Manipulation of the apoplastic pH of intact plants mimics stomatal and growth responses to water availability and microclimatic variation


Manipulation of the apoplastic pH of intact plants mimics stomatal and growth responses to water availability and microclimatic variation

Sally Wilkinson* and William J. Davies

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


An open access article from Journal of Experimental Botany 2008 59(3):619-631.

The apoplastic pH of intact Forsythiaxintermedia (cv. Lynwood)and tomato (Solanum lycopersicum) plants has been manipulatedusing 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 thegs and/or leaf growth rate subsequently measured. The most alkalinepH that was applied corresponds to that measured in sap extractedfrom 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 gsdetermined in well-watered Forsythia plants exposed to a naturallyvarying summer microclimate was eliminated by spraying the plantswith relatively alkaline but not acidic buffers, providing evidencefor a novel pH-based signalling mechanism linking the aerialmicroclimate with stomatal aperture. Increasing the pH of thefoliar spray only reduced gs in plants of the abscisic acid(ABA)-deficient flacca mutant of tomato when ABA was simultaneouslysprayed onto leaves or injected into stems. In well-wateredForsythia(variable PFD, VPD, and temperature), xylem pH and leaf ABAconcentration fluctuated but were positively correlated. Manipulationof foliar apoplastic pH also affected the response of gs andleaf growth to ABA injected into stems of intact ForsythiaThe techniques used here to control physiology and water usein intact growing plants could easily be applied in a horticulturalcontext. plants exposed to a naturally varying summer microclimate plants.

Key words: Abscisic acid (ABA), apoplast, leaf growth, pH, soil drying, stomatal conductance, stomatal guard cells, temperature, vapour pressure deficit (VPD), xylem




Plants in drying soil and/or air must limit water loss to sustaina positive water balance in shoots and roots. Stomata are inducedto close, and leaf growth is reduced in order to limit the surfacearea from which water can be lost, and these changes occur verysensitively in response to changes in rhizospheric (Sobeih et al., 2004)and aerial microclimatic (Tardieu and Davies, 1992, 1993) conditions.An increase in xylem and/or bulk leaf abscisic acid (ABA) concentrationis often associated with the drying of the soil around the root(Zhang and Davies, 1989, 1990), or of the air around the shoot[measured as an increase in vapour pressure deficit (VPD), Trejo et al., 1995;Nejad and Van Meeteren, 2007]. The synthesis of ABA is stimulatedby the dehydration of root and/or leaf cells (Zhang and Tardieu, 1996;Nambara and Marion-Poll, 2005), and/or ABA can be translocatedaround the plant in response to environmental perturbation (seebelow). Roots sensing a loss of soil moisture often transportmore ABA to the shoot via the xylem vessels before the waterstatus of the shoot becomes reduced (Zhang and Davies, 1989).In the shoot, ABA sourced from roots, stems, and/or leaves interactswith guard cells to close stomata (Israelsson et al., 2006),and with the growing cells of the leaf to reduce expansion (Bacon, 1999),although there is still some controversy over the growth-regulatoryrole of ABA (Sharp, 2002).

However, it is important to note the variability in the apparentsensitivity of stomatal conductance (gs) and growth to a givenconcentration of ABA in the xylem stream (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 immediately adjacentto a single guard cell pair were correlated with the stomatalresponse to mild stress in Vicia faba L. in the absence of morewidespread changes in ABA concentration. Trejo et al. (1993)detected a linear relationship between the ABA concentrationin the leaf epidermal subcompartment and the stomatal responsein Commelina communis L., whereas only a very poor relationshipexisted between the bulk leaf ABA concentration and stomatalaperture. Such sensitive and localized changes in ABA concentration,to which stomata respond, arise partly as a result of environment-inducedchanges in the ability of the cells of the stem and of the differentleaf tissues to filter out and remove ABA from, or to releaseABA into the apoplastic stream as it travels from the root tothe leaf, or as it traverses a single leaf (Slovik and Hartung, 1992a,b; Slovik et al., 1995; Wilkinson and Davies, 1997).

