mutants cannot be submitted to drought stressunless grafted onto wild-type rootstock (Cornish & Zeevaart 1988; Fambrini et al. 1995). The combination ofmultiple experimental conditions (exogenous versusdrought-induced ABA; detached leaves versus rooted

478 C. Borel et al.

plants; grafts on a common rootstock) together with the useof transgenic plants with altered rates of ABA synthesisoffers a unique opportunity to evaluate the contribution ofABA in controlling stomatal response to soil drought.

MATERIALS AND METHODS

Gene constructions and plant transformations

Nicotiana plumbaginifolia var. viviani and the stable zep(previously named aba2) mutant (aba2-s1, Marin et al.1996) were transformed with different sense or antisenseZEP cDNA constructs fused to a neomycin phosphotrans-ferase sequence which conferred kanamycin resistance(Frey et al. 1999). Four transgenic lines together with wildtype (WT) and zep were retained in this study: MS-3 andMS-8 were obtained by transformation of zep with a full-length sense ZEP cDNA; AS-7 and AS-15 were obtainedby transformation of WT with a ZEP cDNA in the anti-sense orientation. All ZEP constructs were placed betweenthe CaMV 35S promoter and the nos3¢ end (details in Borelet al. 2001). All transgenic lines contained a single copy ofthe ZEP construct (Borel 1999).

Overall, all transgenic lines presented a reduction in theircapacity to accumulate ABA in response to drought. Thiscapacity has been evaluated for each line (except zep) bycalculating the slope of the relationship between [ABA]xyl

and leaf water potential measured at pre-dawn (Ypre-dawn)on the same plant (Borel et al. 2001). In comparison withWT, the capacity to accumulate ABA with drought wasreduced to 28·6 ± 4% in MS-3 and MS-8, 28·9 ± 3% in AS-7, and 22·4 ± 3% in AS-15 (Fig. 1). [ABA]xyl measured inwell-watered plants was about three-fold lower in zep thanin WT (Borel 1999), and dehydrating detached organs ofzep did not increase ABA content either in leaves (Audranet al. 1998) or in roots (Borel et al. 2001).

Drought experiment with intact plants

Seeds were germinated over a period of 4 to 6 weeks (25 °Cday/17 °C night, 16 h photoperiod) on solid nutrient solu-tion (agar 7 g L-1 in Hoagland N/10) (Borel 1999) withkanamycin for selection of transgenic plants. Greenplantlets (20–30 of each line) were transferred to a green-house and transplanted into 6 dm3 cylindrical pots (0·5 mhigh) in June 1996 (WT, MS-8), August 1996 (MS-3, AS-7,AS-15) and June 1997 (WT).The pots were filled with a 1 : 1(v : v) mixture of sieved peat and clay-sandy-loam soil froma field near Montpellier (France), and they were wrappedin aluminium foil to minimize soil heating. Plants werewatered daily with nutrient solution (Hoagland N/10) untilthe stem was about 50 mm high. Typical growth conditionsin the greenhouse were 26 °C (day)/18 °C (night) mean airtemperature, 70–90% mean daytime relative humidity anda mean daily cumulated photosynthetic photon flux densityof 16 (for plants transplanted in August 1996 and measuredin October 1996) to 21 mol m-2 d-1 (for plants transplantedin June 1996 and measured in August 1996).

© 2001 Blackwell Science Ltd, Plant, Cell and Environment, 24, 477–489

Irrigation was withheld at the onset of flowering (whenthe stem was about 0·4 m high), and the soil was coveredwith white perlite to avoid heating and direct evaporation.Each day during soil drying, stomatal conductance (gs) ofthree to five plants per line was measured on the twelfthleaf (first above the rosette) from 1100 to 1400 h (solartime) using a ventilated closed-cuvette (volume 1 dm3;contact area 5000 mm2) coupled to a gas-exchange analyser(LI-6200; LI-COR, Lincoln, NE, USA). Relative humidityinside the cuvette was maintained constant during mea-surement. Care was taken to ensure direct light exposureof the whole leaf for at least 30 min before and during mea-surement. Photosynthetic photon flux density (PPFD) wasmeasured using the PPFD sensor (LI-190SA; LI-COR) ofthe LI-6200 chamber, and ranged from 800 to 1200 mmolm-2 s-1 during measurements. The boundary layer con-ductance in the cuvette was determined every second hourby inserting a replica of the measured leaves made of wetblotting paper.

Immediately after the measurement of stomatal con-ductance, the leaf was excised, enclosed in a pressurechamber (Soil Moisture Equipment Corp., Santa Barbara,CA, USA) for measurement of leaf water potential (YL),and approximately 70 mm3 of xylem sap were extracted bypressurizing the leaf at about 0·4 MPa above the balancingpressure. The sap was stored at -80 °C for subsequentanalysis of ABA and pH determination. Plants were thenplaced in a dark room until the following morning whenpre-dawn leaf water potential was measured on a leaf adja-cent to that used for the measurement of stomatal conduc-tance. Some plants were depotted to check that roots haddeveloped in the whole pot.

An additional experiment was performed with 15 WTand 20 AS-15 plants that were transplanted in April 1998and grown as above except that they were acclimated to an increased level of xylem ABA. From the seventh leaf appearance, daily irrigation was complemented with2 mmol m-3 of (±)-ABA (Fluka, Buchs, Switzerland) addedto the nutrient solution. At the end of the thirteenth leafexpansion (about 4 weeks later) irrigation was withheld,and measurements of gs, YL, and extraction of xylem sapwere performed at different levels of soil drying on the thir-teenth leaf as described above.

