evidence for an extracellular reception site for abscisic

Plant Physiol. (1994) 104: 1177-1 183

Evidence for an Extracellular Reception Site for Abscisic Acid in Commelina Cuard Cells'

Britt E. Anderson, John M. Ward*, and Julian 1. Schroeder

Department of Biology and Center for Molecular Genetics, University of California, San Diego, La Jolla, California 92093-01 16

bounded by pairs of guard cells. Stomatal aperture is controlled by guard cell turgor, which is mediated by ionic fluxes across p a r d cell plasma membranes.

Although the physiology of ABA action has been studied in detail, the molecular mechanisms of ABA reception and initial events in ABA-induced signal transduction remain unknown. In guard cells, severa1 key ion channels responsible for ABA-induced 'tomata' closure have been characterized

reviewsj see Schroeder and He&chr 1989; Keams and mann, 1993). Whether ABA or other plant hormones exert their primary actions via activation of extracellular receptors or by binding to intracellular receptors or both are funda- mental unanswered questions in plant biology.

Evidence for the plant hormone auxin has shown that extracellular binding sites exist that may contribute to auxin action (Venis et al., 1990; Barbier-Brygoo et al., 1991; Marten et al., 1991; Jones and Herman, 1993). Results conceming the primary site Of ABA reception~ howeverg have so far been equivocal. As Yet unconfirmed data from exPeriments using immunohistochemical staining or photoaffinity labeling have suggested that ABA binds to proteins in the plasma membrane of guard cells (Homberg and Weiler, 1984; Curvetto and Delmastro, 1986). However, evidente exists that ABA uptake may be required for stomatal regulation. Cellular ABA accumulation is known to depend on extracellular pH (Kaiser and Hartung, 1981). Studies in which the extracellular pH was varied showed that ABA was less effective at alkaline pH (Ogunkami et al., 1973), indicating that ABA uptake may be necessary for the regulation of stomatal aperture. A different study showed a lower threshold concentration for ABA at an acidic pH of 5.5 but a more rapid ABA response at a neutra1 pH of 6.8 (Paterson et al., 1988). In addition, other studies show that alkaline pH does not reduce ABA effects (Hartung, 1983).

Extracellular application of ABA to guard cells produces early and rapid increases in cytosolic Ca2+ (McAinsh et al., 1990; Schroeder and Ha@wara, 1990), together with rapid activation of nonselective ion channels, which provide a rnechanism for ca2+ influx and, even in the absence of extracellular Ca2+, for depolarization (Schroeder and Hagi- wara, 1990). Concomitantly, ABA triggers a rapid transient

tion. In Vite Of the rapid nature Of these early responses~ which occur within seconds, these data have not excluded the possibility that ABA uptake into guard cells may be required for their activation.

The phytohormone abscisic acid (ABA) triggers stomatal closing as a physiological response to drought stress. Severa1 basic questions limit an understanding of the mechanism of ABA reception in guard c e k Whether primary ABA receptors are located on the extracellular side of the plasma membrane, within the intracellular space of guard cells, or both remains unknown. Furthermore, it is not clear whether ABA must be transported into guard cells to exert control over stomatal movements. In the present study, a

assays of stomatal movements have been performed to determine primary sites of ABA reception in guard cells. Microinjedion of ABA into guard cells of Commelina communis 1. resulted in injected cytosolic concentrations of 50 to 200 PM ABA and in additional experiments in lower concentrations Of approximately 1 PM ABA. Stomata with ABA-loaded guard cells (n > 180) showed opening similar to stomata with uninjected guard cek. The viability of guard cells following ABA injedion was demonstrated by neutra1 red staining as well as monitoring of stomatal opening. Extracellular application of 10 p~ ABA inhibited stomatal opening by 98% at pH 6.15 and by 57% at pH 8.0. The pH dependenceof extracellular ABA action may suggest a contribution of an intracellular ABA receptor to stomatal regulation. The findings presented here show that intracellular ABA alone does not suffice to inhibit stomatal opening under the imposed conditions. Furthermore, these data provide evidence that a reception site for ABA-mediated inhibition of stomatal opening i s on the extracehlar Side of the Plasma membrane of guard cells.

combination of microinjedion into guard cells and physi0logic-l (Schroeder et a1.j 1987; Schroeder and Ha@wara, 1989; for