The amount of ABA that is removed by the symplast (i.e. thatenters the cells to become stored or metabolized) from the xylemand the leaf apoplast, before the transpiration stream reachesits target cells, depends in part on the pH of these compartments(Kaiser and Hartung, 1981; Hartung and Radin, 1989), which hasalso been shown to be sensitive to the environment in some species(see below). Apoplastic pH can thereby determine the concentrationof ABA that finally arrives at the guard cells or growing cells(Slovik and Hartung, 1992a, b; and see Wilkinson and Davies, 2002;Wilkinson, 2004). In general, a more acidic xylem/apoplasticpH (usually between 5.0 and 6.0) exists in the sap of unstressedplants and allows the greatest removal of ABA from the xylemand leaf apoplast, such that less reaches the guard cells. Morealkaline sap pH values have been detected when plants are exposedto ‘stress’ (see below), usually between 6.4 and7.2. Under these circumstances, the pH gradient over the cellmembrane that normally drives ABA removal from the apoplastis reduced, and ABA can accumulate to concentrations high enoughto affect stomatal guard cells by the time that the transpirationstream reaches their distant locality, even in the absence ofde novo synthesis. Thus pH changes generated by the root thatare propagated along the xylem vessels to penetrate to the leafapoplast (Jia and Davies, 2007), or that are generated withinthe leaf (see below), can function as chemical messengers thatalert the shoot of the need to conserve water, by adjustingthe amount of ABA that finally reaches the guard cells or thegrowing cells of the leaf.

Increases in root and/or shoot xylem sap pH have most commonlybeen shown to occur in response to soil drying (Gollan et al., 1992;Wilkinson and Davies, 1997; Bacon et 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 one of thefirst chemical changes measurable in xylem sap from plants exposedto drying soil (Bahrun et al., 2002; Sobeih et al., 2004), evenwhen moisture tensions are low enough that a supply of wateris still freely available. Sap pH also increases in plants withroots that are exposed to soil flooding (Jackson et al., 2003;Else et al., 2006), to changes in soil nutrient status (Kirkby and Armstrong, 1980;Dodd et al., 2003; Jia and Davies, 2007; and see Wilkinson et al., 2007),and in response to salt stress (Gao et al., 2004). These changescan occur before the shoot water status of the plant is affectedby perturbation at the roots (Schurr et al., 1992; Dodd et al., 2003),within 1–2 d of the onset of perturbation (Mingo et al., 2003),or even within a few hours (Jackson et al., 2003; Gao et al., 2004;Else et al., 2006), often prior to, or coincident with, theassociated environmentally induced change in plant physiology(gs or growth).

That xylem pH can alter shoot physiology via an ABA-mediatedmechanism has been experimentally demonstrated by Wilkinson and Davies (1997),Wilkinson et al. (1998), and Bacon et al. (1998). Artificialbuffers adjusted to relatively alkaline pH values, equivalentto those found in the xylem of plants experiencing soil drying,did not reduce transpiration or growth when supplied to thexylem stream of detached shoots or leaves of ABA-deficient mutantsor ABA-depleted wild-type plants, unless ABA was also suppliedvia the transpiration stream, even at a concentration whichwas not sufficient alone to affect physiology.

More recently, changes in shoot or leaf xylem/apoplastic sappH have also been detected in response to natural or imposedchanges in the aerial environment (Savchenko et al., 2000; Hedrich et al., 2001—CO2;Felle and Hanstein, 2002—light, CO2; Mühling and Lauchli, 2000;Stahlberg et al., 2001—light; Davies et al., 2002; Wilkinson and Davies, 2002—light,VPD, and/or temperature; Jia and Davies, 2007—VPD). Photonflux density (PFD—a measure of light intensity), VPD,and air temperature are positively correlated under most ambientconditions, and as stated above stomatal closure is often associatedwith high VPD. High PFD/VPD/temperature was associated withincreased xylem pH and reduced gs in Forsythiaxintermedia (cv.Lynwood) and Hydrangea macrophylla (cv. Bluewave) when intactplants were exposed to natural fluctuations in the summer microclimateover the course of several weeks (Davies et al., 2002; Wilkinson and Davies, 2002).In related work, Tardieu and Davies (1992, 1993) observed thatstomata and growing leaves became more responsive to the ABAconcentration in the xylem as the VPD increased around leavesof field-grown maize. Since it is known that ABA can be involvedin the stomatal closure response to low humidity (Xie et al., 2006;but see Assmann et al., 2000), it is tempting to suggest a rolefor ABA-based pH signalling in stomatal regulation when theaerial microclimate becomes potentially stressful. As well asmediating changes in foliar compartmentalization of incomingroot-sourced xylem-borne ABA, microclimate-induced changes inpH may also regulate ABA release from symplastic stores in stemsand/or leaves, and/or ABA removal from the leaf via the phloem,and recirculation via the root to the 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 pHcan function as ABA-mediated signals of perturbations in therhizospheric and/or the aerial environment that can be adaptivein the face of stress in the intact plant. Evidence is providedfor a novel pH-based link between the aerial environment andstomatal aperture. Foliar sprays of phosphate buffer iso-osmoticallyadjusted to a range of pH values are used to manipulate leafapoplastic pH and plant physiology artificially in intact unstressedForsythia and tomato plants, and in intact plants of the ABA-deficientflacca mutant of tomato (Imber and Tal, 1970).