Drought experiment on plants grafted ontowild-type tobacco stocks

Three successive experiments were performed on graftedplants in the greenhouse. In each experiment, a graft of onetransgenic line among MS-8, AS-15 and zep was associatedwith WT (15 replicates of each graft type) onto a commonNicotiana tabacum cv. Xanthi (tobacco) stock plant.Tobacco was sown and grown in 6 dm3 cylindrical pots(0·5 m high) filled with the peat : soil mixture describedabove, whereas N. plumbaginifolia plantlets were grown in1·5 dm3 pots filled with peat. All the plants were watereddaily until the grafting. When the tobacco plants reachedthe flowering stage, their single shoots were trimmed below

Stomatal control in plants with modified ABA synthesis rates 479

the tenth leaf and grafting was performed by incising thestem of tobacco at two locations, 10 mm below the fourthand tenth leaves, and inserting one plantlet of N.plumbaginifolia into each incision (a wild type and a trans-genic plant). At this time the N. plumbaginifolia plantletswere four to five visible leaves. Grafts were wrapped in wet


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Figure 1. Relationship between pre-dawn leaf water potentialand concentration of ABA in leaf xylem sap measured beforedawn on pot-grown Nicotiana plumbaginifolia submitted to soildrying. Each point represents coupled measurements on one leaf.(a) Wild-type; (b) complemented zep mutants (MS-3, closedsymbols; MS-8, open symbols); (c) antisense plants (AS-7, closedsymbols and full line; AS-15, open symbols and dashed line).Straight lines and coefficients of correlation were obtained bylinear regression for each line [except in (b), where theregression holds for both MS-3 and MS-8]. Dotted lines areintervals of confidence at 95% [in (c), only the upper limit wasshown for AS-7, and the lower one for AS-15].

tissues and covered by plastic bags for 7 d, after whichleaves of the stock plant were trimmed and grafts devel-oped without protection. Six to 10 weeks after the grafting,grafts presented seven to 10 newly grown and fullyexpanded leaves. Plants were then subjected to drought bywithholding irrigation as described above for intact plants.Measurements of stomatal conductance, of YL, and extrac-tion of xylem sap for ABA analysis were carried out asdescribed above, at varying soil dehydration levels, on theyoungest fully expanded leaf (fifteenth or sixteenth) of eachgraft.

ABA feeding into the xylem sap of well-watered plants

Plants of WT, MS-8 and AS-15 were prepared as describedabove for intact plants, except that the pots (12 dm3) werefilled with peat, they contained three plants (one of eachline) and were continuously irrigated (eight times a day)until the morning of experiments, when ABA was fed to the plants in the greenhouse. During growth, plants weretrimmed to allow for the development of a unique stem.On the morning prior to measurements, a plastic funnel(100 mm high, 100 mm in diameter) was sealed with epoxyresin around the basis of the stem, and filled with degassedbuffer solution (0·4 mol m-3 Ca(NO3)2, 2 mol m-3 KH2PO4,pH 6·0) without ABA or with varying concentrations(0·1–0·8 mol m-3) of synthetic (±)-ABA (Fluka). The stemwas drilled radially with a 1-mm-diameter bit below thesurface of the solution to avoid cavitation. The funnel wascovered to protect the solution against light and pollutants.The plants were then submitted to high PPFD (above800 mmol m-2 s-1) for 2 h in the greenhouse to allow ab-sorption of the ABA solution into the xylem stream. Mea-surements of stomatal conductance, of YL, and extractionof xylem sap for ABA analysis were carried out on the first leaf above the funnel as described for the droughtexperiment.

Detached-leaf experiments

Leaves were sampled on well-watered plants (WT, MS-3,AS-7 and AS-15) that were transplanted in August 1996 andgrown in the greenhouse in 1·5 dm3 pots filled with peat(one plant per pot). When the plants were at the onset offlowering stage (0·40 m high stems), about 15 pots for eachline were placed in a dark room. On the next day, thetwelfth leaf of 10 plants per line was cut under water inreduced light conditions, and immediately placed intoplastic vials (0·1 dm3) containing 40 g of the degassed buffersolution described above, with varying concentrations (0,100, 200, 500, 1000 mol m-3) of synthetic (±)-ABA (Fluka).Concentrations of (+)-ABA were calculated as half of theapplied concentrations of (±)-ABA. Vials were sealed withfoam and covered with parafilm. Leaves in the vials weremaintained inclined at 45° to the horizontal, and exposedto direct light in the glasshouse (600 mmol m-2 s-1 PPFD,27 °C daytime air temperature, and about 50% relative

480 C. Borel et al.

humidity) or placed 0·7 m below metal halide lamps in thelaboratory (800 mmol m-2 s-1 PPFD, 28 °C air temperature,40% relative humidity). Every 20 min during a 3 h period,vials were weighed and leaf temperature was measuredusing an infrared thermometer (IR-74007 THI-300, Tasco,Osaka, Japan). Air temperature and relative humidity weremeasured every 20 s (HMP35A,Vaisala, Finland), averagedand stored in a data-logger every 600 s. The boundary layerconductance (ga) was determined by measuring water evaporation from three wet replicas of the leaves madefrom blotting paper and using the same gravimetric methodas for the determination of leaf evaporation rate. Areas ofleaves and leaf replicas were measured at the end of theexperiment. Stomatal conductance was calculated using theequation:

where Jw is the leaf transpiration rate (kg s-1), A is theleaf area (m2), e(TL) is the saturation vapour pressure atleaf temperature (Pa), ea is the water vapour pressure in thebulk air (Pa), T is the air temperature (K) and ¬ is the gasconstant.