ABA is a vital plant hormone, which, among other functions, is known to induce dormancy in seeds and buds, inhibit root elongation, and reduce the effects of drought stress on plants (Walton, 1980). Studies of Arabidopsis show that two genetic loci each affect the control of processes such as seed gennination, seedling growth, gas exchange regulation, and protein synthesis by ABA, indicating common initial signaling mechanisms (Koomneef et al., 1984; Finkelstein and Somer- d l e , 1990). During drought conditions, ABA acts on stomatal pores in the leaf epidermis to cause closure or to inhibit the , opening of stomata. Stomatal pores, which regulate transpirational H20 loss and COz uptake from the atmosphere, are

' This research was supported by grant MCB-9004977 from the

Research Expenence for Undergraduates supplement, U.S. Depart- ment of Agriculture grant 92-37304-7757 u.1.s.1, and a Nationa1 Science Foundation Plant Biology postdoctoral fellowship U.M.W.).

~ ~ t i ~ ~ ~ l science Foundation, a ~ ~ t i ~ ~ ~ l science Foundation- of K+ efflux (MacRobbie, 19901, which m p i t F S depolariza-

* Corresponding author; fax 1-619-534-7108. 1177

1178 Anderson et ai. Plant Physiol. Vol. 104, 1994

Stomatal pores provide an opportune system to study ABA reception and its initial signal transduction mechanisms for several reasons: (a) the response of single guard cells can be readily monitored by measuring stomatal aperture, (b) the delivery of various physiological stimuli can be made to guard cells within epidermal strips, and (c) several molecular mechanisms that control ABA-induced stomatal movements have been identified. Furthermore, microinjections of dyes and second messengers into guard cells have recently shown that the cytosol of guard cells can be accessed using microelectrodes (Blatt et al., 1990; Gilroy et al., 1990, 1991; McAinsh et al., 1990, 1992). In the present study we have used iontophoretic microinjection to load ABA into the cytosol of guard cells. In conjunction with monitoring the aperture of stomatal pores in leaf epidermal strips, a potent cell biological system is provided to gain initial insight into the question of whether extracellular ABA reception is required for control of stomatal movements.

MATERIALS AND METHODS

Plant Material

Commelina communis L. plants were grown in a controlled environment growth chamber (Conviron E15) at 25OC with a 16-h light/8-h dark daylnight cycle, under fluorescent and incandescent illumination at a photon fluence rate of 100 pE m-2 s-'. Plants were grown in potting soil (Uni-Gro; L&L Nursery Supply Inc., Chino, CA) and watered with deionized water. Epidermal strips were isolated from the third fully expanded leaf of 4- to 6-week-old plants.

Epidermal Strip Preparation and lncubation

Variability in the extent of stomatal responses to physiological stimuli has been reported (Gorton et al., 1989). To minimize experimental variation, pairs of epidermal strips from the same leaf were used for control experiments and treatments. Furthermore, injected and uninjected guard cells (approximately 10 injected and >20 uninjected guard cells per epidermal strip) were mapped to allow repeated aperture measurements of individual stomatal pores over time (Gorton et al., 1989). Each leaf was halved vertically, and two identical strips of abaxial epidennis were gently isolated under water to minimize epidermal cell damage and desiccation. The epidermal strips were anchored on two sides with high- vacuum grease (Dow Coming, Midland, MI) in perfusion chambers prior to microinjection. The epidermal strips were incubated in 50 or 100 m KCl with either 10 mM Mes-Tris (pH 6.15) or 10 m Hepes-KOH (pH 8.0) containing either O or 10 p~ ABA ([a] cis-trans isomer, Sigma). In a11 experiments stomata were initially closed. To promote stomatal opening, the epidermal strips were incubated under white light with a photon fluence rate of 500 pE m-2 s-'. In a11 experiments epidermal strips from the same leaves were used for control treatments. Data from experiments in which stomata of control treatments did not open in response to light were omitted from further analysis. During the incubation period (up to 135 min), the chambers were perfused with COz-free air (21% 0 2 , 79% N2; Parsons Medical, San Diego, CA), and the temperature was controlled at 27OC. Measurements of sto-

mata1 aperture were made using an ocular microrneter and high-resolution, differential interference microscopy at the times indicated in the figure legends.

