ph manipulation for stomatal and growth

Journal of Experimental Botany, Vol. 59, No. 3, pp. 619–631, 2008

doi:10.1093/jxb/erm338 Advance Access publication 13 February, 2008This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

RESEARCH PAPER

Manipulation of the apoplastic pH of intact plants mimicsstomatal and growth responses to water availability andmicroclimatic variation

Sally Wilkinson* and William J. Davies

The Lancaster Environment Centre, Lancaster University, Bailrigg, Lancaster LA1 4YQ, UK

Received 17 October 2007; Revised 28 November 2007; Accepted 30 November 2007

Abstract

The apoplastic pH of intact Forsythia3intermedia (cv.

Lynwood) and tomato (Solanum lycopersicum) plants

has been manipulated using buffered foliar sprays,

and thereby stomatal conductance (gs), leaf growth

rate, and plant water loss have been controlled. The

more alkaline the pH of the foliar spray, the lower

the gs and/or leaf growth rate subsequently measured.

The most alkaline pH that was applied corresponds to

that measured in sap extracted from shoots of tomato

and Forsythia plants experiencing, respectively, soil

drying or a relatively high photon flux density (PFD),

vapour pressure deficit (VPD), and temperature in the

leaf microclimate. The negative correlation between

PFD/VPD/temperature and gs determined in well-

watered Forsythia plants exposed to a naturally vary-

ing summer microclimate was eliminated by spraying

the plants with relatively alkaline but not acidic buffers,

providing evidence for a novel pH-based signalling

mechanism linking the aerial microclimate with stoma-

tal aperture. Increasing the pH of the foliar spray only

reduced gs in plants of the abscisic acid (ABA)-

deficient flacca mutant of tomato when ABA was

simultaneously sprayed onto leaves or injected into

stems. In well-watered Forsythia plants exposed to

a naturally varying summer microclimate (variable

PFD, VPD, and temperature), xylem pH and leaf ABA

concentration fluctuated but were positively corre-

lated. Manipulation of foliar apoplastic pH also affected

the response of gs and leaf growth to ABA injected into

stems of intact Forsythia plants. The techniques used

here to control physiology and water use in intact

growing plants could easily be applied in a horticultural

context.

Key words: Abscisic acid (ABA), apoplast, leaf growth, pH,

soil drying, stomatal conductance, stomatal guard cells,

temperature, vapour pressure deficit (VPD), xylem.

Introduction

Plants in drying soil and/or air must limit water loss tosustain a positive water balance in shoots and roots.Stomata are induced to close, and leaf growth is reducedin order to limit the surface area from which water can belost, and these changes occur very sensitively in responseto changes in rhizospheric (Sobeih et al., 2004) and aerialmicroclimatic (Tardieu and Davies, 1992, 1993) condi-tions. An increase in xylem and/or bulk leaf abscisic acid(ABA) concentration is often associated with the drying ofthe soil around the root (Zhang and Davies, 1989, 1990),or of the air around the shoot [measured as an increase invapour pressure deficit (VPD), Trejo et al., 1995; Nejadand Van Meeteren, 2007]. The synthesis of ABA isstimulated by the dehydration of root and/or leaf cells(Zhang and Tardieu, 1996; Nambara and Marion-Poll,2005), and/or ABA can be translocated around the plant inresponse to environmental perturbation (see below). Rootssensing a loss of soil moisture often transport more ABAto the shoot via the xylem vessels before the water statusof the shoot becomes reduced (Zhang and Davies, 1989).In the shoot, ABA sourced from roots, stems, and/orleaves interacts with guard cells to close stomata (Israelssonet al., 2006), and with the growing cells of the leaf to

* To whom correspondence should be addressed. E-mail: [email protected]: ABA, abscisic acid; cv, cultivar; gs, stomatal conductance; LER, leaf elongation rate; PFD, photon flux density; RIA, radioimmunoassay;VPD, vapour pressure deficit.

ª 2008 The Author(s).This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) whichpermits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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reduce expansion (Bacon, 1999), although there is stillsome controversy over the growth-regulatory role of ABA(Sharp, 2002).However, it is important to note the variability in the

apparent sensitivity of stomatal conductance (gs) andgrowth to a given concentration of ABA in the xylemstream (Trejo and Davies, 1991; Gollan et al., 1992;Schurr et al., 1992; Tardieu and Davies, 1992, 1993).Zhang and Outlaw (2001a) determined that changes in theABA concentration of the apoplastic fluid immediatelyadjacent to a single guard cell pair were correlated withthe stomatal response to mild stress in Vicia faba L. in theabsence of more widespread changes in ABA concentra-tion. Trejo et al. (1993) detected a linear relationshipbetween the ABA concentration in the leaf epidermalsubcompartment and the stomatal response in Commelinacommunis L., whereas only a very poor relationshipexisted between the bulk leaf ABA concentration andstomatal aperture. Such sensitive and localized changes inABA concentration, to which stomata respond, arisepartly as a result of environment-induced changes in theability of the cells of the stem and of the different leaftissues to filter out and remove ABA from, or to releaseABA into the apoplastic stream as it travels from the rootto the leaf, or as it traverses a single leaf (Slovik andHartung, 1992a, b; Slovik et al., 1995; Wilkinson andDavies, 1997).The amount of ABA that is removed by the symplast