ABA analysis and pH of the xylem sap

Xylem pH was measured in sap samples with a microelec-trode (INGOLD: Mettler-Toledo, Nänikon, Switzerland)after thawing and equilibration to room temperature. ABAconcentration was then analysed in crude samples of xylemsap by radio-immunoassay (Quarrie et al. 1988) as previ-ously described (Barrieu & Simonneau, 2000). Specificityfor ABA of the monoclonal antibody (MAC 252, providedby Dr S.A. Quarrie, Cambridge Laboratory, John InnesCentre, UK) was verified in xylem sap of N. plumbaginifo-lia by comparing radio-immunoassay of crude sap sampleswith radio-immunoassay of sap fractions recovered fromthin layer chromatography (Borel 1999).

Statistical analysis

Fitting of non-linear relationships (stomatal response to[ABA]xyl) and parameter estimation were carried out using the least squares method (GRG2 algorithm, Excel;Microsoft, Redmond, WA, USA). Differences among lineswere tested by comparing the residual sums of squares forthe individual fittings to each line, to the residual sum ofsquares for the common fitting to the whole of the lines,using an F-test (Borel et al. 2001).

RESULTS

Common response of stomatal conductance toexogenous ABA was observed for whole plantsand detached leaves of all lines

Stomatal response to exogenous ABA was studied in well-watered conditions in both whole plants and detachedleaves. Perfusing well-watered plants with deionized and

J M T e T e g g gw w L a a s a sA g= ( ) -[ ] +( )�



degased water (with no ABA) hardly modified the maximalvalue of stomatal conductance observed in intact plants just before perfusion (0·50 mol m-2 s-1 compared with0·53 mol m-2 s-1). Feeding detached leaves with artificialsap (without ABA) resulted in slightly lower values than inintact plants with differences among lines (0·45 in WT, 0·32in MS-3, 0·38 in AS-7 and 0·55 mol m-2 s-1 in AS-15).For further comparisons, relative stomatal conductance wascalculated as the ratio of gs to maximal gs, the latter beingdetermined for each line within each experiment in well-watered conditions and without applied ABA.

Supplying exogenous ABA to whole plants greatlyincreased the concentration of ABA in the xylem sap. The[ABA]xyl measured at the same time as stomatal con-ductance in ABA-fed plants increased up to 9000 mmolm-3 whereas it was about 10–40 mmol m-3 in well-wateredplants without exogenous ABA. Stomatal conductance neg-atively correlated with [ABA]xyl and was minimal when[ABA]xyl exceeded 200 mmol m-3 (Fig. 2a–c). The responseof stomatal conductance to exogenous ABA was similar inall lines. A unique decreasing exponential relationshipfitted all data without significant difference among lines(P < 0·05) (Fig. 2).

Similarly, although with more data scattering, detachedleaves of the four studied lines presented a commondecrease in relative gs as the concentration of ABA in-creased in the feeding solution (Fig. 3a–c). Although datascattering hindered statistical analyses, the response of relative gs to exogenous ABA appeared similar fordetached leaves and perfused plants. However, for all lines,stomatal closure at the highest ABA concentration was lesspronounced in detached than in intact leaves. Stomatal con-ductance thus appeared slightly less sensitive to high[ABA]xyl in detached leaves (notably that of antisenselines) than in perfused plants (Figs 2 & 3).

Grafts of WT and transgenic lines onto tobaccostocks, presented a common response ofstomatal conductance to changes in [ABA]xyl

induced by soil drying

Relationships between gs and [ABA]xyl were analysed inthree successive experiments performed on grafted plants.In each experiment, one transgenic line among MS-8, AS-15 and zep was associated with WT on a same tobacco root-stock. The maximal value of gs measured in well-wateredconditions differed among experiments but was similarbetween WT and the transgenic line within each experi-ment (1·0 mol m-2 s-1 when MS-8 and WT were graftedtogether, and 0·6 mol m-2 s-1 in all other experiments). Inall cases, a common exponential decrease in gs (relative tomaximal gs) with increasing [ABA]xyl was noticed for alltransgenic lines and WT (Fig. 4a–c). A unique relationshipwas fitted without significant difference among lines(P < 0·05). Interestingly, zep, which could not be subjectedto drought on its own roots, presented the same responseof relative gs to [ABA]xyl as the other lines (Fig. 4c) whenit was grafted onto tobacco stock.


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Figure 2. Stomatal conductance as a function of (+)-ABAconcentration in leaf xylem sap ([ABA]xylem) of well-wateredNicotiana plumbaginifolia fed with exogenous ABA. A funnelwas sealed around the stem below the last fully expanded leaf,and filled with artificial sap containing ABA. Artificial sap wasabsorbed via a 1 mm hole drilled in the stem several hoursbefore measurements in the greenhouse. Stomatal conductance(gs) was expressed as percentage of the mean maximum value(0·53 mol m-2 s-1, mean of all lines) measured on plants perfusedwith degassed water containing no ABA. (a) Wild-type; (b)complemented zep mutant MS-8; (c) antisense line AS-15. Thesolid line [same in (a), (b) and (c)] was obtained by non-linearfitting to the whole set of data (individual fittings for each linewere not significantly different, P < 0·05) and corresponds to thefollowing equation: gs (% of control) = 9 + 94 exp(-4·6 ¥ 10-3

[ABA]xylem).