Microinjection

Microelectrodes were pulled from filamented glass (Sutter Instrumerits Co., San Rafael, CA, catalog No. QF 100-70-10) to a tip diameter of approximately 250 nm. The electrode tip was filled with 3 pL of a filtered solution of 10 mM ABA (free acid), 20 I ~ M KOH (from a stock of 100 mM ABA dissolved in 200 mM KOH), and 10 mM Mes. The final pH of the microinjected solution was adjusted to 7.2 by alidition of approximately 6 mM HCl. The osmolality was adjusted to 525 mmol kg-' by addition of D-mannitol. For experiments in which the final injected cytosolic ABA concenhation was approximately 1 p ~ , the electrode tip was filled with a solution containing 100 PM ABA, 100 mM KCl, 10 mM Mes (pH 7.5) (suggested by A. Schwartz and S.M. Assrriann). The electrode was backfilled with 1 M KCl. Electrode maneuvering and guard cell impalement were accomplished with a micro- manipularor (Narishige, Tokyo, Japan) and an inverted microscope (Diaphot; Nikon, Tokyo, Japan) under >:400 mag- nification using high-resolution, differential interference- contrast o'ptics. Impalement of guard cells became apparent both microscopically and by the simultaneous appearance of hyperpolairized membrane potentials. One or two guard cells per closed stoma were iontophoretically injected (approximately 10 stomata injected per epidermal strip). Iontophoretic injection 'was accomplished using alternating 5-s pulses of -200 and O pA of current applied through the electrode for 2 min (Gilroy et al., 1991) using an Axopatch-ID patch- clamp amplifier in current clamp mode (Axon Instruments, Inc., Foster City, CA). To avoid extemal effects of any ABA possibly leaking from the injection microelectrode tluring the microinjection procedure, the epidermal strip wa:; continu- ously perfused with incubation medium using a peristaltic pump (Rainin; Woburn, MA). Epidermal strips were incubated in neutra1 red to verify the viability of injected guard cells at the end of a11 experiments (Gilroy et al., 1991).

Carboxyfluorescein Experiments

To estirnate the approximate cytosolic concentration range of ABA achieved by iontophoretic injection, microelectrodes were filled with 10 p~ to 20 mM carboxyfluorescein adjusted to pH 7.2 with KOH and with the osmolality adjusted to 525 mmol k g ' by the addition of mannitol. Guard cells were iontophoretically injected (as above). Microelectrode and cell fluorescence were recorded by passing light from a 100-W mercury lamp through a fluorescein isothiocyanate filter set (Omega Optical, excitation 480 nm, emission 535 nm). Fluo- rescence measurements were made using a pinhole dia- phragm and a photo multiplier (Thom EM1 Electron Tubes, Inc., Rockaway, NJ) and recorded and analyzed using an Axolab interface (Axon Instruments, Inc.) and a 25-MHz 386- AT microcomputer (Schroeder and Hagiwara, 1990). Fluores- cence intensities were measured from 9.6 pm2 of guard cell cytoplasmic regions exposed through a pinhole ciiaphragm (the less-Ihorescent areas directly over the vacuole and the

Extracellular Guard Cell ABA Receptor 1179

brightly fluorescent nucleus were avoided). The fluorescenceintensities of known concentrations, between 10 ^M and 20mM, of carboxyfluorescein were measured in microelectrodetips as above and were used to estimate cytoplasmic carboxy-fluorescein concentration following iontophoretic microinjec-tion. This calibration provided a coarse correction for fluo-rescence quenching in a volume of similar optical thicknessas guard cells. Based on the similarity of ABA and carboxy-fluorescein in charge (both have a pKa of 4.7) and mol wt(264.3 and 376.3, respectively), the mobility of these twocompounds during iontophoretic microinjection should besimilar (Pusch and Neher, 1988). Furthermore, experimentswere performed at a constant temperature of 25 °C in allexperiments to reduce variation in diffusion of dyes andABA. Therefore, estimates of the efficiency of carboxyflu-orescein microinjection were used to determine the concen-tration range of injected cytosolic ABA concentrations follow-ing microinjection. The efficiency of ABA microinjection wasnot determined by co-injection of carboxyfluorescein andABA in the same guard cells, since high concentrations ofinjected carboxyfluorescein resulted in guard cells that didnot remain viable for the time required for our experimentsand showed stomatal closing of the half of the stomatalapertures flanked by the injected guard cells in all experi-ments (n > 40).

