Use of the pressure probe in studies of stomatal function

DOI: 10.1093/jxb/erg162

REVIEW ARTICLE: FIELD TECHNIQUES

Use of the pressure probe in studies of stomatal function

Peter J. Franks1

School of Tropical Biology, James Cook University, PO Box 6811, Cairns, QLD 4870, Australia

Received 5 November 2002; Accepted 4 March 2003

Abstract

Over the past few decades the pressure probe has

been used extensively in studies of the hydro-

mechanical and osmotic properties of plant cells.

However, although pressure probe techniques have

been employed successfully in the study of stomatal

function, there is no detailed account of this special

application of the pressure probe technique. This

paper describes the construction and use of the

pressure probe in studies relating to stomatal func-

tion, and reviews the current state of knowledge of

stomatal function in relation to guard cell and leaf

hydromechanical properties.

Key words: Guard cell turgor pressure, pressure probe,

stomata, stomatal mechanics, transpiration.

Introduction

Stomatal guard cells, with their ability to sense andintegrate many internal and external environmental signalsdirectly, are amongst the most physiologically complexcells in higher plants (Cowan, 1977; Raschke, 1979;Farquhar et al., 1980; Willmer and Fricker, 1996;MacRobbie, 1998; Assmann and Shimazaki, 1999;Schroeder et al., 2001; Zeiger et al., 2002). Althoughthere have been signi®cant advances in understandingstomatal function over the past few decades, there is stillmuch that is not understood. Stomata are self-poweredturgor-operated valves and, although operating as a co-ordinated population within the leaf epidermis, eachprecisely controlled stomatal pore is the result of acontinuously shifting balance between forces generatedwithin the guard cells and those in neighbouring epidermalcells. Until only recently, data on the basic relationshipbetween guard cell pressure and stomatal aperture wereunavailable, largely because of technical dif®culties. Thisconsiderably limited the development of mechanistic

models of stomatal function. However, development ofthe pressure probe has enabled exploration of thebiophysical principles of stomatal function at the cellularlevel. The purpose of this article is to describe in detailhow the pressure probe is used to study stomata, and todiscuss some of the main ®ndings.

The cell pressure probe was ®rst used in the study ofhydromechanical properties of plant cells by HuÈsken et al.(1978). It has since been used in the study of water andsolute relations of a variety of plant tissues, including leafepidermis (Zimmermann et al., 1980; Tomos et al., 1981;Tyerman and Steudle, 1982; Shackel, 1987), mesophyll(Steudle et al., 1980; Nonami and Schulze, 1989), xylem(Balling, 1990; Wei et al., 1999) and roots (Steudle andFrensch, 1989; Azaizeh, 1991; Zimmermann et al., 1992).There are several excellent technical and review papers onthe general use of the modern cell pressure probe. Steudle(1993) provides a detailed description of theory andapplication of the cell pressure probe to studies of waterand solute relations in various plant tissues. Boyer (1995)also presents a detailed chapter on cell pressure probedesign and application. More recently, Tomos and Leigh(1999) provided a comprehensive review of the pressureprobe literature. However, use of the pressure probe instudies of stomatal function represents a special case thatto date has not been well described.

Although some of the most useful data on thebiomechanics of stomatal movement have come fromdirect measurement and manipulation of guard cellpressure, application of the cell pressure probe tostudies of stomatal function is not limited to the probingof guard cells alone. Although guard cells can senseand respond independently to several environmentalstimuli, their functioning in the intact leaf is intricatelylinked via hydraulic, mechanical and chemical interactionsto the whole plant. Therefore, use of the pressure probein studies of stomtal function can involve measurementsnot only on stomata, but on tissues located throughoutthe plant.

1E±mail: [email protected]

Journal of Experimental Botany, Vol. 54, No. 387, ã Society for Experimental Biology 2003; all rights reserved

Journal of Experimental Botany, Vol. 54, No. 387, pp. 1495±1504, June 2003

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Pressure probe design and application

As with most physiological measurement techniquesinvolving precision instruments, experiments using thepressure probe are best carried out in the air-conditionedand dust-free environment of a standard laboratory.However, this need not limit its use to specialized researchlaboratories. Most ®eld stations are equipped with suchfacilities, and thus there is no real impediment to using thepressure probe in remote locations. Furthermore, thepressure probe itself (excluding microscope and dataacquisition hardware) meets several additional criteria thatmake it worthy of consideration for use in ®eld labora-tories: it is light, compact, robust, and cheap.