Moreover, the response of relative gs to [ABA]xyl wassimilar in grafts subjected to soil water deficit onto tobaccorootstocks (Fig. 4) and in well-watered plants supplied withexogenous ABA (Fig. 2).

482 C. Borel et al.

Stomatal conductance of intact plantssubmitted to soil water deficit correlated with[ABA]xyl for all lines, but with a slightly highersensitivity in transgenic lines than in WT

In experiments with intact plants, maximal gs measured inwell-watered conditions differed among WT and transgeniclines: 1·55 mol m-2 s-1 for WT, 0·55 mol m-2 s-1 for MS-3and MS-8 (without significant difference between bothlines), 1·0 mol m-2 s-1 for AS-7, and 1·3 mol m-2 s-1 for AS-15.As the soil dried, Ypre-dawn decreased from -0·2 to about-1·0 MPa and gs decreased from maximal values to lessthan 0·1 mol m-2 s-1. A decrease in Ypre-dawn down to -0·6 MPa induced stomatal closure in all lines. Concomi-tantly, mid-day [ABA]xyl increased up to 800 mmol m-3 inWT although it remained lower than 300 mmol m-3 in alltransgenic lines.

Stomatal conductance correlated with [ABA]xyl in alllines (Fig. 5a–c). However, up to two-fold differencesappeared among transgenic lines and WT (P < 0·05).Under-producing lines exhibited a higher sensitivity of gs to[ABA]xyl than WT. This conclusion was not statisticallysupported in MS-8, where scattering of data was observedfor low values of [ABA]xyl (Fig. 5b). A higher stomatal sensitivity to [ABA]xyl was apparent in AS-7 and AS-15 for which almost all data points were distributed below themean response of WT (Fig. 5c). Overall, half stomatalclosure was observed at about 40 mmol m-3 ABA in thexylem sap of antisense transgenic lines, compared with80 mmol m-3 in WT.

For WT and AS-15, the experiments were repeated undertwo contrasted evaporative demands that varied with airvapour pressure deficit: 0·9 and 2·2 kPa on two separateexperiments with WT, and 0·9 and 3·1 kPa on two otherexperiments with AS-15. In both lines, the same relativeresponse of gs to [ABA]xyl was obtained (not detailed)whatever the evaporative demand although minimal valuesof gs (less than 0·1 mol m-2 s-1) were obtained at differenttimes from the last irrigation (4 d with the lowest evapora-tive demand as opposed to 7 d with the highest evaporativedemand).

Acclimating intact plants to elevated [ABA]xyl

did not influence stomatal response to [ABA]xyl

An experiment was performed on WT and AS-15 whereplants were irrigated with 1 mmol m-3 of (+)-ABA duringthe development of the studied leaf (thirteenth from theplant basis). At the end of this acclimation period, theincrease in [ABA]xyl measured at mid-day was significantin WT (36 ± 2 mmol m-3 in treated plants compared with18 ± 4 mmol m-3 in control plants) but not significant in AS-15 (26 ± 4 mmol m-3).Acclimation was followed by a periodof soil drying without irrigation during which stomatalresponse to drought-induced ABA was studied in accli-mated leaves. Maximal gs in acclimated plants was lowerthan in plants cultivated without ABA. However acclima-tion to elevated ABA did not affect the relative response


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Figure 3. Stomatal conductance of detached Nicotianaplumbaginifolia leaves as a function of concentration of (+)-ABAin assay solutions. The solutions were synthetic ABA in artificialxylem sap. Stomatal conductance is expressed as percentage ofmaximal value determined for each line on plants fed withartificial xylem sap only (no ABA). Values are means ± standard-errors of four observations on independent leaves. (a) Wild-type;(b) complemented zep mutant MS-3; (c) antisense plants (AS-7,closed symbols; AS-15, open symbols). The solid line [same in(a), (b) and (c)] was obtained by non-linear fitting to the wholeset of data and corresponds to: gs (% of control) = 30 + 70 exp(-4·7 ¥ 10-3 [ABA]) (no significant difference was observedbetween individual fittings to each line, P < 0·05).


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Figure 4. Stomatal conductance (gs) as a function of ABAconcentration in the leaf xylem sap ([ABA]xylem) of graftedNicotiana plumbaginifolia submitted to soil drying onto pot-grown tobacco stocks. Each point represents coupled values of gs

and [ABA]xyl measured on one leaf around midday in thegreenhouse. Stomatal conductance was expressed as a percentageof the mean maximal value measured on grafts of each line ontowell-watered tobacco stocks. (a) Wild-type; (b) complementedzep mutants MS-8; (c) antisense plants AS-15 (open symbols)and zep (stars). Non-linear fitting did not significantly differedamong lines (P < 0·05). The solid line [same in (a), (b) and (c)]represents the non-linear fitting obtained in Fig. 5a for intact WTplants (not significantly different, P < 0·05) with thecorresponding equation: gs (% of control) = 10 + 90 exp(-1·1 ¥ 10-2 [ABA]xylem).