RESULTS

Iontophoretic Microinjection of Commelina Guard Cells

To study the primary reception site of ABA in guard cells,it was necessary to apply known concentrations of ABA eitherextracellularly or to the guard cell cytosol. Single-cell microin-jection was used to access the guard cell cytosol. The effi-ciency of the iontophoretic microinjection technique wasanalyzed using the fluorescent dye carboxyfluorescein. Uponimpalement of guard cells with the microelectrode and injec-tion of negative current, rapid appearance of the dye in thecytoplasm was observed (Fig. 1). Localization of dye in thecell periphery and in chloroplasts and the nucleus indicatedcytoplasmic rather than vacuolar injection. The observedresults of more than 40 injections of carboxyfluorescein intostomatal guard cells indicated that the iontophoretic injectiontechnique used in this study allowed reproducible deliveryinto the cytosol.

Quantitation of carboxyfluorescein microinjection by sin-

Figure 1. Carboxyfluorescein was iontophoretically microinjectedinto the cytosol of one stomatal guard cell in an epidermal stripfrom the abaxial side of a Commelina leaf.

Figure 2. Iontophoretic microinjection of ABA does not inhibitstomatal opening. One guard cell (indicated by arrow) was microin-jected using a microelectrode containing ABA. A, Stomata followingmicroinjection at time 0 min. B, The same stomata after 135 min ofincubation under light. The abaxial epidermal strip was incubatedin 50 HIM KCI, 10 mM Hepes-HCI (pH 8.0). This experiment isrepresentative of more than 100 guard cells microinjected with ABAunder these conditions. Bars indicate 20 ̂ m.

gle-cell fluorescence photometry was used to estimate theefficiency of iontophoretic microinjection of ABA. From theseexperiments it was estimated that iontophoretic injection ofABA resulted in a concentration of 50 to 200 JIM ABA in theguard cell cytosol. The efficiency of ABA microinjectiondetermined here correlates closely with microinjection effi-ciencies reported for guard cells in recent studies (Gilroy etal., 1990, 1991; McAinsh et al., 1990; Lee et al., 1993). Theseestimates give the cytosolic concentration of microinjectedABA and do not include endogenous ABA in guard cells(Harris et al., 1988). The injected concentration range of ABAis severalfold higher than the concentration of ABA found inVicia faba guard cells following water stress, which waswithin the range of 7 to 13 ^M (Harris et al., 1988).

To test whether guard cells microinjected with ABA wouldremain viable for the duration of these experiments, epider-mal strips were stained with the viability stain neutral red atthe completion of all experiments. Up to 2.5 h after microin-jection of ABA, 80 to 90% of the cells remained viable asdetermined by the ability to accumulate neutral red in thevacuole and sustain visible cytoplasmic streaming. In addi-tion, stomata with microinjected guard cells were competentto open, which requires guard cell viability, as describedbelow. Approximately 10% of the injected guard cells showedimmediate cessation of cytoplasmic streaming during mi-croinjection and a visible deflation in turgor, indicating avacuolar location of the electrode tip. Data from these cellswere not included in this study.

Microinjection of ABA

The question of whether ABA, applied directly to the guardcell cytoplasm, could inhibit stomatal opening was addressedusing the iontophoretic microinjection technique. Aperturemeasurements of noninjected stomata and of stomata withinepidermal strips from the same leaf externally treated withABA were made in parallel and served as controls for allexperimental trials. Guard cells were microinjected with ABA(50-200 JIM, one guard cell per stoma) and exposed to light

1180 Anderson et al. Plant Physiol. Vol. 104, 1994

in the absence of external ABA (n > 180 ABA-injected guardcells in this study). Figure 2A shows two initially closedstomata following ABA microinjection and prior to exposureto light. In Figure 2B, the same two stomata are shownfollowing incubation under white light for 135 min. In allexperimental trials, the stomata with injected guard cells (n> 100) opened similarly to the uninjected control stomata.Stomata in which both surrounding guard cells were microin-jected with ABA (n = 10) also opened similarly to uninjectedcontrol stomata (data not shown). These results indicated thatintracellular ABA alone could not inhibit stomatal opening.In additional control experiments, stomata microinjected with50 to 200 MM ABA and incubated in the presence of 10 MMextracellular ABA at pH 6.15 remained closed (n = 20).