Materials and Methods

Plant material
Seeds of tomato (Solanum lycopersicum L. cv. Ailsa Craig), ofboth the wild type and its ABA-deficient flacca mutant (Imber and Tal, 1970),were sown in Levington F2 compost (Fisons, Ipswich, UK). Seedlingswere transplanted to 1.0 l pots in a greenhouse with a variableday/night temperature, with supplemental lighting (providedby 600 W sodium Plantastar lamps, Osram, Germany) giving a photoperiodof 16 h. They were watered daily with tap water to the drippoint, and weekly with full-strength Hoagland nutrient solution.Flacca plants were sprayed twice weekly with 0.2 mmol m–3ABA (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 Forsythiaxintermedia (cv. Lynwood) plantswere potted in the spring into a medium comprising 100% sphagnumpeat with 6.0 g l–1 Osmocote Plus 12–14 month controlled-releasefertilizer and 1.5 g l–1 MgCO3. Plants were grown to ~100cm in height and experimentally manipulated in polythene tunnelsexposed to a naturally varying summer microclimate throughout,in 3.0 or 5.0 l pots supplied with ~300–500 cm3 water daily(depending on pot size and prevailing conditions) by hand orvia 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 overthe entire foliated region, with water or potassium phosphatebuffer (10 mol m–3 KH2PO4/K2HPO4). Buffers were adjustedto a range of pH values (5.0–6.7) by altering the ratioof the two salts, such that different treatments were iso-osmotic.In some experiments using the flacca tomato mutant, ABA (0.06–0.15mmol m–3) was added to the buffer spray. It is assumedthat the foliar spray penetrates to the interior of the leafvia ingress through stomatal pores (Kosegarten et al., 2001).In some cases, stems of Forsythia plants and flacca mutant tomatoplants were injected between 10.00 h and 11.00 h daily withwater or ABA (0.3 cm3 water or 0.06 mmol m–3 ABA for flacca;1–10 mmol m–3 ABA for Forsythia). The injectionpoint was immediately sealed with lanolin. It is assumed thatat least a portion of the injected solution penetrated to thexylem vessels of the stem, and was thereby transported to theleaves above this point. gs, PFD, and leaf temperature weremeasured daily at ~14.00–15.00 h with a porometer (AP-4,Delta-T Devices Ltd, UK) in 4–6 replicates of Forsythiaand tomato wild-type and flacca mutant plants that had beensprayed with buffers adjusted to a range of pH values, and injectedwith water or ABA where stated. The gs was measured in the mostrecently fully expanded leaf/leaflet, ensuring that the measurementleaf was above the ABA/water injection point (in the same branch)where appropriate. These positions were ~10–20 cm apart.Forsythia leaf lengths were measured daily with a ruler at ~16.00h to establish a rate of elongation in expanding leaves, again10–20 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 monitoredevery 2–3 d over the course of several weeks exposureto natural summer variations in the local microclimate (withrespect to PFD, VPD, and temperature) using the porometer. Measurementswere taken at the same time of day (~14.00 h) from the firstfully expanded leaf (usually the fourth from the apex), andthe ambient PFD incident on each leaf, and its surface temperaturewere also measured using the porometer. In addition, equivalentleaves were also harvested from 4–5 equivalent plantson six occasions throughout the experiment (every 4–5d), to determine bulk leaf ABA concentration (see below). Xylemsap was also collected from stems cut from these plants usinga Scholander pressure bomb (SKPM 1400; Skye Instruments Ltd,Powys, UK). Sap was expressed from the apical 10–20 cmof detached shoots at overpressures of –0.4 to –0.8MPa. 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 apoplasticsap from a portion of the shoot apex, as opposed to micro-samplingof the apoplast local to the guard cell, allows measurementof larger scale 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, finelyground, and extracted overnight at 5.0 °C with distilleddeionized water using an extraction ratio of 1:50 (g DW:cm3water). The ABA concentration of the extract was determinedusing a radioimmunoassay (RIA) following the protocol of Quarrie et al. (1988),using [G-3H](±)-ABA at a specific activity of 2.0 TBqmmol–1 (Amersham International, Bucks, UK), and the monoclonalantibody AFRC MAC 252 (generously provided by Dr SA Quarrie;Institute of Plant Science Research, Norwich, UK) which is specificfor (+)-ABA. Any ABA present in the tissue extract inhibitsthe binding of the tritiated ABA to the antibody. This concentrationis quantified using a series of standards of known non-radioactiveABA concentration in the assay such that sample counts can becalibrated from the resultant standard curve. However, the possibilityexists in any species that compounds with a similar structureto ABA but without ABA activity may contaminate the tissue extract.Every species must be tested for these contaminants using aspike dilution test. The standard curve is tested in the assayin the presence and absence of an increasing dilution of tissueextract. If the resulting standard curves at each dilution remainparallel, then the only compound present in the tissue extractthat affects the relationship between the binding of the addednon-radioactive ABA and the tritiated ABA to the antibody isendogenous ABA itself. Spike tests on leaf tissue collectedfrom well-watered Forsythia plants were carried out, and nocontaminants were found (not shown).