(i.e. that enters the cells to become stored or metabolized)from the xylem and the leaf apoplast, before the tran-spiration stream reaches its target cells, depends in part onthe pH of these compartments (Kaiser and Hartung, 1981;Hartung and Radin, 1989), which has also been shown tobe sensitive to the environment in some species (seebelow). Apoplastic pH can thereby determine the concen-tration of ABA that finally arrives at the guard cells orgrowing cells (Slovik and Hartung, 1992a, b; and seeWilkinson and Davies, 2002; Wilkinson, 2004). Ingeneral, a more acidic xylem/apoplastic pH (usuallybetween 5.0 and 6.0) exists in the sap of unstressed plantsand allows the greatest removal of ABA from the xylemand leaf apoplast, such that less reaches the guard cells.More alkaline sap pH values have been detected whenplants are exposed to ‘stress’ (see below), usually between6.4 and 7.2. Under these circumstances, the pH gradientover the cell membrane that normally drives ABA removalfrom the apoplast is reduced, and ABA can accumulate toconcentrations high enough to affect stomatal guard cellsby the time that the transpiration stream reaches theirdistant locality, even in the absence of de novo synthesis.Thus pH changes generated by the root that are propa-gated along the xylem vessels to penetrate to the leafapoplast (Jia and Davies, 2007), or that are generatedwithin the leaf (see below), can function as chemicalmessengers that alert the shoot of the need to conserve

water, by adjusting the amount of ABA that finallyreaches the guard cells or the growing cells of the leaf.Increases in root and/or shoot xylem sap pH have most

commonly been shown to occur in response to soil drying(Gollan et al., 1992; Wilkinson and Davies, 1997; Baconet al., 1998; Wilkinson et al., 1998; and see Wilkinson,2004), although this is not a universal phenomenon(Thomas and Eamus, 2002). Alterations in pH can be oneof the first chemical changes measurable in xylem sapfrom plants exposed to drying soil (Bahrun et al., 2002;Sobeih et al., 2004), even when moisture tensions are lowenough that a supply of water is still freely available. SappH also increases in plants with roots that are exposed tosoil flooding (Jackson et al., 2003; Else et al., 2006), tochanges in soil nutrient status (Kirkby and Armstrong,1980; Dodd et al., 2003; Jia and Davies, 2007; and seeWilkinson et al., 2007), and in response to salt stress (Gaoet al., 2004). These changes can occur before the shootwater status of the plant is affected by perturbation at theroots (Schurr et al., 1992; Dodd et al., 2003), within 1–2 d of the onset of perturbation (Mingo et al., 2003), oreven within a few hours (Jackson et al., 2003; Gao et al.,2004; Else et al., 2006), often prior to, or coincident with,the associated environmentally induced change in plantphysiology (gs or growth).That xylem pH can alter shoot physiology via an ABA-

mediated mechanism has been experimentally demon-strated by Wilkinson and Davies (1997), Wilkinson et al.(1998), and Bacon et al. (1998). Artificial buffers adjustedto relatively alkaline pH values, equivalent to those foundin the xylem of plants experiencing soil drying, did notreduce transpiration or growth when supplied to the xylemstream of detached shoots or leaves of ABA-deficientmutants or ABA-depleted wild-type plants, unless ABAwas also supplied via the transpiration stream, even ata concentration which was not sufficient alone to affectphysiology.More recently, changes in shoot or leaf xylem/apo-

plastic sap pH have also been detected in response tonatural or imposed changes in the aerial environment(Savchenko et al., 2000; Hedrich et al., 2001—CO2; Felleand Hanstein, 2002—light, CO2; Muhling and Lauchli,2000; Stahlberg et al., 2001—light; Davies et al., 2002;Wilkinson and Davies, 2002—light, VPD, and/or temper-ature; Jia and Davies, 2007—VPD). Photon flux density(PFD—a measure of light intensity), VPD, and air tem-perature are positively correlated under most ambientconditions, and as stated above stomatal closure is oftenassociated with high VPD. High PFD/VPD/temperaturewas associated with increased xylem pH and reduced gs inForsythia3intermedia (cv. Lynwood) and Hydrangeamacrophylla (cv. Bluewave) when intact plants wereexposed to natural fluctuations in the summer microcli-mate over the course of several weeks (Davies et al.,2002; Wilkinson and Davies, 2002). In related work,

620 Wilkinson and Davies



Tardieu and Davies (1992, 1993) observed that stomataand growing leaves became more responsive to the ABAconcentration in the xylem as the VPD increased aroundleaves of field-grown maize. Since it is known that ABAcan be involved in the stomatal closure response to lowhumidity (Xie et al., 2006; but see Assmann et al., 2000),it is tempting to suggest a role for ABA-based pHsignalling in stomatal regulation when the aerial microcli-mate becomes potentially stressful. As well as mediatingchanges in foliar compartmentalization of incoming root-sourced xylem-borne ABA, microclimate-induced changesin pH may also regulate ABA release from symplasticstores in stems and/or leaves, and/or ABA removal fromthe leaf via the phloem, and recirculation via the root tothe shoot (Jia and Zhang, 1997; Sauter and Hartung, 2002;Else et al., 2006; see Wilkinson and Davies 2002).Here it is demonstrated that changes in foliar apoplastic

pH can function as ABA-mediated signals of perturba-tions in the rhizospheric and/or the aerial environment thatcan be adaptive in the face of stress in the intact plant.Evidence is provided for a novel pH-based link betweenthe aerial environment and stomatal aperture. Foliar spraysof phosphate buffer iso-osmotically adjusted to a range ofpH values are used to manipulate leaf apoplastic pH andplant physiology artificially in intact unstressed Forsythiaand tomato plants, and in intact plants of the ABA-deficient flacca mutant of tomato (Imber and Tal, 1970).