To quantify the effect of internal ABA on stomatal opening,apertures were measured for stomata with guard cells injectedwith 50 to 200 MM internal ABA throughout a 135-minincubation. As shown in Figure 3, stomata with one guardcell microinjected with ABA consistently had rates and max-imum stomatal opening that were similar to uninjected con-trols. When guard cells were injected with lower cytosolicABA concentrations of approximately 1 MM (n = 24 guardcells), stomatal pores also opened similarly to controls.

It is possible that microinjected ABA requires the presenceof additional CaCl2 in the bath solution for inhibition ofstomatal opening (Schwartz et al., 1988). Stomata of guardcells injected with 50 to 200 MM ABA, with 0.5 HIM CaCl2added to the bath solution, also opened similarly to controls(n = 10). These data further support the observation (Fig. 2)that internal ABA alone was not sufficient to inhibit stomatalopening.

Extracellular Application of ABA

The ability of low concentrations of externally applied ABAto inhibit stomatal opening was tested. To exclude the pos-sibility that externally applied ABA could be taken up byguard cells, the experiments were conducted at an extracel-lular pH of 8.0. ABA predominates in the deprotonated format pH 8.0 and uptake is eliminated (Kaiser and Hartung,

Unln|«ct*dABA InJccMd

50 100Time (mln)

150

Figure 3. Stomata with guard cells microinjected with 50 to 200 MMABA open at a similar rate and to a maximum aperture as uninjectedcontrol stomata. Guard cells were microinjected and abaxial epi-dermal strips were incubated in 50 ITIM KCI, 10 HIM Mes-Tris (pH6.15). D, Uninjected guard cells (n = 20); •, stomata with one guardcell injected with ABA (50-200 MM; n = 10). Data are from onerepresentative experiment of nine. Error bars denote SE.

Figure 4. Extracellular application of ABA inhibits stomatal opening.Abaxial epidermal strips containing uninjected stomata were incu-bated as described in the legend to Figure 2 with the addition of10 MM ABA to the perfusion chamber. A and B show the same twostomatal complexes before ABA exposure (A) and 135 min afterABA addition to the bath and exposure to white light (B). This figuredepicts an epidermal strip that showed a maximum response toexternal ABA. Although 10 MM external ABA allowed partial stomatalopening in most experiments at pH 8.0, stomata not exposed toexternal ABA opened to a greater extent in all experiments (see textand Fig. 5). Bars indicate 20 Mm.

1981; Baier and Hartung, 1991). Epidermal strips with ini-tially closed stomata (Fig. 4A) were incubated under light inthe presence of 10 MM external ABA. As shown in Figure 4B,after 135 min the stomata exposed to 10 MM external ABAremained closed or did not open as wide as nontreatedcontrols (n > 400 stomata in 20 experiments). Parallel controlexperiments in the absence of external ABA showed widerstomatal opening than ABA-treated stomata. This confirmedthat ABA in the external solution contributes to inhibition ofstomatal opening.

Taken together, the above experiments (Figs. 2-4) dem-onstrate a requirement for external ABA in the inhibition ofstomatal opening under the imposed conditions. To furtheranalyze whether the uptake of externally applied ABA mayalso contribute to the inhibition of stomatal opening, theeffect of ABA on stomatal aperture was quantitatively anddirectly compared at pH 6.15 and 8.0 using epidermal stripsfrom the same leaves. Guard cells were exposed to 0 or 10MM external ABA at pH 6.15 or 8.0, placed under light, andperfused with CO2-free air for 2 h. External ABA at both pH6.15 and 8.0 inhibited stomatal opening (Fig. 5), and inhibi-tion was more effective at pH 6.15 in all experiments. Extra-cellular ABA inhibited stomatal opening without addition ofexogenous Ca2+ to the bath solution in all experiments. Thesedata suggest that basal free Ca2+ levels in the cell wall spaceof guard cells and in the unbuffered bath solutions weresufficient for ABA-induced inhibition of stomatal openingunder the imposed conditions and/or that Ca2+-independentevents contribute to inhibition of stomatal opening (Mac-Robbie, 1990). Stomatal opening was inhibited an average of98% by 10 MM external ABA at pH 6.15 and 57% at pH 8.0.These results may suggest that, in addition to an extracellularABA requirement, the pH-dependent uptake of ABA contrib-utes to the inhibition of stomatal opening and/or that otherprocesses involved in stomatal regulation are pH dependent.