Transpiration bioassays
The effects of exogenous ABA concentration were tested on therate of transpirational water loss through stomata (a measureof stomatal openness) from detached leaves of greenhouse-cultivatedForsythia (growth conditions as for tomato). Leaves (third toseventh below growing apices) were detached from plants whichhad been kept for 1.0 h in the dark, and petioles were re-cutunder water before immediate transfer to treatment solutions,in order to prevent embolism (blockage of the xylem vesselswith air). Treatment solutions were 5.0 cm3 water±ABAat the appropriate concentration, in 6.0 cm3 plastic vials coveredin aluminium foil to prevent evaporation from the solution surface.Leaves were introduced through slits in the foil so that petioleswere submerged. The vials containing the leaves were placedunder lights (PFD 400 µmol m–2 s–1) at 24°C before 11.00 h. They were weighed approximately every30 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 expressedon a per unit leaf area per second basis. Means and standarderrors from five replicates were determined.


Effects of foliar sprays adjusted to a range of pH values on gs and growth in intact Forsythia and tomato plants
Foliar sprays adjusted to relatively alkaline ‘stressful’pH values (6.4–6.7) reduced the gs of intact well-wateredForsythia and wild-type tomato plants, and reduced leaf elongationrates (LERs) in Forsythia plants, in comparison with controlssprayed with water (Fig. 1A–C). These pH values are equivalentto those detected in sap expressed from well-watered Forsythiaplants experiencing a high PFD (Davies et al., 2002; and seebelow), and from tomato plants exposed to drying soil (Wilkinson et al., 1998).Foliar sprays adjusted to more acidic pH values, equivalentto those detected in sap from plants experiencing lower PFDs(Forsythia) or well-watered soil (tomato), did not reduce gsor LERs. The effect on gs was induced after 2.0 d (one 09.00h foliar spray application per day), and the effect on LER wasinduced within 5.0 d of the start of treatment.

Effects of foliar sprays adjusted to a range of pH values on the relationship between PFD and gs in intact Forsythia plants
There was a negative correlation between current PFD and morninggs sampled between 09.30 h and 10.30 h approximately every 1–2d in intact Forsythia plants that were exposed to natural summervariations in the aerial microclimate over the course of 8 d(Fig. 2A). It is assumed that the correlation between gs andVPD, and between gs and leaf temperature would be the same,given that these climatic variables are positively correlatedunder most ambient conditions. The value of the r2 coefficientof the second order regression between PFD and gs (Sigmaplot2001) was reduced as the pH of the foliar spray applied to theplants increased from 5.0 to 6.7 (Fig. 2A–D). Controls(Fig. 2A) were sprayed daily with water. Manipulating an increasein the foliar apoplastic pH effectively removed the correlationbetween PFD (and, by inference, VPD and leaf temperature) andgs.

Effects of foliar sprays adjusted to a range of pH values on gs and its response to ABA in intact plants of 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), did notreduce gs in leaves of flacca plants compared with water-sprayedcontrols (Fig. 3). However, the wild-type response to alkalinepH was restored in the flacca plants when ABA was simultaneouslysprayed onto the leaves or injected into the stems (Fig. 3A, B)at a concentration approximating 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 be detected2 d after the start of treatment (one 09.00 h application perday). More acidic foliar sprays did not reduce gs in eitherthe presence or absence of ABA. The different rates of controlgs in the two experiments (Fig. 3A, B) were a result of differingambient conditions in the greenhouse (temperatures differedover a range of 5 °C and PFDs over a range of 150 µmolm–2 s–1), and of the differing stages of the measurementleaf (immature—Fig. 3A; mature—Fig. 3B). In someexperiments foliar sprays adjusted to pH 6.0 increased gs incomparison with water-sprayed controls, especially in the absenceof ABA (not shown), presumably as the endogenous apoplasticpH of the control plants was >6.0.