There is no commercially available pressure probesuitable for general stomatal studies. However, the instru-ment is simple to construct if one has access to machiningfacilities. The pressure probe operates on the principle ofregulated volume displacement. Once the probe is insertedinto a cell, a closed, elastic-walled, ¯uid-®lled system isformed, where the oil within the pressure probe interfaceswith the cellular contents. The pressure is regulated bysliding a steel piston into or out of the oil-®lled reservoir ofthe pressure probe.

Main components

A schematic diagram of a pressure probe suitable forstudies of stomatal function is shown in Fig. 1. A list ofequipment suppliers is provided in the appendix. Inprinciple, this instrument is similar to pressure probesdescribed elsewhere (Steudle, 1993), except for two mainfeatures. First, the piston and transducer housing areseparated from the glass capillary holder by a length of¯exible HPLC (PEEK) tubing. This adaptation, ®rstdescribed by Murphy and Smith (1994), greatly facilitateshigh precision orientation of the glass capillary, which isimportant because much of the success in taking measure-ments directly on guard cells depends on where and howthe capillary is inserted into the guard cell. Second, thestomatal pressure probe needs to be capable of operation atrelatively high pressures. Guard cells can require up to5 MPa to achieve full opening (Franks et al., 1998). Thetwo seals most likely to leak are the o-rings around thesliding steel piston and around the glass capillary.However, provided these ®ttings are machined to ®netolerances and shaped appropriately, a high-pressure sealcan be maintained.

The transducer and piston housing (Fig. 1a, A±F) formsthe bulk of the instrument and is machined from a block ofPlexiglass. The simplest design is to bore a `T' and then ateach opening tap threads to hold the transducer, pistonguide and PEEK tube connector. The micrometer screwmoves the piston in and out to regulate pressure. It may beoperated by an electric motor (HuÈsken et al., 1978) or, if

®tted with a wheel of about 7 cm diameter, can be operatedeasily by hand.

A suitable pressure transducer is the piezoresistive straingauge type (e.g. XTL-190-1000-A, rated at 1000 psi,Kulite Semiconductor Products, Inc., Leonia, USA). Apartfrom being small and rugged, these sensors offer highsignal output, excellent repeatability and in®nite resolution(limited only by that of the recording instrument). They arealso temperature compensated and tolerant of considerableoverpressure. The recommended silicone oil (Wacker AS4, Wacker-Chemi, Berlin) is a phenylmethyl polysiloxanewith low kinematic viscosity (4 mm±2 s±1) and lowcompressibility.

To observe the probe tip during insertion into cells andto monitor the oil±sap meniscus, a high-powered micro-scope is necessary. If working with epidermal peels aninverted microscope allows unhindered probe access.When working with intact plant material a standard

Fig. 1. (a) Schematic diagram of pressure probe apparatus used forstomatal studies and suitable for most cell pressure probe applications:A, piston and transducer housing, incorporating pressure transducer(B), piston guide and o-ring seal (C), piston shaft (D), micrometerscrew (E); F, HPLC ®tting; G, HPLC tubing; H, glass capillary holder;I, borosilicate glass capillary pulled to a ®ne pointed tip; J, specimen(epidermal peel in well slide or intact leaf); K, hydraulicmicromanipulator; L, hydraulic line; M, microscope stage; N, longworking distance microscope objective lens necessary for intactleaves; O, microscope objective lens on inverted microscope (suitablefor epidermal peels); P, image monitoring and capture; Q, transduceroutput; R, transducer power supply (high precision ®xed voltage).(b) Detail of the micro-capillary holder in Fig. 1(a), showing theo-ring (J) and glued section (K) which prevents the capillary fromcreeping out under high pressure.

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compound microscope with epi-illumination is required. Inthis case, a long working distance objective is required toenable probing of cells while viewing (403 magni®cationwith a working distance of 7 mm is suf®cient). Amicroscope with a ®xed stage is best.