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Figure 5. Stomatal conductance (gs) as a function of ABAconcentration in the leaf xylem sap ([ABA]xylem) of intact pot-grown Nicotiana plumbaginifolia submitted to soil drying. Eachpoint corresponds to coupled values of gs and [ABA]xylem

measured on the same leaf around midday in the greenhouse. (a)Wild-type; (b) complemented zep mutants (MS-3, closedsymbols; MS-8, open symbols); (c) antisense plants (AS-7, closedsymbols; AS-15, open symbols). Cross-symbols in (a) and (c)correspond to measurements on plants irrigated with ABA-enriched solution in order to increase [ABA]xylem during thewhole development of the measured leaf. Stomatal conductance(gs) was expressed as percentage of the mean maximum valuedetermined for each line on well-watered plants (1·55 mol m-2

s-1 for wild-type, 0·5 for MS-3 and MS-8, 1·0 for AS-7, and 1·3 forAS-15). Three experiments (three sowing dates) were merged forwild-type and AS-15 (one sowing date for each other line).Exponential fitting was significantly different among lines(except MS-8 where data were scarce and scattered). For sake ofcomparison, the same solid line (fitted to WT data set) wasshown in (a), (b) and (c) corresponding to: gs (% ofcontrol) = 10 + 90 exp(-1·1 ¥ 10-2 [ABA]xylem).

of gs to drought-induced ABA, either in WT and AS-15(Fig. 5a & c, crossed symbols). In particular, acclimating thelow-ABA line AS-15 (Fig. 1) to elevated ABA levels didnot decrease its stomatal sensitivity to [ABA]xyl.

The relationship between gs and [ABA]xyl wasindependent of YL and of xylem sap pH

Leaf water potential (YL) measured at the same time as gs

on intact plants submitted to soil drying varied from -0·7to -1·4 MPa (Fig. 6d) with a concomitant decrease in Ypre-dawn from -0·2 to -1·0 MPa. No significant differencesin YL appeared among lines when dehydrated to a givenYpre-dawn and experimented in similar conditions (i.e. sownand measured on the same day) with the exception of MS-3 and MS-8, which presented lower but also very scatteredvalues of YL (Fig. 6d). The differences in YL were more pronounced for a given line among experimental condi-tions than among lines in a given experimental condition.Notably, feeding detached leaves with artificial sap inducedstomatal closure and consequently raised YL from about -0·8 to -0·2 MPa (Fig. 6a & b). Intermediate YL (-1·1 to -0·5 MPa) was observed in plants grafted onto tobacco andsubmitted to soil drying (Fig. 6c). Contrary to intact plants,grafts of MS-8 exhibited slightly but not significantly higherYL than did other lines.

Within each experiment, stomatal conductance corre-lated with leaf water potential (Fig. 6). However, the relationship between gs and YL was loose and varied con-siderably among experiments. Notably, in one experimenton WT plants that had been subjected to drought, all theleaves but one (twelfth) of half the plants were wrapped inaluminium foil. This increased YL by approximately0·2 MPa in comparison with control plants without an alu-minium envelope (Fig. 7b).The relationship between gs andYL notably differed between wrapped and control plants(Fig. 7b). Furthermore, in plants that had been subjected todrought (either intact or grafted, Fig. 6c & d), gs was posi-tively correlated with YL whereas it correlated negativelywith YL in ABA-fed plants (Fig. 6a). This indicated that gs

was not directly controlled by YL and that the correlationbetween gs and YL was the result of their concomitant variation, which occurred differently in plants subjected todrought and in ABA-fed plants. Soil drying decreased YL

at the same time as gs declined. In contrast, feeding ABAin well-watered plants induced decreases in gs and hencereduced water flux through the plant, leading to a rise inYL up to the value observed at pre-dawn in well-wateredconditions (Fig. 6a).

The effects of YL and xylem sap pH on stomatal responseto [ABA]xyl were further tested in WT and AS-15 by com-paring the relationships between gs and [ABA]xyl for dif-ferent intervals of YL and of xylem sap pH (Fig. 8). Both inWT and AS-15, the relationship between gs and [ABA]xyl

was similar whatever the values of YL (from -1·2 to -0·5 MPa) and of xylem sap pH (from 5·5 to 6·8) (Fig. 8).In AS-15, relationships fitted for the different YL intervalsdid not differ (P < 0·05, Fig. 8b). Stomatal conductance of

484 C. Borel et al.


100

50

0

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of c

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ith n

o A

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Figure 6. Relationship between stomatal conductance (issuedfrom Figs 2–5) and leaf water potential measured at the sametime (midday) on the same leaf of Nicotiana plumbaginifolia. (a)Whole plants supplied with exogenous ABA; (b) detached leavesinfused with exogenous ABA; (c) plants grafted onto tobaccosubmitted to soil drying; (d) intacts plants submitted to soildrying onto their own roots. Each symbol represents a differentline (as in Figs 1–5). wild-type, open circles; MS-3, closedtriangles; MS-8, open triangles; AS-7, closed squares; AS-15, opensquares and zep, closed stars.

WT appeared to be slightly less sensitive to [ABA]xyl forintermediate YL (from -0·8 to -0·7 MPa), but the responseof gs to [ABA]xyl was unique for other intervals of YL

including intervals below -0·8 and above -0·7 MPa(Fig. 8a). Xylem sap pH and YL varied in the same rangesfor WT and AS-15.