Forsythia plants were able to generate and respond to ABA
When the soil around the roots of intact Forsythia plants wasallowed to dry, increases in bulk leaf ABA could be detectedwithin 3 d (not shown). When leaves were harvested and shootxylem/apoplastic sap was expressed approximately every 4–5d from intact Forsythia plants exposed to a naturally varyingsummer aerial microclimate (with respect to PFD, VPD, and temperature),sap pH and bulk leaf ABA concentration varied but were positivelycorrelated (Fig. 4). Stomatal closure was associated with morealkaline xylem pH at relatively high PFD/VPD/temperatures (Davies et al., 2002;Wilkinson and Davies, 2002), and with higher bulk leaf ABA concentrations(not shown). Stomata of detached Forsythia leaves were competentto close in response to ABA in a concentration-dependent mannerwhen this was supplied via the xylem at the cut petiole, andsubsequent rates of transpiration were measured (Fig. 5).

Effects of foliar sprays adjusted to a range of pH values on the response of gs and leaf growth to ABA injected into the stems of intact Forsythia plants
ABA injected into the stems of intact Forsythia plants reducedgs and LER in a concentration-dependent manner 4–6 h laterwhen leaves were sprayed with buffers adjusted to pH 5.0 andpH 5.8, but not when these were sprayed with a more alkalinebuffer (pH 6.7; Figs 6, 7). gs and leaf growth were most sensitiveto ABA at pH 5.8. At pH 6.7, gs and leaf growth rate were alreadyreduced even in water-injected controls, such that ABA couldnot induce any further reductions in these variables.



A technique is reported here that allows manipulation of foliarapoplastic pH in intact plants, such that it has been possibleto assess the effects of this variable on shoot physiology invivo. Intact plants were sprayed with iso-osmotic phosphatebuffers adjusted to a range of pH values. Kosegarten et al. (2001)demonstrated that a similar technique (spraying sunflower leaveson intact plants with diluted citric and sulphuric acids) indeedresulted in changes in apoplastic pH, as measured with a fluorescentdye loaded into the apoplast of the leaves.

For the first time it has been demonstrated that applicationsof buffers adjusted to a ‘stressful’ pH of between6.4 and 7.0 can close stomata and reduce leaf growth in theintact plant (Fig. 1). These changes could be induced in theabsence of the environmental perturbation that normally generatesan equivalent endogenous pH change, in this case soil drying(tomato—Wilkinson et al., 1998) or a stressful aerialleaf microclimate (ForsythiaDavies et al., 2002). Implementationof this technique in a horticultural/agricultural context couldbe of great benefit with regard to water conservation (see below).That ABA was either necessary for the induction of stomatalclosure by relatively alkaline buffers (in ABA-deficient flaccamutants of tomato—Fig. 3) or that responses of gs andleaf growth to injected ABA were modulated by the pH application(Figs 6, 7Forsythia) indicated that the effect of thefoliar buffer was to adjust the compartmentation of ABA withinthe leaf, thereby altering the concentration that finally penetratedto the apoplastic micro-compartment around the stomatal guardcells or the growing cells of the leaf (see Introduction; Wilkinson and Davies, 2002;Wilkinson, 2004). When the foliar spray was more alkaline, moreof the ABA in the transpiration stream (whether this was sourcedfrom the root or the shoot, see below) was presumably allowedto by-pass the cells of the stem and of the leaf without beingsequestered, such that more finally penetrated to the guardcells and the growing 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 loading into the xylem lumen from existingstores in stem and/or petiole parenchyma can occur when thepH of sap perfused through the lumen is more alkaline (Sauter and Hartung, 2002;Else et al., 2006; and see Fig. 4). ABA concentrations in sapcollected from leaf petioles were higher than those in sap collectedfrom stem bases of flooded tomato plants exhibiting increasesin sap alkalinity (Else et al., 2006). Another pH-based effecton shoot ABA redistribution has been demonstrated by Jia and Zhang (1997).Movement of ABA out of maize leaves via the phloem was reducedwhen the leaf apoplast was adjusted to a relatively high pH,such that ABA accumulated in the leaf. Thus there is also acontribution to the alkalinity-induced enrichment of xylem andleaf apoplastic 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 aerial microclimate (high PFD,VPD, temperature) correlates with xylem alkalization (Forsythia,Hydrangea) and with stomatal closure (Forsythia). It was suggestedthat a 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, thisis the first time that a causal link between aerial perturbation,apoplastic pH, and stomatal response has been demonstrated.That applying relatively alkaline buffers to the leaves removedthe correlation between decreasing stress and stomatal opening(Fig. 2) indicates that an acidic milieu contributes to stomatalopening as the environment becomes less stressful. When thisis considered alongside the findings that (i) the aerial environmentcan change the pH and the stomatal response in parallel (Davies et al., 2002;Wilkinson and Davies, 2002) and (ii) xylem pH and leaf ABA concentrationare positively correlated (Fig. 4), it is evident that the environmentcan affect stomata through a change in pH and thereby via achange in the amount of root- and/or leaf-sourced ABA that isable to penetrate to the guard cells. These pH changes do notact directly on stomata, and require ABA to do so in intactleaves (Fig. 3). Opposing, and presumably direct effects ofpH have been observed on stomatal behaviour when stomatal apertureswere measured after incubation of epidermal peels on solutionsbuffered to a range of pH values (Wilkinson and Davies, 1997;Wilkinson, 1999). The more potent effect of a propagated pHchange in the intact leaf is on the distribution of ABA betweenleaf compartments, and this must over-ride any direct effectsof pH on guard cell biochemistry.