Due to the highly delicate nature of plant cells,accidental mechanical shocks and vibration due to nearbymachinery can be a problem. These disturbances are notalways present, but at some locations can be severe enoughto impede work with the pressure probe or any other type ofmicroprobe. In many cases the vibrations and mechanicalshocks can be dampened suf®ciently with a `steady bench'.Depending on the severity of the vibration problem, thesebenches can vary in type from a simple slab of rock (about6036035 cm), to a slab of rock or other high-densitymaterial mounted on air cushions that can be tuned foroptimal dampening.

There are several different types of micromanipulatorsuitable for pressure probe work, and the choice can be amatter of personal preference. However, highly recom-mended is the hydraulic type in which the controls areseparated from the moving probe holder by a length of ®nehydraulic tubing. This ensures that random hand move-ments do not hinder probe manipulations.

When working with stomata it is often desirable tocapture images for later analysis of stomatal apertures. Forthis purpose a standard CCD camera and image capturesoftware can be used. Similarly, although in manyinstances a chart recorder or voltmeter may be suf®cientfor transducer output recordings, time-series measure-ments and analyses involving pressure and volumemanipulations will require a data acquisition card andcomputer software.

Glass microcapillaries

Borosilicate glass capillaries, typically 1.2 mm OD,0.69 mm ID, are pulled into a ®ne point with a pipettepuller. A programmable pipette puller (e.g. Model P-87,Flaming/Brown Micropipette Puller, Sutter InstrumentCo., Novato, USA) allows tips with almost identicalshape to be pulled repeatedly with ease. A suitable workingtip diameter is typically around 1±2 mm. Often the probetips are narrower than this when initially pulled and can beeasily enlarged by scraping gently against the rough edgeof a glass slide (using a micromanipulator and micro-scope). A probe tip that is either blocked or too narrow toallow the rapid movement of ¯uid in and out could lead toerroneously high pressures being recorded. However, sucha constriction will be immediately apparent to the user.Before any measurements are made, the user shouldroutinely check for unrestricted ¯ow through the probe tipby ensuring that quick adjustments of pressure areaccompanied by equally rapid movement and re-equilibra-tion of the oil/sap meniscus.

Filling the probe system

After construction, the entire pressure probe reservoir mustbe ®lled with clean silicone oil, being careful to excludebubbles. With the piston removed, the chambers in theplexiglass block and the PEEK tubing are ®lled. The pistonis then inserted and tightened into place. When not holdinga class capillary, the capillary holder attached to the end ofthe PEEK tubing should be capped to keep the contentsclean and to prevent oil from evaporating or leaking. Aftera glass microcapillary is pulled (several may be pulled andkept in a container for later use), it is loaded with silicon oilusing a ®ne syringe that can slide inside the capillary,®lling it from the bottom up. When full, the capillary isscrewed into the capillary holder, ensuring that no airbubbles are trapped when the oil in the capillary and holderare mated together. When tightening the capillary intoplace, it is important to monitor pressure and wind out thepiston to avoid overpressure.

Calibration

Although transducers often come with a calibrationcerti®cate, a calibration check (transducer output voltageversus pressure) should be made. For this purpose aregulated high pressure air line and test gauge can be ®ttedvia an adaptor to the capillary holder. The user shouldcheck for linearity and absence of hysteresis, and ensurethat the performance conforms to the manufacturer'sspeci®cations.

Making measurements

Although ideally suited to obtaining data on the relation-ship between guard cell turgor and stomatal aperture(Franks et al., 1995, 1998), the stomatal pressure probedescribed here is suitable for a variety of water relationsstudies on other types of cells. As mentioned earlier,stomatal function involves the co-ordination of manydifferent types of cells, so measurements which aim toexplore stomatal function are not limited to stomatal guardcells. What is limiting, however, is the size of the cellssuitable for pressure probe measurements. Due to thedependence of several factors, including the wall proper-ties of the cell, the optical and mechanical precision of thesystem being used, and the skill of the operator, there is noexact minimum size, but many researchers would agreethat cells smaller than about 20 mm are extremely dif®cultto work with. Many plants have cells (including stomatalguard cells) that are this size or smaller, so this must beconsidered when planning experiments.