(a) (b) (c)1.5

1.0

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0.00 1 2 3 4 –0.5 –0.6 –0.7 –0.8 –0.9 –1.0 10 100 1000

Leaf water potential (MPa)Days from last irrigation

Sto

mat

al c

ondu

ctan

ce(m

ol m

–2 s

–1)

Figure 7. Stomatal conductance of wild-type N. plumbaginifolia plotted as a function of (a) the time since irrigation was withheld;(b) midday leaf water potential; and (c) xylem ABA concentration ([ABA]xyl). Stomatal conductance was measured around midday on atranspiring leaf while the rest of the plant was either also submitted to evaporative demand (closed symbols), or not transpiring (wrappedin aluminium foil, open symbols).

–0.7 < YL < –0.5 MPa

–0.8 < YL < –0.7 MPa

–0.9 < YL < –0.8 MPa

–1.2 < YL < –0.9 MPa

6.6 < pH < 6.8

6.4 < pH < 6.6

6.2 < pH < 6.4

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[ABA]xylem (mmol m–3)[ABA]xylem (mmol m–3)

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al c

ondu

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ce (

mol

m–2

s–1

)

Figure 8. Relationship betweenstomatal conductance and ABAconcentration in leaf xylem sap([ABA]xylem) of pot-grown Nicotianaplumbaginifolia submitted to soil drying(same data as in Fig. 5a & b, except whenleaf water potential or xylem sap pHwere not determined). Several classes ofleaf water potential (YL) (a, b) and xylemsap pH (c, d) were distinguished. Eachpoint represents values of stomatalconductance, [ABA]xylem, YL and xylemsap pH, from one leaf of wild-type (a, c)or the antisense line AS-15 (b, d).

WT response of stomatal conductance to [ABA]xyl was the same with various sources of ABA and under contrastingevaporative demands

The response of relative gs to [ABA]xyl was similar for WTplants either intact (Fig. 5a), grafted onto tobacco (Fig. 4a)or fed with exogenous ABA (Fig. 2a). Additionally, gs

response of intact plants to drought-induced ABA was conserved under various evaporative demands, whether itwas varied by measuring plants on days with contrastingvapour pressure deficits (from 0·8 to 2·3 kPa), or by wrap-ping all leaves but one with aluminium foil (Fig. 7). A 50%decrease in stomatal conductance was observed at about80 mmol ABA m-3 in the xylem sap whatever the treatment(grafted or intact plants, various evaporative demands) andthe origin of ABA (artificially fed or drought-induced).

DISCUSSION

Manipulating capacities to synthesize ABA didnot affect stomatal sensitivity to ABA indetached leaves and whole plants fed withABA and in grafts submitted to soil drying ontotobacco stocks

For all lines and for these three experimental conditions,relative gs always correlated with [ABA]xyl following aunique relationship (Figs 2–4). This was independent of theorigin of ABA, either exogenously fed to intact plants anddetached leaves, or endogenously supplied to grafts by thetobacco rootstocks in response to soil drying.

The present study reports the first case where mani-pulation of ABA biosynthesis is shown not to influenceintrinsic sensitivity of stomatal conductance to xylem ABA.Similar conclusions were suggested with reciprocal grafts of wild-type sunflower and the low-ABA mutant w-1(Fambrini et al. 1995). This excludes any strong genetic orphysiological linkage between the capacity to synthesizeABA and the stomatal sensitivity to ABA, suggesting thatboth processes may be manipulated independently. Consis-tently, genes involved in stomatal response to ABA on onehand, and in the control of ABA synthesis rate on the otherhand, continue to be cloned in abi and low-ABA genotypes(Cutler & Krochko 1999; Li et al. 2000) and to date, thefunction of these genes do not support any interactionbetween both processes. In the same way, plant capacity to synthesize ABA presumably does not interact with stomatal sensitivity of Arabidopsis to air humidity since thesame sensitivity to air humidity has been evidenced in wild-type and the ABA-deficient mutant aba1 (Assmann, Snyder& Lee, 2000). By contrast, stomatal insensitivity to light hasbeen observed in ABA-deficient mutants (Nagel, Konings& Lambers 1994) including non-grafted zep whose stomataremain opened in darkness (Borel 1999). This abnormalphenotype was suggested to result from undevelopedstomata lacking a functional closing mechanism (Popova &Riddle 1996). Delivery of sufficient amounts of ABA duringleaf development in ABA-deficient mutants is possibly

486 C. Borel et al.

needed for the restoration of a normal stomatal sensitivityto light and other environmental conditions. In the presentstudy, stomatal sensitivity of the ABA-deficient zep mutantwas analysed on leaves that received slight amounts ofABA from the tobacco rootstock throughout their devel-opment and therefore may differ from stomatal sensitivityof non-grafted zep plants.

Relative gs (calculated as the ratio of actual to maximalgs measured on well-watered plants of each line within each experiment) was used to compare lines in differentexperiments because of the variability in maximal gs amongexperiments. The exact origin of this variability remains tobe elucidated although it probably resulted from differ-ences in leaf age across experiments and lines. Rapiddecrease in stomatal conductance with leaf age has beenevidenced after leaf growth ceased (Solarova 1980) andsuch a behaviour has been observed in N. plumbaginifolia(not shown). Differences in vapour pressure deficit amongexperiments also slightly changed gs of well-watered plants, but with a much lower effect than leaf positionand/or leaf age (Borel 1999). By contrast, it is unlikely thatdifferences in transpiration rates at the plant level influ-enced maximal gs: covering all the leaves but one did notsignificantly modify the gs of well-watered plants (1·34 ±0·13 mol m-2 s-1 in seven WT plants with only one leaf transpiring, compared with 1·35 ± 0·14 mol m-2 s-1 in 11control plants).