Materials and methods

Plant material

Seeds of tomato (Solanum lycopersicum L. cv. Ailsa Craig), of boththe wild type and its ABA-deficient flacca mutant (Imber and Tal,1970), were sown in Levington F2 compost (Fisons, Ipswich, UK).Seedlings were transplanted to 1.0 l pots in a greenhouse witha variable day/night temperature, with supplemental lighting (pro-vided by 600 W sodium Plantastar lamps, Osram, Germany) givinga photoperiod of 16 h. They were watered daily with tap water tothe drip point, and weekly with full-strength Hoagland nutrientsolution. Flacca plants were sprayed twice weekly with 0.2 mmolm�3 ABA (Sigma, UK) to maintain water relations. This treatmentwas withdrawn 1 week prior to experimentation. Plants of 4–8weeks of age were used in the experiments, which were conductedin the greenhouse (conditions as above).Pruned, 1-year-old Forsythia3intermedia (cv. Lynwood) plants

were potted in the spring into a medium comprising 100%sphagnum peat with 6.0 g l�1 Osmocote Plus 12–14 monthcontrolled-release fertilizer and 1.5 g l�1 MgCO3. Plants weregrown to ;100 cm in height and experimentally manipulated inpolythene tunnels exposed to a naturally varying summer microcli-mate throughout, in 3.0 or 5.0 l pots supplied with ;300–500 cm3

water daily (depending on pot size and prevailing conditions) byhand or via drip irrigation, to maintain them in a well-watered state.

Manipulation of apoplastic pH in intact plants using

foliar sprays

Forsythia and tomato (wild-type or flacca mutant) plants weresprayed daily for up to 8 d between 09.00 h and 10.00 h over the

entire foliated region, with water or potassium phosphate buffer(10 mol m�3 KH2PO4/K2HPO4). Buffers were adjusted to a rangeof pH values (5.0–6.7) by altering the ratio of the two salts, suchthat different treatments were iso-osmotic. In some experimentsusing the flacca tomato mutant, ABA (0.06–0.15 mmol m�3) wasadded to the buffer spray. It is assumed that the foliar spraypenetrates to the interior of the leaf via ingress through stomatalpores (Kosegarten et al., 2001). In some cases, stems of Forsythiaplants and flacca mutant tomato plants were injected between10.00 h and 11.00 h daily with water or ABA (0.3 cm3 water or0.06 mmol m�3 ABA for flacca; 1–10 mmol m�3 ABA forForsythia). The injection point was immediately sealed withlanolin. It is assumed that at least a portion of the injected solutionpenetrated to the xylem vessels of the stem, and was therebytransported to the leaves above this point. gs, PFD, and leaftemperature were measured daily at ;14.00–15.00 h with a poro-meter (AP-4, Delta-T Devices Ltd, UK) in 4–6 replicates ofForsythia and tomato wild-type and flacca mutant plants that hadbeen sprayed with buffers adjusted to a range of pH values, andinjected with water or ABA where stated. The gs was measured inthe most recently fully expanded leaf/leaflet, ensuring that themeasurement leaf was above the ABA/water injection point (in thesame branch) where appropriate. These positions were ;10–20 cmapart. Forsythia leaf lengths were measured daily with a ruler at;16.00 h to establish a rate of elongation in expanding leaves,again 10–20 cm above the point of injection where appropriate.

Measuring environmental and physiological variables in intact

Forsythia plants exposed to a naturally varying microclimate

In a separate experiment, gs of intact plants was monitored every2–3 d over the course of several weeks exposure to natural summervariations in the local microclimate (with respect to PFD, VPD, andtemperature) using the porometer. Measurements were taken at thesame time of day (;14.00 h) from the first fully expanded leaf(usually the fourth from the apex), and the ambient PFD incident oneach leaf, and its surface temperature were also measured using theporometer. In addition, equivalent leaves were also harvested from4–5 equivalent plants on six occasions throughout the experiment(every 4–5 d), to determine bulk leaf ABA concentration (seebelow). Xylem sap was also collected from stems cut from theseplants using a Scholander pressure bomb (SKPM 1400; SkyeInstruments Ltd, Powys, UK). Sap was expressed from the apical10–20 cm of detached shoots at overpressures of –0.4 to –0.8 MPa.The pH was determined in each extract within 10 min (MicroCombination pH electrode, Lazar Research Laboratories Inc., CA,USA). Bulk extraction of a mixture of xylem and leaf apoplastic sapfrom a portion of the shoot apex, as opposed to micro-sampling ofthe apoplast local to the guard cell, allows measurement of largerscale more widespread changes in sap pH and ABA concentration(see Discussion).