The basic procedure for measuring cell turgor is shownin Fig. 2. Upon insertion of the probe into a cell, the cellsap is forced back a short distance into the glass capillary.The pressure must then be increased via movement of thesteel piston so that the sap±oil meniscus is brought back tothe surface of the cell. The pressure measured in the system

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at this point is equal to the turgor pressure of the cell priorto probe insertion. For stomata, the technique of Frankset al. (1995) involves the manipulation of pressure in guardcells to obtain aperture versus pressure relationships. Inthis method, the probe is inserted into closed (de¯ated)guard cell pairs at their common ends, and the guard cellsare pressurized (in¯ated) by extruding oil into them. In thiscase, the oil±sap meniscus is in the guard cells and itsposition is either advancing away from the probe tip, ®xed,or retreating back toward the probe tip, depending onwhether the operator is increasing pressure, holding itconstant, or decreasing pressure. Detailed instructions onthe measurement of cell wall elasticity, cell membranehydraulic conductivity, osmotic pressure, and re¯ectioncoef®cients are given in Steudle (1993), Boyer (1995) andFranks et al. (2001).

Discussion

Guard cell wall properties

Stomatal pores form because guard cells behave in aunique manner when internally pressurized. When theturgor pressure within a mature plant cell increases, thewalls will, in most cases, stretch almost equally in alldirections (isotropic expansion). With guard cells, how-ever, stretching is not directionally uniform. Instead,pressurization results in greater stretching in the longitu-dinal direction than in the tangential. This unequal(anisotropic) cell wall stretching, which is dominated bythe longitudinal expansion of the dorsal wall, is what bendsthe guard cells away from each other and creates the

stomatal pore. All guard cells, regardless of shape, operateon the basis of this pattern of anisotropic wall expansion. Itis thought that a combination of differential wall thicken-ings (thinner dorsal walls, thicker ventral walls), wallchemical composition and radial arrangement of micro-®brils in the walls contributes to the unique mode ofexpansion of guard cells (Haberlandt, 1884; Aylor et al.,1973; Majewska-Sawka et al., 2002). However, thestructure of guard cell walls is not well understood, andlittle is known about the physical changes that take placewithin the walls during stomatal opening and closing. Thepressure probe is becoming an important tool for exploringthese properties.

Stomatal aperture versus pressure relationship

Although it has long been known that stomata open andclose by increasing and decreasing their turgor pressure(von MoÈhl, 1856; Heath, 1938; Meidner, 1982), there areonly a limited number of studies in which the relationshipbetween guard cell pressure and stomatal aperture hasbeen measured. Using the apparatus and techniquesdescribed here, data have been obtained for several species(Franks et al., 1995, 1998). Typical characteristics forTradescantia virginiana are shown in Fig. 3. While thesedata are far from comprehensive, the emerging pattern isthat under the in¯uence of the turgor pressure fromneighbouring epidermal cells, the relationship betweenstomatal pore width (a) and guard cell turgor pressure (Pg)is sigmoidal. As epidermal turgor pressure (Pe) reduces tozero, this relationship tends toward a simple saturatingcurve. For an isolated guard cell, the change in volume Vwith a small change in Pg (dV/dPg) always decreases withincreasing Pg (Franks et al., 2001; Fig. 4). This explainsthe curve for Pe=0 in Fig. 3, since stomatal aperture is

Fig. 2. The main steps involved in measuring or manipulating turgorpressure in cells: (a) manipulating the oil-®lled glass capillary into theappropriate orientation for puncturing the cell; (b) the cell ispunctured and sap under pressure forces the sap±oil meniscus backinto the capillary; (c) pressure is increased in the system until the sap±oil meniscus is brought back to the cell wall. The pressure required todo this equals the turgor pressure prior to puncturing the cell.

Fig. 3. Relationship between stomatal aperture and guard cellhydrostatic pressure for Tradescantia virginiana, adapted from Frankset al. (1998). Arrows indicate the reduction in aperture with increasingepidermal pressure and constant guard cell pressure.