It is important to distinguish here between (at least) threedifferent groups of environmentally induced pH change that seemto occur. First, rapid (inducible within minutes), often transientchanges in pH are apparently localized to the immediate guardcell apoplast. These may be induced by environment-led changesin stomatal aperture and the accompanying ionic fluxes in andout of the guard cells themselves (Bowling and Edwards, 1984;Edwards et al., 1988; Hedrich et al., 2001; Felle and Hanstein, 2002).Stomatal closure, induced by various imposed environmental changes,correlated with short-term localized alkalization. Secondly,light/dark transitions and changes in atmospheric CO2 concentrationcan induce rapid short-term oscillations in apoplastic pH whichseem to result from changes in photosynthetic leaf cell H+-ATPaseactivity and/or changes in photosynthetically derived malateconcentration, which can propagate a short distance within theleaf apoplast (Mühling and Lauchli, 2000; Hedrich et al., 2001;Stahlberg et al., 2001; Felle and Hanstein, 2002). Thirdly,and more relevant here, are those which arise more slowly afterperturbation (~4–48 h, Sobeih et al., 2004; Else et al., 2006)at a point distant 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 distances, often betweenorgans (Hoffmann and Kosegarten, 1995; Else et al., 2006; Jia and Davies, 2007).The latter are more likely to be those involved in long-distanceABA-based signalling, whilst the rapid transient pH changeslocalized to the apoplast around guard cells and photosyntheticcells are likely to be non-message-carrying by-products of aprior stomatal or photosynthetic cell response, and/or to beinvolved in localized changes in ion transport activity requiredto generate the biochemical driving force for nutrient uptakeor stomatal movement in response to light/CO2 (Zeiger and Zhu, 1998;Assmann and Shimazaki, 1999).

There has been previous speculation about the mechanism behindthe more widespread xylem sap pH change that can occur in responseto variations in the leaf microclimate, with respect to individualeffects of PFD, VPD, and/or temperature (Wilkinson and Davies, 2002;Wilkinson, 2004). Mechanisms whereby soil drying, flooding,and variations in nutrient availability can lead to an increasein xylem sap pH have been discussed elsewhere (Wilkinson, 1999,2004; Wilkinson and Davies, 2002; Else et al., 2006; Wilkinson et al., 2007).It was suggested that VPD may underlie the aerial environment-ledalterations in xylem sap pH (Wilkinson and Davies, 2002), inorder to explain how stomata can become more responsive to agiven dose of ABA in the xylem stream as VPD increases (Tardieu and Davies, 1992,1993). However, Jia and Davies (2007) increased VPD at a fixedtemperature and PFD around C. communis leaves and found that,alone, this actually acidified the apoplast, which would tendto increase ABA retention by the symplast. The authors suggestedthat fewer protons were removed from the transpiration streamwhen it by-passed the symplast more rapidly at high VPD. Thusit would seem that increasing VPD acts more directly on guardcell biochemistry (Assmann et al., 2000; Bunce, 2006) to increasestomatal and/or growth sensitivity to ABA (Grantz, 1990; Tardieu and Davies, 1992,1993; Bunce, 1996; Xie et al., 2006), and that PFD and/or airtemperature (see below) remain the most likely effectors ofthe pH changes described above. Nevertheless, it must be notedthat evidence still exists to show that high VPD can limit stomatalaperture by increasing the apoplastic ABA concentration in thevicinity of the guard cells (Zhang and Outlaw, 2001b).