Radioimmunoassay for measuring leaf ABA concentration

Upon harvesting leaf tissue this was immediately frozen in liquidnitrogen. Frozen leaf tissue was freeze-dried for 48 h, finely ground,and extracted overnight at 5.0 �C with distilled deionized waterusing an extraction ratio of 1:50 (g DW:cm3 water). The ABAconcentration of the extract was determined using a radioimmuno-assay (RIA) following the protocol of Quarrie et al. (1988), using[G-3H](6)-ABA at a specific activity of 2.0 TBq mmol�1 (AmershamInternational, Bucks, UK), and the monoclonal antibody AFRCMAC 252 (generously provided by Dr SA Quarrie; Institute ofPlant Science Research, Norwich, UK) which is specific for (+)-ABA. Any ABA present in the tissue extract inhibits the binding ofthe tritiated ABA to the antibody. This concentration is quantified

pH and ABA signalling in intact plants 621



using a series of standards of known non-radioactive ABAconcentration in the assay such that sample counts can be calibratedfrom the resultant standard curve. However, the possibility exists inany species that compounds with a similar structure to ABA butwithout ABA activity may contaminate the tissue extract. Everyspecies must be tested for these contaminants using a spike dilutiontest. The standard curve is tested in the assay in the presence andabsence of an increasing dilution of tissue extract. If the resultingstandard curves at each dilution remain parallel, then the onlycompound present in the tissue extract that affects the relationshipbetween the binding of the added non-radioactive ABA and thetritiated ABA to the antibody is endogenous ABA itself. Spike testson leaf tissue collected from well-watered Forsythia plants werecarried out, and no contaminants were found (not shown).

Transpiration bioassays

The effects of exogenous ABA concentration were tested on the rateof transpirational water loss through stomata (a measure of stomatalopenness) from detached leaves of greenhouse-cultivated Forsythia(growth conditions as for tomato). Leaves (third to seventh belowgrowing apices) were detached from plants which had been kept for1.0 h in the dark, and petioles were re-cut under water beforeimmediate transfer to treatment solutions, in order to preventembolism (blockage of the xylem vessels with air). Treatmentsolutions were 5.0 cm3 water6ABA at the appropriate concentra-tion, in 6.0 cm3 plastic vials covered in aluminium foil to preventevaporation from the solution surface. Leaves were introducedthrough slits in the foil so that petioles were submerged. The vialscontaining the leaves were placed under lights (PFD 400 lmol m�2

s�1) at 24 �C before 11.00 h. They were weighed approximatelyevery 30 min for up to 5 h, after which time leaf area was measuredin a leaf area meter (Li-3000A; Li-Cor Inc., Lincoln, NE, USA).Water loss was converted from weight to mmol, and expressed ona per unit leaf area per second basis. Means and standard errorsfrom five replicates were determined.

Results

Effects of foliar sprays adjusted to a range of pHvalues on gs and growth in intact Forsythia andtomato plants

Foliar sprays adjusted to relatively alkaline ‘stressful’ pHvalues (6.4–6.7) reduced the gs of intact well-wateredForsythia and wild-type tomato plants, and reduced leafelongation rates (LERs) in Forsythia plants, in compari-son with controls sprayed with water (Fig. 1A–C). ThesepH values are equivalent to those detected in sapexpressed from well-watered Forsythia plants experienc-ing a high PFD (Davies et al., 2002; and see below), andfrom tomato plants exposed to drying soil (Wilkinsonet al., 1998). Foliar sprays adjusted to more acidic pHvalues, equivalent to those detected in sap from plantsexperiencing lower PFDs (Forsythia) or well-watered soil

Fig. 1. The effect of pH on mean gs (n¼6 6SE; A, C) and leafelongation rate (LER; n¼5 6 SE; B) when intact pot-grown Forsythia(A, B) or tomato (C) plants were sprayed once daily over the foliatedregion with water (controls) or phosphate buffers (10 mol m�3

KH2PO4/K2HPO4) iso-osmotically adjusted to a range of pH values.




(tomato), did not reduce gs or LERs. The effect on gs wasinduced after 2.0 d (one 09.00 h foliar spray applicationper day), and the effect on LER was induced within 5.0 dof the start of treatment.

Effects of foliar sprays adjusted to a range of pHvalues on the relationship between PFD and gs inintact Forsythia plants

There was a negative correlation between current PFD andmorning gs sampled between 09.30 h and 10.30 happroximately every 1–2 d in intact Forsythia plants thatwere exposed to natural summer variations in the aerialmicroclimate over the course of 8 d (Fig. 2A). It is assumedthat the correlation between gs and VPD, and between gsand leaf temperature would be the same, given that theseclimatic variables are positively correlated under mostambient conditions. The value of the r2 coefficient of the

second order regression between PFD and gs (Sigmaplot2001) was reduced as the pH of the foliar spray applied tothe plants increased from 5.0 to 6.7 (Fig. 2A–D). Controls(Fig. 2A) were sprayed daily with water. Manipulatingan increase in the foliar apoplastic pH effectively removedthe correlation between PFD (and, by inference, VPD andleaf temperature) and gs.