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linearly related to guard cell volume in isolated guard cells(Raschke, 1979; Fig. 5). However, the sigmoidal relation-ship between stomatal pore width and Pg in the presence ofepidermal turgor is due, in part, to the pressure-dependentin¯uence of epidermal cell turgor on stomatal aperture. Atlow Pg, the closing force exerted on stomata by turgidepidermal cells is at its greatest, but diminishes withincreasing guard cell turgor. Thus the mode of response ofstomatal conductance to any environmental signal willdepend signi®cantly upon the magnitude of Pg at the timethe signal is received, such that responses to perturbationsat low Pg may differ in form from those occurring at highPg. Evidence for this can be seen in stomatal responses toCO2 (Wong et al., 1978) and leaf-to-air vapour pressuredifference (Nonami et al., 1990) under high versus lowlight.

Guard cell osmotic pressure

The driving force behind active stomatal opening andclosure is the regulation of guard cell osmotic pressure. Aswith most other living cells, guard cells conform to thegeneral water potential model. Guard cell water potential,Yg, will passively equilibrate with that of its surroundingsthrough osmotic uptake or release of water. At any point intime guard cell water potential is de®ned by the differencebetween its turgor (or hydrostatic) pressure, Pg, andosmotic pressure, Pg:

Yg=Pg±Pg (1)

By actively adjusting osmotic pressure, guard cell turgorincreases or decreases so as to balance equation 1. WhenYg is close to zero, such as in well-watered plants at highhumidity, Pg will approach Pg in magnitude. Using rapid

plasmolytic techniques, Raschke (1979) measured around4.0 MPa osmotic pressure in Vicia faba guard cells, andMacRobbie (1980) measured a similar value in Commelinacommunis guard cells. Using the same technique, Meidnerand Bannister (1979) measured around 3.0 MPa osmoticpressure in Vicia faba guard cells, and 4.0 MPa inCommelina communis. Using freezing-point depressiontechniques, Bearce and Kohl (1970) report similar valuesfor Chrysanthemum and Pelargonium guard cells. There-fore, osmotic pressures of around 3±4 MPa appear to becommon in open stomata, suggesting that guard cell turgorpressures of this magnitude must also be common. This isnot to say that they are always required. However, using acell pressure probe technique, Franks et al. (1998) foundthat indeed, guard cell turgor pressures of about 4.0 MPawere required to achieve maximum stomatal opening inVicia faba, Tradescantia virginiana, Nephrolepis exaltataand Ginkgo biloba.

Despite signi®cant progress toward understanding theprocess by which guard cell osmotic pressure is generated,much remains unknown about how it is regulated, or therelative quantities of osmotica that it comprises. Byutilizing the pressure probe as a microsampling device,and applying micro-analytical techniques (Tomos andSharrock, 2001) the nature and regulation of guard cellosmotic contents may be explored.

Mechanical advantage of epidermal cells

The term `mechanical advantage', denoted here as m,refers to the extent to which forces in the epidermal cellscounteract the movement of guard cells. An expression forthe mechanical advantage was formalized by Cook et al.(1976) as follows:

Fig. 4. Relationship between guard cell volume and guard cellhydrostatic pressure for Vicia faba, as measured simultaneously usingconfocal microscopy and the guard cell pressure probe (redrawn fromFranks et al., 2001).

Fig. 5. Linear relationship between relative stomatal aperture, a*, andguard cell volume, Vg, in Vicia faba. Data were calculated from®gures in Franks et al. (2001), using a*=(a±a0)/(a1±a0), where a ispore width, a0 is pore width at initial volume, and a1 is pore width at®nal volume.


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m � ÿ

@a

@Pe

� �Pg

@a

@Pg

� �Pe

�2�

which de®nes m as the ratio of sensitivities of a to Pe andPg. Due to this mechanical advantage, an equal increase inturgor pressure in both guard and epidermal cells willresult in a reduction in stomatal aperture, and vice versa.For example, starting at the point Pe=0, Pg=1 MPa anda=10 mm in Fig. 3, if Pe and Pg both increase by 0.9 MPa,the net change in aperture will be a decrease from 10 mm to1.3 mm. What is also evident from Fig. 3 is that thismechanical advantage is not constant, but varies widelywith Pg and Pe. It should be noted also that, although agiven aperture can be achieved with a lower guard cellturgor, if epidermal turgor is reduced, the widest stomatalapertures still require high guard cell turgor in combinationwith low epidermal turgor.