Whatever the cause of variability in maximal gs acrossexperiments, the use of relative gs could not have biasedcomparisons between lines in all conditions. Notably, inexperiments with the grafted plants, maximal gs was similarfor all lines in a given experiment associating one of thetransgenic line and the wild-type, and then comparison inabsolute and relative gs led to the same conclusion. Addi-tionally, expressing stomatal conductance relative to themaximal value observed in well-watered plants resulted forthe wild-type in a unique relationship between relative gs and [ABA]xyl in all experimental conditions, includingintact plants. This strongly supports the use of relative gs tocompare experiments with contrasting maximal gs.

Similar responses of stomatal conductance to[ABA]xyl among lines and under contrastingconditions reinforce the key role of ABAin controlling gs response to drought in N. plumbaginifolia

In plants submitted to soil drying, leaf water potential,[ABA]xyl, and xylem sap pH measured either at pre-dawnor at the same time (‘mid-day’) as gs, all co-evolved withthe decrease in stomatal conductance, but only mid-day[ABA]xyl correlated with relative gs with a unique relation-ship for contrasting experimental conditions (Fig. 7). Aunique relationship between mid-day [ABA]xyl and relativegs also held for wild-type leaves whether they were attachedto plants grafted onto tobacco stocks that had been sub-jected to drought, or attached to intact plants and submittedto changes in stress-induced or exogenous ABA (Figs 2a,


3a & 4a). The relationship was still conserved (i) amonglines despite contrasting time-courses of [ABA]xyl afterwithholding irrigation, and (ii) for WT plants whether theytranspired from all or only one of their leaves (Fig. 7).

This makes it unlikely that any other component of thexylem sap significantly influences the control of gs by ABAin N. plumbaginifolia. Consistently, no effect of xylem pHwas detected in stomatal response to [ABA]xyl of WT andAS-15 (Fig. 8c & d). Xylem pH weakly varied with droughtin N. plumbaginifolia, compared for example with the two-unit change induced by drought in tomato plants(Wilkinson et al. 1998). This may explain why pH influenceon stomatal conductance in the present experiments with N.plumbaginifolia was not as important as suggested intomato (Wilkinson et al. 1998) or in Commelina communis(Thompson et al. 1997). However high concentrations ofexogenous ABA failed to reduce stomatal conductance ofdetached leaves to the same extent as comparable concen-trations of endogenous ABA did in plants grafted ontotobacco or in intact plants. In this latter case, the whole com-position of the xylem sap, which originated from the tobaccostock or the intact roots, was influenced by the droughttreatment, whereas only [ABA]xyl was varied in ABA-fedleaves. Thus, any chemical signal of soil drying, which mayhave accompanied the action of ABA on stomatal con-ductance can explain why ABA-fed leaves were less sensi-tive to ABA than plants subjected to drought.

The role of YL, which modulates stomatal sensitivity toABA in maize (Tardieu & Davies 1992) and apparentlycontrols stomatal conductance in some woody species(Fuchs & Livingston 1996), was clearly excluded in N.plumbaginifolia (Fig. 8a & b). Co-evolutions of YL and gs

during drought led to relationships between these two vari-ables (Fig. 6c & d), but the relationship notably changedwhen evolutions of gs and YL were uncoupled. This wasachieved in WT by covering all but one leaf of plants thatwere subjected to drought (Fig. 7b), or by injecting ABA inall lines (Fig. 6a & b). In WT, gs correlated with [ABA]xyl

following a unique relationship for all treatments exceptdetached leaves (Figs 2a, 4a, 5a & 7c), whereas relationshipbetween gs and YL notably differed between intact andABA-fed plants (Fig. 6) and between wrapped and controlplants (Fig. 7b). Strictly, this conclusion should be restrictedto the experienced range of YL which may appear limited(-0·5 to -1·2 MPa) compared with other species (seeTardieu & Simonneau 1998) but this corresponded to awide range of evaporative demands (vapour pressure de-ficit from 0·9 to 3·1 kPa, all experiments included).

Nicotiana plumbaginifolia exhibited the same responseof gs to drought as other anisohydric species in which stom-atal sensitivity to ABA was also shown to be independentof YL (Tardieu, Lafarge & Simonneau 1996; in sunflower;Borel et al. 1997; in barley). Such stomatal control was pro-posed to be at the origin of the anisohydric behaviour thatcharacterizes species which exhibit parallel reductions inmidday YL (measured under non-limiting PPFD for stom-atal opening) and in Ypre-dawn as soil water is depleted(Tardieu & Simonneau 1998). Consistently with this inter-


pretation, typical decreases in midday YL from -0·7 to -1·4 MPa were observed in N. plumbaginifolia when Ypre-dawn decreased from -0·2 to -1·0 MPa, without markeddifferences among lines (Borel 1999).