Effects of foliar sprays adjusted to a range of pHvalues on gs and its response to ABA in intact plantsof the ABA-deficient flacca mutant of tomato

Foliar sprays adjusted to relatively alkaline pH values, towhich gs was responsive in the wild type (Fig. 1C), didnot reduce gs in leaves of flacca plants compared withwater-sprayed controls (Fig. 3). However, the wild-typeresponse to alkaline pH was restored in the flacca plantswhen ABA was simultaneously sprayed onto the leaves or

Fig. 2. Ambient PFD incident at ;10.00 h on the first fully expanded leaf of intact pot-grown Forsythia plants in polythene tunnels, plotted againstthe gs of the same leaf, in plants which were sprayed daily over the foliated region with water or phosphate buffers (10 mol m�3 KH2PO4/K2HPO4)iso-osmotically adjusted to a range of pH values. A bar indicating maximum and minimum leaf surface temperature is also shown. Points representmeasurements taken every 1–2 d over 8 d in each of four plants per pH treatment. Second-order regressions are shown at each pH, with 95%confidence intervals (dotted lines) and r2 curve coefficients, as calculated on Sigmaplot 2001.




injected into the stems (Fig. 3A, B) at a concentrationapproximating that measured in sap from well-wateredwild-type plants that alone did not reduce gs. The effect ofABA in combination with pH 6.8 to reduce gs could bedetected 2 d after the start of treatment (one 09.00 happlication per day). More acidic foliar sprays did notreduce gs in either the presence or absence of ABA. Thedifferent rates of control gs in the two experiments (Fig.3A, B) were a result of differing ambient conditions in thegreenhouse (temperatures differed over a range of 5 �Cand PFDs over a range of 150 lmol m�2 s�1), and of thediffering stages of the measurement leaf (immature—Fig.3A; mature—Fig. 3B). In some experiments foliar spraysadjusted to pH 6.0 increased gs in comparison with water-sprayed controls, especially in the absence of ABA (not

shown), presumably as the endogenous apoplastic pH ofthe control plants was >6.0.

Forsythia plants were able to generate andrespond to ABA

When the soil around the roots of intact Forsythia plantswas allowed to dry, increases in bulk leaf ABA could bedetected within 3 d (not shown). When leaves wereharvested and shoot xylem/apoplastic sap was expressedapproximately every 4–5 d from intact Forsythia plantsexposed to a naturally varying summer aerial microcli-mate (with respect to PFD, VPD, and temperature), sappH and bulk leaf ABA concentration varied but werepositively correlated (Fig. 4). Stomatal closure wasassociated with more alkaline xylem pH at relatively highPFD/VPD/temperatures (Davies et al., 2002; Wilkinsonand Davies, 2002), and with higher bulk leaf ABAconcentrations (not shown). Stomata of detached Forsythialeaves were competent to close in response to ABA ina concentration-dependent manner when this was suppliedvia the xylem at the cut petiole, and subsequent rates oftranspiration were measured (Fig. 5).

Effects of foliar sprays adjusted to a range of pHvalues on the response of gs and leaf growth to ABAinjected into the stems of intact Forsythia plants

ABA injected into the stems of intact Forsythia plantsreduced gs and LER in a concentration-dependent manner

Fig. 3. The effect of pH6ABA on mean immature (A, n¼6 6SE) ormature (B, n¼6 6SE) leaf gs in intact flacca tomato mutant plants. Thefoliated region was sprayed with water (controls) or phosphate buffers(10 mol m�3 KH2PO4/K2HPO4) iso-osmotically adjusted to pH 6.0 orpH 6.8. (A) The effect of ABA on the response to pH when the ABA(0.06 mmol m�3) was supplied to the leaf surface in the water/bufferspray. (B) The effect of ABA on the response to pH when plants wereinjected with water or 0.15 mmol m�3 ABA (0.30 cm3) ;5–10 cmbelow the measurement leaf. Spraying and injecting took place 4–6 hprior to measurement of gs.

Fig. 4. The pH of xylem/apoplastic sap expressed from the apical 10–20 cm of shoots severed from pot-grown Forsythia plants, plottedagainst the bulk leaf ABA concentration of leaves detached from thesame plant at the same time (;15.00–16.00 h). Measurements weretaken every 4–5 d over the course of several weeks from 4–5 plantsexperiencing variable mid-summer ambient conditions in polythenetunnels with respect to PFD/VPD/temperature (PFD ranging from;130 lmol m�2 s�1 to 1100 lmol m�2 s�1). A second-orderregression is shown, with a 95% confidence interval (dotted line) andan r2 coefficient, calculated using Sigmaplot 2001.




4–6 h later when leaves were sprayed with buffersadjusted to pH 5.0 and pH 5.8, but not when these weresprayed with a more alkaline buffer (pH 6.7; Figs 6, 7). gsand leaf growth were most sensitive to ABA at pH 5.8. AtpH 6.7, gs and leaf growth rate were already reduced evenin water-injected controls, such that ABA could notinduce any further reductions in these variables.

Discussion

A technique is reported here that allows manipulation offoliar apoplastic pH in intact plants, such that it has beenpossible to assess the effects of this variable on shootphysiology in vivo. Intact plants were sprayed with iso-osmotic phosphate buffers adjusted to a range of pHvalues. Kosegarten et al. (2001) demonstrated thata similar technique (spraying sunflower leaves on intactplants with diluted citric and sulphuric acids) indeedresulted in changes in apoplastic pH, as measured witha fluorescent dye loaded into the apoplast of the leaves.For the first time it has been demonstrated that

applications of buffers adjusted to a ‘stressful’ pH ofbetween 6.4 and 7.0 can close stomata and reduce leafgrowth in the intact plant (Fig. 1). These changes could beinduced in the absence of the environmental perturbationthat normally generates an equivalent endogenous pHchange, in this case soil drying (tomato—Wilkinson et al.,1998) or a stressful aerial leaf microclimate (Forsythia—Davies et al., 2002). Implementation of this technique in

Fig. 5. The effect of water or ABA concentration on the mean rate ofwater loss via transpiration from mature, detached Forsythia leaves(n¼5, means with standard error bars shown) severed from greenhouse-raised plants. Water or ABA solutions were fed to the xylem bysubmerging the petiole in the treatment solution.