This knowledge of m suggests that, if hydraulic couplingis close between guard and epidermal cells, any increase inepidermal water potential will, in the ®rst instance,promote stomatal closure, and any decrease in epidermalwater potential will promote opening. This seeminglycounterproductive characteristic alludes to the specialregulatory properties of stomata. In practice, the net resultof such perturbations is usually the opposite. The almostuniversal observation is that in response to conditions thattend to reduce bulk leaf water potential, such as increasingtranspiration rate (see reviews by Monteith, 1995; Buckleyand Mott, 2002b) or decreased xylem hydraulic conduct-ance (Hubbard et al., 2001; Cochard et al., 2002), stomataexhibit in the steady-state a net reduction in aperture.

Stomatal control of transpiration rate

Due to the pronounced diurnal and seasonal variations intranspiration potential (leaf-to-air vapour pressure differ-ence, VPD), one of the key regulatory roles played bystomata is that of limiting transpirational water loss or,more precisely, minimizing transpiration-induced waterde®cit. The underlying mechanism of the so-called`humidity response', where stomatal conductance decrea-ses with increasing VPD, remains unknown (Jones, 1998;Meinzer, 2002), but the pressure probe is a tool that willcontinue to make some important inroads into this subject(see below). Different types of plants are known to vary inthe sensitivity of stomatal conductance to VPD (Tardieuand Simonneau, 1998; Franks and Farquhar, 1999). Thosethat are most sensitive are capable of maintaining a moreconstant leaf water potential, since they minimize anyincrease in transpiration rate with increasing VPD. Sincestomatal conductance appears to be positively correlatedwith whole plant, leaf-speci®c, hydraulic conductanceacross species (Meinzer et al., 1995; Saliendra et al.,

1995), diffferences in ability to minimize transpiration-induced reductions in bulk leaf water potential cannot beeasily attributed to differences in xylem hydraulicconductance. Indeed, there is, as yet, little evidence tosuggest a mechanistic link between xylem hydraulicconductance and stomatal sensitivity to VPD.

The pressure probe has contributed much towardunravelling the mechanistic basis of the humidity response.Figure 6 is a block diagram of the essential mechanisticelements of the stomatal response to humidity, adaptedfrom the negative feedback model presented in Franks andFarquhar (1999). The two branches of the feedback looprepresent the combined actions of epidermal and guardcells, whereby a common increase or decrease in turgorpressure leads to opposing effects on stomatal conduct-ance. The net change in stomatal conductance (Dg) is theresult of these stomatal and epidermal effects. Details ofthe model are explained in Franks and Farquhar (1999), butits operation may be summarized as follows: immediatelyafter a change in VPD (before stomata have had time torespond) there is a change in transpiration rate (DE)g. Thissets in train a series of interrelated responses in thestomatal apparatus, as determined by speci®c hydraulic,osmotic and mechanical properties. The resulting changein stomatal conductance, Dg, is the sum of the change dueto guard cell effects alone (Dg)Pe, and the change due toepidermal cell effects alone (Dg)Pg. This gives a change intranspiration rate (DE)w that is determined entirely by thisconductance change. The actual change in transpirationrate as a result of the VPD change, DE, is the sum of (DE)g

and (DE)w. With this mechanism, an increase in VPD willgive a positive change in (DE)g and a negative change in

Fig. 6. Block diagram of essential functional elements in a steady-state feedback model of stomatal response to increasing leaf-to-airvapour pressure difference, VPD (adapted from Franks and Farquhar,1999). Ye, Pe and Pe are epidermal water potential, osmotic pressureand turgor pressure, respectively. Yg, Pg and Pg are guard cell waterpotential, osmotic pressure and turgor pressure, respectively. E istranspiration rate, g is stomatal conductance. The diagram illustratesthe combined roles of hydraulic, osmotic and mechanical forces, andthe dual involvement of both epidermis and stomatal guard cells. Seetext for further explanation.