Further experiments were conducted to determine the ef-


7

12

T

O 2

Time (hr) Figure 5. Externa1 ABA at pH 6.1 5 and 8.0 inhibits stomatal opening. Epidermal strips were incubated at pH 6.1 5 or 8.0 in the presence and absence of 10 PM external ABA. Apertures of mapped stomata (n = 40 for each bar) were measured before the addition of ABA and after 2 h of exposure to extracellular ABA and incubation under white light. Data are from one representative experiment of five. Error bars denote SE.

fects of microinjected ABA on the time course of stomatal opening at an extracellular pH of 8.0. Guard cells were injected with ABA and incubated for 135 min under white light at an externa1 pH of 8.0. Stomata with guard cells that were microinjected with ABA opened at a rate and to an extent that was similar to uninjected control stomata (Fig. 6). These data may indicate that the decreased effectiveness of external ABA in the inhibition of stomatal opening at pH 8.0 (Fig. 5) was not solely due to a decrease in ABA uptake and a lack of intracellular ABA receptor activation. Although a possibility of functional intracellular receptors for ABA was not excluded, the presented data demonstrate a requirement for extracellular ABA in the inhibition of stomatal opening.

DISCUSSION

Phytohormones such as auxin, GAs, and ABA have im- portant functions in the control of plant growth, develop- ment, and responses to the environment. Functional reception sites at the extracellular side of the plasma membrane have been implicated for the hormones auxin (Venis et al., 1990; Barbier-Brygoo et al., 1991; Marten et al., 1991; Jones and Herman, 1993) and GAs (Hooley et al., 1991). However, the cellular location of ABA receptors has remained equivocal. Stomatal guard cells provide a well-suited system to study ABA receptor location.

ABA Reception by Guard Cells

Under drought stress conditions, ABA accumulates rapidly in leaves (for review, see Walton, 1980) and can decrease transpirational water loss by triggering stomatal closure. The physiological response of stomatal guard cells to exogenously applied ABA is well characterized. Studies have shown that an extracellular concentration of 1 ~ L M ABA is sufficient to

induce maximum stomatal closure in Commelina (McAinsh et al., 1990). Although externally applied ABA triggers stomatal closure in excised epidermal strips, it has remained unclear if binding to an extracellular receptor contributes to this response. High-affinity ABA-binding sites have been indicated in the plasma membrane of V. faba guard cells (Hornberg and Weiler, 1984; Curvetto and Delmastro, 1986). However, it is not clear if these binding sites represent ABA uptake transporters or ABA receptors. Severa1 distinct mechanisms for ABA reception are therefore possible, including: (a) intracellular ABA receptors requiring ABA uptake into guard cells and cytosolic ABA accumulation, (b) ABA receptors at the extracellular side of the plasma membrane that trigger intracellular signaling events, and (c) extracellular ABA receptors acting in parallel with intracellular receptors, resulting in a requirement for both extracellular and cytosolic ABA. To gain insight into which of these ABA reception mechanisms can be excluded and which mechanisms function in the inhibition of stomatal opening, we utilized the technique of microinjection of ABA into the cytosol of guard cells and the physiological assay of stomatal aperture measurement.

Extracellular Location of Guard Cell ABA Receptors

As reported here, stomata with one or both guard cells microinjected cytosolically with ABA consistently opened at a similar rate and to a similar maximum aperture as uninjected stomata (Figs. 2, 3, and 6). These data indicate that intracellular ABA alone was not sufficient to inhibit stomatal opening. Externally applied ABA (10 PM) inhibited stomatal opening (Figs. 4 and 5). Taken together, these data demonstrate that extracellular ABA was required to inhibit stomatal opening under the conditions imposed here. However, the possibility that, following external application, ABA uptake had an additional role in the inhibition of opening could not be ruled out by these observations (Fig. 5). The experimental approach presented here focuses on whether extracellular ABA is required. Data using co-injection of ABA and carboxyfluorescein have indicated a contribution of intracellular ABA to stomatal regulation using a complementary approach

o + . I O 5 0 1 O0 150

Time (min)

Figure 6. Microinjected ABA does not inhibit stomatal opening at an extracellular pH of 8.0. Guard cells were microinjected with ABA and abaxial epidermal strips were incubated as described in the legend to Figure 2. O, Uninjected guard cells (n = 20); m, stomata with one guard cell injected with ABA (50-200 PM; n = 10). Data are from one representative experiment of seven. Error bars denote SE.