Fig. 6. The effect of pH on the response of gs to water or ABA inintact Forsythia plants, when the foliated region was sprayed once daily(09.00 h) with phosphate buffers (10 mol m�3 KH2PO4/K2HPO4) iso-osmotically adjusted to a range of pH values, and the stems wereinjected once daily (10.00 h) with water or ABA (0.30 cm3) ;6–10 cmbelow the measurement leaf. gs was measured in the most recentlymature leaf in each of six plants, and means with standard errors areshown.




a horticultural/agricultural context could be of great bene-fit with regard to water conservation (see below). ThatABA was either necessary for the induction of stomatal

closure by relatively alkaline buffers (in ABA-deficientflacca mutants of tomato—Fig. 3) or that responses of gsand leaf growth to injected ABA were modulated by thepH application (Figs 6, 7—Forsythia) indicated that theeffect of the foliar buffer was to adjust the compartmen-tation of ABA within the leaf, thereby altering theconcentration that finally penetrated to the apoplasticmicro-compartment around the stomatal guard cells or thegrowing cells of the leaf (see Introduction; Wilkinson andDavies, 2002; Wilkinson, 2004). When the foliar spraywas more alkaline, more of the ABA in the transpirationstream (whether this was sourced from the root or theshoot, see below) was presumably allowed to by-pass thecells of the stem and of the leaf without being sequestered,such that more finally penetrated to the guard cells and thegrowing cells in the leaf (Fig. 4; Slovik and Hartung,1992a, b; Wilkinson and Davies, 1997; Bacon et al.,1998; Wilkinson et al., 1998). In addition, ABA loadinginto the xylem lumen from existing stores in stem and/orpetiole parenchyma can occur when the pH of sapperfused through the lumen is more alkaline (Sauter andHartung, 2002; Else et al., 2006; and see Fig. 4). ABAconcentrations in sap collected from leaf petioles werehigher than those in sap collected from stem bases offlooded tomato plants exhibiting increases in sap alkalin-ity (Else et al., 2006). Another pH-based effect on shootABA redistribution has been demonstrated by Jia andZhang (1997). Movement of ABA out of maize leaves viathe phloem was reduced when the leaf apoplast wasadjusted to a relatively high pH, such that ABA accu-mulated in the leaf. Thus there is also a contribution to thealkalinity-induced enrichment of xylem and leaf apo-plastic sap with ABA that is sourced from the stem andthe leaves.In previous work (Davies et al., 2002; Wilkinson and

Davies, 2002) it was observed that a stressful aerialmicroclimate (high PFD, VPD, temperature) correlateswith xylem alkalization (Forsythia, Hydrangea) and withstomatal closure (Forsythia). It was suggested thata change in apoplastic pH could be generated within theleaf/shoot by some aspect of the aerial microclimate(VPD, PFD, and/or temperature; see also Wilkinson,2004). However, this is the first time that a causal linkbetween aerial perturbation, apoplastic pH, and stomatalresponse has been demonstrated. That applying relativelyalkaline buffers to the leaves removed the correlationbetween decreasing stress and stomatal opening (Fig. 2)indicates that an acidic milieu contributes to stomatalopening as the environment becomes less stressful. Whenthis is considered alongside the findings that (i) the aerialenvironment can change the pH and the stomatal responsein parallel (Davies et al., 2002; Wilkinson and Davies,2002) and (ii) xylem pH and leaf ABA concentration arepositively correlated (Fig. 4), it is evident that theenvironment can affect stomata through a change in pH

Fig. 7. The effect of pH on the mean response of leaf elongation rate(LER; n¼6 6SE) to water or ABA in intact Forsythia plants, when thefoliated region was sprayed daily (09.00 h) with phosphate buffers(10 mol m�3 KH2PO4/K2HPO4) iso-osmotically adjusted to a range ofpH values, and the stems were injected once daily (10.00 h) with wateror ABA (0.30 cm3) ;6–10 cm below the measurement leaf.




and thereby via a change in the amount of root- and/orleaf-sourced ABA that is able to penetrate to the guardcells. These pH changes do not act directly on stomata,and require ABA to do so in intact leaves (Fig. 3).Opposing, and presumably direct effects of pH have beenobserved on stomatal behaviour when stomatal apertureswere measured after incubation of epidermal peels onsolutions buffered to a range of pH values (Wilkinson andDavies, 1997; Wilkinson, 1999). The more potent effectof a propagated pH change in the intact leaf is on thedistribution of ABA between leaf compartments, and thismust over-ride any direct effects of pH on guard cellbiochemistry.It is important to distinguish here between (at least)