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(DE)w, such that DE will be smaller than it would bewithout the feedback mechanism.

By manipulating the components of the model in Fig. 6,observed responses to humidity can be simulated.However, such an exercise in itself is of little valuewithout valid empirical data for each of the modelparameters and, unfortunately, there is to date only limitedinformation on these. The pressure probe studies byShackel and Brinkmann (1985), Nonami and Schulze(1989) and Nonami et al. (1990) have provided importantinformation on the hydraulic properties of the leafepidermis. Their experiments with Tradescantia virgini-ana showed that the humidity response in T. virginiana isaccompanied by a signi®cant drawdown in water potentialbetween xylem and subsidiary cells (about 0.1 MPa perkPa VPD under the experimental conditions; Fig. 7), whileno comparable drawdown was observed in the xylem. Therelatively constant xylem water potential, in this case, maybe attributed to a relatively high xylem hydraulic con-ductance, since the relatively moderate stomatal responsestill allowed a considerable increase in transpiration rate.However, the results do indicate that a partial hydraulicdecoupling of the epidermis from the xylem may form thebasis of the, as yet unknown, sensor for the stomatalhumidity response.

With the above hydraulic information, and the hydro-mechanical information obtained for T. virginiana byFranks et al. (1998), some of the blocks in the model inFig. 6 can be parameterized. This in turn allows thecon®rmation of one further important requirement. Giventhe now known hydraulic and mechanical constraints ofT. virginiana leaf tissue and stomatal guard cells, thestomatal response to humidity, in this plant at least, must

include either or both of the following: (1) in addition tothe drawdown in water potential between xylem andsubsidiary cells, a further and substantial drawdown inwater potential between subsidiary cells and sites ofevaporation on guard cells, (2) an active change in guardcell osmotic pressure that is mediated by the ratio andmagnitude of these two effects. There is no directinformation on whether changes in stomatal conductancewith VPD are accompanied by changes in guard cellosmotic pressure. However, if guard cell osmotic pressurewere to be maintained constant, the hydraulic conductivitybetween xylem and guard cell evaporative sites wouldhave to be around ten times lower than that between xylemand subsidiary cells just to overcome the effects of themechanical advantage and induce stomatal closure ratherthan opening in response to increasing VPD (Fig. 8). Theabsence of data on subsidiary cell to guard cell hydraulicconductivity and VPD-induced changes in guard cellosmotic pressure leave only speculation on this topic.However, given that in most cases the stomatal closure inresponse to increasing VPD is much more dramatic than inFig. 8 (dotted line), and in some cases beyond thetheoretical limits of a ®xed-gain negative feedback system,it is unlikely that guard cell osmotic pressure remainsunchanged during the response. Furthermore, to explainsome of the more extreme and less reversible stomatalhumidity responses, there have been suggestions ofdiurnally regulated shifts in stomatal sensitivity character-istics (Franks et al., 1997; Mencuccini et al., 2000), altered

Fig. 7. Effect of an increase in leaf-to-air vapour pressure difference(VPD) on the water potential of xylem, epidermis and subsidiary cells,as measured with a cell pressure probe (epidermal and subsidiarycells) and an in situ psychrometer (xylem). (Adapted from Nonamiet al., 1990.)

Fig. 8. Simulation of stomatal conductance in response to increasingVPD, using the model in Fig. 6, and pressure probe data forTradescantia virginiana from Figs 3 and 5. The simulation is toillustrate, for the simple case of constant guard cell osmotic pressure,the requirements for stomatal closure (rather than opening) in responseto increasing VPD. Solid line: equal hydraulic conductivity fromxylem to subsidiary cells and xylem to evaporative sites in guard cells(negligible hydraulic resistance between subsidiary cells andevaporative sites guard cells); dotted line: hydraulic conductivity fromxylem to evaporative sites in guard cells is ten times lower than fromxylem to subsidiary cells (high hydraulic resistance betweensubsidiary cells and evaporative sites in guard cells).