Is PFD the driver for the changes in pH measured in apoplasticsap expressed from plants experiencing a range of microclimaticconditions (Davies et al., 2002; Wilkinson and Davies, 2002)?Bunce (2006) found that at a fixed temperature and fixed highVPD, increasing the PFD from 300 to 1500 µmol m–2s–1 re-opened stomata in four different species. Kaiser and Hartung (1981)provided evidence that saturating light can induce chloroplasticalkalization, causing ABA to be retained inside these organellesand reducing stomatal sensitivity to externally supplied ABA.Heckenberger et al. (1996) found that gs was less sensitiveto ABA fed to sunflower leaves at a PFD of 450 µmol m–2s–1 as opposed to 200 µmol m–2 s–1 whenthe temperature was fixed at 21 °C. It would seem, therefore,that pH, leaf ABA concentration, and stomatal aperture are mostlikely to be responding to the high temperature that occurswhen Forsythia plants are exposed to a stressful aerial microclimate,rather than to the associated high PFD or VPD.

High temperature may increase CO2 removal from the apoplastfor photosynthesis, which will tend to alkalize apoplastic sapby virtue of its low buffering capacity (Savchenko et al., 2000).High temperature will also increase nitrate transport into theleaf (Aslam et al., 2001; Castle et al., 2006), via effectseither on the transpiration rate or on the activation stateof uptake and transport proteins. Increases in xylem nitrateconcentration have been shown to increase apoplastic pH in severalspecies (see Wilkinson et al., 2007). Co-transport of nitrateinto the symplast with two protons depletes the apoplast ofpositive charge (Ullrich, 1992). The reduction of nitrate toammonium in the cells of the leaf (which increases with substrateavailability), and the subsequent synthesis of ammonium to organicmaterial generates OH anions (Raven and Smith, 1976).Both effects will tend to increase xylem sap pH. Changes inxylem sap nitrate concentration above the deficient range haverecently been shown to be powerful modulators of stomatal apertureand leaf growth (Jia and Davies, 2007; Wilkinson et al., 2007).Relatively high xylem nitrate concentrations, within the physiologicalrange, closed stomata and/or reduced leaf growth synergisticallywith ABA and/or alkalinity, in maize, tomato, and Commelina.The effect could be removed by supplying the nitrate to thexylem of detached leaves in a more acidic buffer. Nitrate mayonly be 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 photosynthesisinduced by high temperature (to an optimum) may influence leafapoplastic pH by inducing an increase in sugar uptake into thephloem, which can occur via proton co-transport across the plasmamembrane of the sieve tube cells, again depleting the apoplastof positive charge (Wilkinson, 1999). Finally it has been shownthat an increase in the apoplastic malate concentration coincideswith alkalization and stomatal closure (Patonnier et al., 1999Fraxinusexcelsior; Hedrich et al., 2001Vicia faba and Solanumtuberosum). Increased photosynthesis at high temperature maylead to greater malate synthesis in leaf cells, and its subsequentrelease to the apoplast. Further research is required to establishwhether temperature can alter apoplastic pH via any of the mechanismsproposed above. It must also be noted here that temperaturecan have other more direct effects on stomatal guard cells (Ilan et al., 1995)which may be overridden by the accumulation of ABA. Stomatalsensitivity to ABA also increases with temperature up to 25°C (Wilkinson et al., 2001), and this effect may act inconjunction with the increase in apoplastic alkalinity.

Whatever the cause of the pH change, it appears to be importantin modulating physiological responses to the aerial environmentin intact plants (Fig. 2) via an ABA-dependent mechanism (Figs 3, 4, 6, 7GoGoGo).It is proposed that environmental factors that affect aerialparts of the plant (e.g. PFD, temperature, VPD, diurnal/seasonalchange, and fungal infection) can interact with factors thataffect underground parts (drought, flooding, salt stress, nutrientavailability, and nodulation) and/or the whole plant (water/nutrientavailability and temperature) by impinging on the ABA concentrationfinally perceived by the guard cells and the growing cells ofthe leaf. The pH of distinct regions of the root, stem, andshoot, each perhaps responding locally to differing levels andtypes of stimuli/perturbation, will govern the amount of ABAthat becomes ‘locked away’ in these localities,or that is free to travel on to the responsive cells at theculmination of the transpiration stream in the leaf apoplast.Perturbations that increase the pH of a particular region willamplify the ABA signal as the transpiration stream traversesit. This effect is depicted in Fig. 8.