three different groups of environmentally induced pHchange that seem to occur. First, rapid (inducible withinminutes), often transient changes in pH are apparentlylocalized to the immediate guard cell apoplast. These maybe induced by environment-led changes in stomatalaperture and the accompanying ionic fluxes in and out ofthe guard cells themselves (Bowling and Edwards, 1984;Edwards et al., 1988; Hedrich et al., 2001; Felle andHanstein, 2002). Stomatal closure, induced by variousimposed environmental changes, correlated with short-term localized alkalization. Secondly, light/dark transi-tions and changes in atmospheric CO2 concentration caninduce rapid short-term oscillations in apoplastic pHwhich seem to result from changes in photosynthetic leafcell H+-ATPase activity and/or changes in photosyntheti-cally derived malate concentration, which can propagatea short distance within the leaf apoplast (Muhling andLauchli, 2000; Hedrich et al., 2001; Stahlberg et al., 2001;Felle and Hanstein, 2002). Thirdly, and more relevanthere, are those which arise more slowly after perturbation(;4–48 h, Sobeih et al., 2004; Else et al., 2006) at a pointdistant from the guard cells, which are of a longerduration (several days, Bahrun et al., 2002; Mingo et al.,2003), and which can be transported over longer dis-tances, often between organs (Hoffmann and Kosegarten,1995; Else et al., 2006; Jia and Davies, 2007). The latterare more likely to be those involved in long-distanceABA-based signalling, whilst the rapid transient pHchanges localized to the apoplast around guard cells andphotosynthetic cells are likely to be non-message-carryingby-products of a prior stomatal or photosynthetic cellresponse, and/or to be involved in localized changes in iontransport activity required to generate the biochemicaldriving force for nutrient uptake or stomatal movement inresponse to light/CO2 (Zeiger and Zhu, 1998; Assmannand Shimazaki, 1999).There has been previous speculation about the mecha-

nism behind the more widespread xylem sap pH changethat can occur in response to variations in the leafmicroclimate, with respect to individual effects of PFD,VPD, and/or temperature (Wilkinson and Davies, 2002;

Wilkinson, 2004). Mechanisms whereby soil drying,flooding, and variations in nutrient availability can lead toan increase in xylem sap pH have been discussedelsewhere (Wilkinson, 1999, 2004; Wilkinson and Davies,2002; Else et al., 2006; Wilkinson et al., 2007). It wassuggested that VPD may underlie the aerial environment-led alterations in xylem sap pH (Wilkinson and Davies,2002), in order to explain how stomata can become moreresponsive to a given dose of ABA in the xylem stream asVPD increases (Tardieu and Davies, 1992, 1993). How-ever, Jia and Davies (2007) increased VPD at a fixedtemperature and PFD around C. communis leaves andfound that, alone, this actually acidified the apoplast,which would tend to increase ABA retention by thesymplast. The authors suggested that fewer protons wereremoved from the transpiration stream when it by-passedthe symplast more rapidly at high VPD. Thus it wouldseem that increasing VPD acts more directly on guard cellbiochemistry (Assmann et al., 2000; Bunce, 2006) toincrease stomatal and/or growth sensitivity to ABA(Grantz, 1990; Tardieu and Davies, 1992, 1993; Bunce,1996; Xie et al., 2006), and that PFD and/or air tem-perature (see below) remain the most likely effectors ofthe pH changes described above. Nevertheless, it must benoted that evidence still exists to show that high VPD canlimit stomatal aperture by increasing the apoplastic ABAconcentration in the vicinity of the guard cells (Zhang andOutlaw, 2001b).Is PFD the driver for the changes in pH measured in

apoplastic sap expressed from plants experiencing a rangeof microclimatic conditions (Davies et al., 2002; Wilkinsonand Davies, 2002)? Bunce (2006) found that at a fixedtemperature and fixed high VPD, increasing the PFD from300 to 1500 lmol m�2 s�1 re-opened stomata in fourdifferent species. Kaiser and Hartung (1981) providedevidence that saturating light can induce chloroplasticalkalization, causing ABA to be retained inside theseorganelles and reducing stomatal sensitivity to externallysupplied ABA. Heckenberger et al. (1996) found that gswas less sensitive to ABA fed to sunflower leaves ata PFD of 450 lmol m�2 s�1 as opposed to 200 lmol m�2

s�1 when the temperature was fixed at 21 �C. It wouldseem, therefore, that pH, leaf ABA concentration, andstomatal aperture are most likely to be responding to thehigh temperature that occurs when Forsythia plants areexposed to a stressful aerial microclimate, rather than tothe associated high PFD or VPD.High temperature may increase CO2 removal from the

apoplast for photosynthesis, which will tend to alkalizeapoplastic sap by virtue of its low buffering capacity(Savchenko et al., 2000). High temperature will alsoincrease nitrate transport into the leaf (Aslam et al., 2001;Castle et al., 2006), via effects either on the transpirationrate or on the activation state of uptake and transportproteins. Increases in xylem nitrate concentration have




been shown to increase apoplastic pH in several species(see Wilkinson et al., 2007). Co-transport of nitrate intothe symplast with two protons depletes the apoplast ofpositive charge (Ullrich, 1992). The reduction of nitrate toammonium in the cells of the leaf (which increases withsubstrate availability), and the subsequent synthesis ofammonium to organic material generates OH– anions(Raven and Smith, 1976). Both effects will tend toincrease xylem sap pH. Changes in xylem sap nitrateconcentration above the deficient range have recently beenshown to be powerful modulators of stomatal aperture and

leaf growth (Jia and Davies, 2007; Wilkinson et al.,2007). Relatively high xylem nitrate concentrations,within the physiological range, closed stomata and/orreduced leaf growth synergistically with ABA and/oralkalinity, in maize, tomato, and Commelina. The effectcould be removed by supplying the nitrate to the xylem ofdetached leaves in a more acidic buffer. Nitrate may onlybe a factor in apoplastic pH alkalization in species whichmetabolize this anion in the shoot as opposed to the root(Wilkinson, 1999; Wilkinson et al., 2007). Alternatively,high rates of photosynthesis induced by high temperature