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leaf hydraulic conductivity (Salleo et al., 2000; Buckleyand Mott, 2002a, b), and ABA-mediated effects onstomatal osmotic pressure (Tardieu, 1993; Zhang andOutlaw, 2001; Wilkinson and Davies, 2002). Outlaw andDe Vlieghere-He (2001) have also shown that underincreased transpiration rate, the accumulation of apoplasticsucrose in the evaporative sites of guard cell walls of Viciafaba may lower the water potential of guard cell wallssuf®ciently to cause stomatal closure. Further advancesand application of pressure probe techniques and relatedmicro-techniques (Outlaw and Zhang, 2001; Zwienieckiet al., 2002) are essential for a more complete understand-ing of the complex cellular mechanisms underlying themulti-sensory behaviour of stomata.

Conclusions and future challenges

The pressure probe is used widely in studies of plant cellwater relations and cell biomechanics. However, itspotential for use in studies speci®c to stomatal functionhas yet to be fully realized. There are many interactingbiochemical and biomechanical processes governingstomatal response to environmental signals, and many ofthese can be studied at the cellular level using the pressureprobe. One of the primary applications of the pressureprobe, in this context, is measuring the relationshipbetween stomatal guard cell turgor, epidermal cell turgor,and stomatal aperture. This information is crucial for thedevelopment of mechanistic models of stomatal move-ment, so that patterns of leaf and canopy gas exchangemight be better predicted.

The above discussion has reviewed the contribution thatpressure probe techniques have made to current under-standing of stomatal function. However, there is still much

that remains unknown about the mechanism of stomatalmovement and gas exchange regulation. Some key areas offuture research focus are listed below.

(1) More species: Data on stomatal biomechanics havebeen gathered for only a handful of species so far, and mostof these are herbaceous plants. The link between stomatalmechanics, stomatal morphology and leaf gas exchangehas barely been explored, but more information is neededon how these properties interrelate across plant life formsand environments.

(2) Guard cell and leaf hydromechanics: In the intactleaf, stomatal aperture is the result of an intricate balanceof forces and ¯uxes in and around the stomatal apparatus.While there is now some idea of the mechanical forcesinvolved, and the dominant ionic and molecular ¯uxes,little is known about water ¯uxes and hydraulic resistancesin the vicinity of the guard cells. Without this information,several current hypotheses about the stomatal mechanismwill remain untested.

(3) Guard cell osmotic pressure: Despite a goodunderstanding of many of the components of guard cellosmotic regulation, much remains unknown about thecontrol of guard cell osmotic pressure. Development ofnovel single-cell sampling and analysis techniques, usingthe pressure probe simultaneously to measure pressure andextract samples, will help advance current understandingin this area.

Acknowledgements

The author thanks Dr Maurizio Mencuccini and organizers of theworkshop on Field Techniques in Environmental Physiology(Tenerierife 2003) for inviting this paper. The author also acknow-ledges helpful comments from an anonymous reviewer.

Table A1. List of equipment and suppliers

Item Manufacturer/supplier Type/part no.

Microscope Carl Zeiss Inverted (for epidermal peels) Axiovert 35MMicroscope Carl Zeiss Compound, with 403 long working distance lens,

for whole leavesCCD camera Diagnostic Instruments Inc., Burlingame, USAImage capture software Diagnostic Instruments Inc., Burlingame, USAMicromanipulator Narishige Scienti®c Instrument Lab, Minamikarasuyama, Japan HydraulicSteady bench Terra Universal Inc., Anaheim, CA, USAPressure transducer Kulite Semiconductor Leonia, NJ, USA 1000 psi ratedSteel piston Small Parts Inc. Miami Lakes, FL, USA 1/32 inch diameter straight stainless steel wireo-rings Small Parts Inc.Miami Lakes, FL, USA 1/32 inch ID, 3/32 inch OD, buna-n typeHPLC PEEK tubing Small Parts Inc. Miami Lakes, FL, USAHPLC ®ttings Small Parts Inc. Miami Lakes, FL, USASilicone oil Wacker-Chemie GmbH, Germany Type AS-4Micrometer screw Mitutoyo America Corporation, USAPipette puller Flaming Brown, Novato, CA, USA Flaming/Brown ProgrammableGlass capillaries Small Parts Inc. Miami Lakes, FL, USA Borosilicate, 1.2 mm OD, 0.69 mm ID

Appendix

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