It is important to note here that the present data provide newevidence for the concept that the shoot may generate and respondto ABA-based signals independently of those sourced from theroot (Wilkinson and Davies, 2002). However, given the dynamicnature of both the rhizospheric and the aerial environment,ABA-based signals from both sources, and interactions betweenthem, are likely to be important. Whilst recently describeddata have been interpreted to rule out a function for root-sourcedABA in communicating water deficit from the root to the shoot(Christmann et al., 2007), recirculation of ABA originally synthesizedin the shoot back into the transpiration stream via the rootwas not accounted for by the authors. This will represent asubstantial proportion of the root-sourced signal (Wolf et al., 1990;Neales and Mcleod, 1991). Stores of ABA within roots can alsobe mobilized when soil begins to dry (Slovik et al., 1995).Thus de novo biosynthesis of ABA in roots is not necessarilya requirement for long-distance root-sourced ABA signallingunder all circumstances, although a wealth of evidence existsin the literature to show that ABA biosynthesis in roots andtransport of this to the shoot does occur in drying soil (seereferences in Davies and Zhang, 1991; Wilkinson, 1999; Davies et al., 2002;Wilkinson and Davies, 2002). Furthermore, use of the root pressurevessel to restore shoot water relations has demonstrated thepotential for root-sourced chemical signals to act independentlyof hydraulic signals (Gollan et al., 1986). It is proposed thatinteractions between ABA and pH will allow the shoot to modifyits response to an ABA-based root signal as a function of localclimatic conditions, in the face of the potential from bothenvironments for dehydration. This may be especially importantin tall woody species where leaves are much further away fromthe root. Preliminary results from our laboratory show thatstomata of several woody species are competent to respond toan increase in apoplastic pH induced by a foliar spray to theintact plant, even when soil drying does not alkalize xylemsap from the same species (Sharp RG and WJ Davies, unpublishedresults). This research could potentially benefit the horticultural and/oragricultural industry. Buffered foliar sprays could be usedto reduce plant water loss during cultivation, and/or to induceplants to grow more slowly or to specified proportions, withoutemploying more traditional deficit irrigation techniques. Thisis particularly pertinent with regard to current global warming.



The authors would like to thank Dr DLR De Silva and Dr J Theobald for conducting the RIA experiments, and Maureen Harrison, Anne Keates, and Philip Nott for technical support. The work was financially supported by DEFRA Horticulture Link project HLO132LHN/HDC HNS 97.



ABA, abscisic acid; cv, cultivar; gs, stomatal conductance; LER, leaf elongation rate; PFD, photon flux density; RIA, radioimmunoassay; VPD, vapour pressure deficit.


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Figure 1   The effect of pH on mean gs (n=6 ±SE; A, C) and leaf elongation rate (LER; n=5 ± SE; B) when intact pot-grown Forsythia (A, B) or tomato (C) plants were sprayed once daily over the foliated region with water (controls) or phosphate buffers (10 mol m–3 KH2PO4/K2HPO4) iso-osmotically adjusted to a range of pH values.

(Click image to enlarge)


Figure 2  Ambient PFD incident at ~10.00 h on the first fully expanded leaf of intact pot-grown Forsythia plants in polythene tunnels, plotted against the 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 represent measurements 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.

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Figure 3  The effect of pH±ABA on mean immature (A, n=6 ±SE) or mature (B, n=6 ±SE) leaf gs in intact flacca tomato mutant plants. The foliated region was sprayed with water (controls) or phosphate buffers (10 mol m–3 KH2PO4/K2HPO4) iso-osmotically adjusted to pH 6.0 or pH 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/buffer spray. (B) The effect of ABA on the response to pH when plants were injected with water or 0.15 mmol m–3 ABA (0.30 cm3) ~5–10 cm below the measurement leaf. Spraying and injecting took place 4–6 h prior to measurement of gs.

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Figure 4  The pH of xylem/apoplastic sap expressed from the apical 10–20 cm of shoots severed from pot-grown Forsythia plants, plotted against the bulk leaf ABA concentration of leaves detached from the same plant at the same time (~15.00–16.00 h). Measurements were taken every 4–5 d over the course of several weeks from 4–5 plants experiencing variable mid-summer ambient conditions in polythene tunnels with respect to PFD/VPD/temperature (PFD ranging from ~130 µmol m–2 s–1 to 1100 µmol m–2 s–1). A second-order regression is shown, with a 95% confidence interval (dotted line) and an r2 coefficient, calculated using Sigmaplot 2001.

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

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Figure 6  The effect of pH on the response of gs to water or ABA in intact 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 were injected once daily (10.00 h) with water or ABA (0.30 cm3) ~6–10 cm below the measurement leaf. gs was measured in the most recently mature leaf in each of six plants, and means with standard errors are shown.

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Figure 7   The effect of pH on the mean response of leaf elongation rate (LER; n=6 ±SE) to water or ABA in intact Forsythia plants, when the foliated region was sprayed 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 were injected once daily (10.00 h) with water or ABA (0.30 cm3) ~6–10 cm below the measurement leaf.

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

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