Drought-induced accumulation of ABA in the xylem sapof WT N. plumbaginifolia was intermediate between that ofmaize (isohydric) and of sunflower (anisohydric) (Tardieu& Simonneau 1998). The anisohydric behaviour was conserved in all lines of N. plumbaginifolia (Borel 1999)although transgenic lines accumulated much less ABA inresponse to drought than maize. Therefore, differences indrought-induced accumulation of ABA were unlikely to be responsible for the contrast between isohydric andanisohydric species.

Slight differences appeared among lines instomatal responses to drought-induced ABA,which did not result from differences in YL,neither in pH, nor in acclimation to elevatedABA levels before drought

Stomatal conductance of intact plants appeared to be less sensitive to drought-induced changes of [ABA]xyl inunder-producing lines than in WT (Fig. 5). This contrastedwith the unique stomatal response to [ABA]xyl that wasobserved in ABA-fed plants and, more intriguingly, inplants of different lines grafted onto the same tobacco root-stock (Figs 2 & 4). It can be questioned whether this con-trast resulted from acclimation of under-producing lines toreduced levels of ABA when they were cultivated on theirown roots. Acclimation may have occurred during leafdevelopment in well-watered conditions, although ABAaccumulation rates were not very contrasted between WTand transgenic lines in well-irrigated plants (Borel et al.2001). Different time-courses of ABA accumulation amonglines more likely occurred during soil drying. Acclimationof wild-type and one of the transgenic lines (AS-15) tomodified levels of ABA during the whole development ofthe measured leaf resulted in a conserved stomatal re-sponse to [ABA]xyl (Fig. 5, crossed-symbols). Therefore,acclimation of stomata to slight differences in ABA supplyinduced by the genetic manipulations of ZEP levels isunlikely to explain the differences among lines in stomatalresponses of intact plants to [ABA]xyl. Such a result, andthe fact that low-ABA lines conserved the same intrinsicstomatal sensitivity to ABA, also suggest that changes instomatal sensitivity to ABA following a dehydration–rehydration cycle (see Peng & Weyers 1994) are probablynot attributable to acclimation to transient rises in ABAcontent, provided that results in N. plumbaginifolia couldbe extrapolated to other species.

Neither leaf water potential, nor xylem pH could explainthe differences in stomatal responses among lines. First,stomatal sensitivity to ABA was independent of YL (in alllines, Figs 6, 8a & b) and of xylem pH (in WT and AS-15,Fig. 8c & d; not tested in other lines). Second, neither YL

nor xylem pH noticeably differed in intact plants amonglines.


A root-sourced signal of soil-drying other thanABA is suggested to modulate stomatalsensitivity to ABA in intact plants

The coupling between drought-induced changes in[ABA]xyl and other changes in the chemical composition ofthe xylem sap probably differed among lines since ABAsynthesis abilities were differently altered. This may be atthe origin of the slight divergences observed in intact plantsamong lines (Fig. 5). An unidentified root-sourced signal of soil drought, transported in the xylem stream, possiblyinteracted with ABA. Such a signal must be of a chemicalnature, since YL did not participate to the regulation of gs

in N. plumbaginifolia.The existence of a signal of soil dryingco-acting with ABA on stomatal conductance has been pre-viously questioned by Blackman & Davies (1985) who sug-gested an effect of cytokinins, and by Wilkinson & Davies(1997) who emphasized the influence of slight drifts inxylem sap pH on the redistribution of ABA betweenapoplast and symplast. No effect of xylem sap pH on stomatal response to ABA was detectable in the presentexperiments with N. plumbaginifolia (Fig. 8c & d). Con-versely, all the results agreed with the possible combined(antagonist) actions of cytokinins (CK) and ABA, as pro-posed by Cheikh & Jones (1994) and Stoll, Loveys & Dry(2000). So far as root signals were supplied by the same rootsystem (as in grafts that were subjected to drought), ABAand CK were presumably the same in all lines, resulting inthe same time-course of ABA/CK ratio among lines asdrought develops and therefore a common response of gs

to [ABA]xyl could be expected. CK were also expected notto modify the gs response to [ABA]xyl among lines in ABA-feeding experiments: detached leaves did not receive CK atall, and whole plants of all lines probably conserved aunique CK level since well-watered conditions were main-tained during ABA feeding. By contrast, in intact plantssubmitted to drought, differential effects of CK on gs

response to [ABA]xyl were expected among lines, becauseof concomitant decreases in CK for all lines (as describedin other species: Bano et al. 1993; Munns & Sharp 1993) butdifferential accumulations of ABA. Soil water deficit had tobe more severe in order to reach a given [ABA]xyl in under-producing lines than in WT. Associated with this given[ABA]xyl, the CK level was therefore probably lower (andhence, so was stomatal conductance) in low-ABA-linesthan in WT, which could explain the lower stomatal con-ductance in transgenic than in WT N. plumbaginifolia(Fig. 5). Strong consistency of the results with an assumedrole of CK merits further studies, considering that any othermediator of drought stress with a similar action on stomatawould have caused the same responses.

ACKNOWLEDGMENTS

C.B. was supported by the Ministère Français de l’Educa-tion Nationale et de la Recherche Scientifique et Technique(Grant no. 95108).We are grateful to Anne Frey for pro-viding transgenic lines. A special mention is due to Benoît

488 C. Borel et al.

Suard, for the clever ‘high hygrometry’ compartment in the greenhouse and for taking care of its electronic com-ponents. Finally, the authors are also grateful to PhilippeBarrieu for friendly assistance with ABA assays.

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Received 3 July 2000; accepted for publication 14 January 2001


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