Fig. 8. Diagrammatic representation of the effects of the rhizosphere and the aerial microclimate on xylem and apoplastic pH in the different regionsof the plant, and on modulation by pH of ABA translocation/sequestration within and between the different plant tissues/organs. The ABAconcentration finally perceived by the stomata is thus representative of the entire plant environment via effects of the environment on pH. Stomatarespond to this ABA concentration by adjusting their aperture and controlling water loss.




(to an optimum) may influence leaf apoplastic pH byinducing an increase in sugar uptake into the phloem,which can occur via proton co-transport across the plasmamembrane of the sieve tube cells, again depleting theapoplast of positive charge (Wilkinson, 1999). Finally ithas been shown that an increase in the apoplastic malateconcentration coincides with alkalization and stomatalclosure (Patonnier et al., 1999—Fraxinus excelsior;Hedrich et al., 2001—Vicia faba and Solanum tuber-osum). Increased photosynthesis at high temperature maylead to greater malate synthesis in leaf cells, and itssubsequent release to the apoplast. Further research isrequired to establish whether temperature can alter apo-plastic pH via any of the mechanisms proposed above. Itmust also be noted here that temperature can have othermore direct effects on stomatal guard cells (Ilan et al.,1995) which may be overridden by the accumulation ofABA. Stomatal sensitivity to ABA also increases withtemperature up to 25 �C (Wilkinson et al., 2001), and thiseffect may act in conjunction with the increase inapoplastic alkalinity.Whatever the cause of the pH change, it appears to be

important in modulating physiological responses to theaerial environment in intact plants (Fig. 2) via an ABA-dependent mechanism (Figs 3, 4, 6, 7). It is proposed thatenvironmental factors that affect aerial parts of the plant(e.g. PFD, temperature, VPD, diurnal/seasonal change,and fungal infection) can interact with factors that affectunderground parts (drought, flooding, salt stress, nutrientavailability, and nodulation) and/or the whole plant(water/nutrient availability and temperature) by impingingon the ABA concentration finally perceived by the guardcells and the growing cells of the leaf. The pH of distinctregions of the root, stem, and shoot, each perhapsresponding locally to differing levels and types of stimuli/perturbation, will govern the amount of ABA thatbecomes ‘locked away’ in these localities, or that is freeto travel on to the responsive cells at the culmination ofthe transpiration stream in the leaf apoplast. Perturbationsthat increase the pH of a particular region will amplify theABA signal as the transpiration stream traverses it. Thiseffect is depicted in Fig. 8.It is important to note here that the present data provide

new evidence for the concept that the shoot may generateand respond to ABA-based signals independently of thosesourced from the root (Wilkinson and Davies, 2002).However, given the dynamic nature of both the rhizo-spheric and the aerial environment, ABA-based signalsfrom both sources, and interactions between them, arelikely to be important. Whilst recently described data havebeen interpreted to rule out a function for root-sourcedABA in communicating water deficit from the root to theshoot (Christmann et al., 2007), recirculation of ABAoriginally synthesized in the shoot back into the transpira-tion stream via the root was not accounted for by the

authors. This will represent a substantial proportion of theroot-sourced signal (Wolf et al., 1990; Neales andMcleod, 1991). Stores of ABA within roots can also bemobilized when soil begins to dry (Slovik et al., 1995).Thus de novo biosynthesis of ABA in roots is notnecessarily a requirement for long-distance root-sourcedABA signalling under all circumstances, although a wealthof evidence exists in the literature to show that ABAbiosynthesis in roots and transport of this to the shootdoes occur in drying soil (see references in Davies andZhang, 1991; Wilkinson, 1999; Davies et al., 2002;Wilkinson and Davies, 2002). Furthermore, use of theroot pressure vessel to restore shoot water relations hasdemonstrated the potential for root-sourced chemicalsignals to act independently of hydraulic signals (Gollanet al., 1986). It is proposed that interactions between ABAand pH will allow the shoot to modify its response to anABA-based root signal as a function of local climaticconditions, in the face of the potential from both environ-ments for dehydration. This may be especially importantin tall woody species where leaves are much further awayfrom the root. Preliminary results from our laboratoryshow that stomata of several woody species are competentto respond to an increase in apoplastic pH induced bya foliar spray to the intact plant, even when soil dryingdoes not alkalize xylem sap from the same species (SharpRG and WJ Davies, unpublished results).This research could potentially benefit the horticultural

and/or agricultural industry. Buffered foliar sprays couldbe used to reduce plant water loss during cultivation, and/or to induce plants to grow more slowly or to specifiedproportions, without employing more traditional deficitirrigation techniques. This is particularly pertinent withregard to current global warming.

Acknowledgements

The authors would like to thank Dr DLR De Silva and Dr JTheobald for conducting the RIA experiments, and MaureenHarrison, Anne Keates, and Philip Nott for technical support. Thework was financially supported by DEFRA Horticulture Linkproject HLO132LHN/HDC HNS 97.

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