1182 Anderson et al. Plant Physiol. Vol. 104, 1994

that focuses on a requirement for intracellular ABA (Schwartz et al., 1993).

In the present study inhibition of stomatal opening by ABA was analyzed rather than ABA-mediated stomatal closing to ensure viability of guard cells after microinjection. In calibration experiments, high levels of microinjected carboxyfluorescein inhibited stomatal opening and reduced cell viability as judged by neutral red staining (n > 40, see "Materials and Methods"). Further experiments would therefore be required to determine if the presented results also apply to ABA- induced stomatal closing.

Dependence of ABA Effectiveness on Extracellular pH

At an extracellular pH of 8.0, a condition that eliminates ABA uptake (Kaiser and Hartung, 1981; Baier and Hartung, 1991), externally applied ABA consistently inhibited stomatal opening (Fig. 5), although to a lesser extent than at pH 6.15. Furthermore, stomata with guard cells microinjected with ABA and incubated at pH 8.0 opened at a rate similar to uninjected control stomata (Fig. 6) . These data further support a requirement for an ABA receptor at the extracellular side of the plasma membrane that functions in the regulation of stomatal aperture.

However, the data reported here do not completely exclude the presence of an additional intracellular ABA receptor. Alternate explanations for the decreased inhibition of stomatal opening at pH 8.0 are pH effects on plasma membrane ion channels and pumps that control stomatal movements or a direct pH dependence of ABA binding to receptors at the plasma membrane. Previous studies have suggested that a decreased effectiveness of extracellular ABA to induce stomatal closure at alkaline pH is correlated with a decreased rate of ABA binding to high-affinity sites at the plasma membrane of guard cells (Hornberg and Weiler, 1984).

Studies of the Arabidopis thaliana ABA-insensitive mutants abil and abi2 have shown that early ABA-induced signaling events are shared in different physiological responses of different cell types (Finkelstein and Somerville, 1990). Recent studies using different techniques to those used here, specif- ically of a-amylase secretion in barley aleurone cells (Gilroy and Jones, 1994) and ABA-induced expression of the wheat Em gene in a transient assay (R. Quatrano, personal com- munication), also suggest an extracellular reception site for ABA.

Stability of Microinjeded ABA

The possibilities that microinjected ABA was either rapidly degraded in guard cells, leaked out of guard cells, or was microinjected at concentrations too high to be effective in this study need to be considered. Stomatal opening was observed in response to ABA injection, which requires guard cell viability. Additional experiments, in which ABA was injected at approximately 100-fold lower concentrations (1 PM), also did not show an inhibition of stomatal opening. Furthermore, there is no evidence for reduced ABA effectiveness on stomatal regulation or other responses at high ABA concentrations (Walton, 1980). For example, induction of P- glucuronidase activity from ABA-inducible promoter-P-glu-

curonidase constructs in transformed rice protoplasts was proportional to ABA concentrations from 1 to 100 PM (Mar- cotte et al., 1988). The stability of ABA in guard cells is demonstrated by its accumulation for up to 8 h in V. faba guard cells to concentrations of 7 to 13 PM following dehydration stress of leaves (Harris et al., 1988; Harris and Out- law, 1991). Therefore, it is unlikely that ABA was microinjected at concentrations that were too high to inhibit stomatal opening.

ABA is metabolized slowly to phaseic acid and 4 '-dihydro- phaseic acid with a half-time of 6 to 8 h in leaf tfiscs of V. faba (Mertens et al., 1982). Upon the remova1 of dehydration stress, the ABA concentration of V. faba guard cells returned to prestress levels with a half-time of 2 h (Harris and Outlaw, 1991). However, responses of guard cells to ABA have been reported to occur more rapidly. Recent data show that a brief (2 min) exposure to extracellular ABA is sufficiení to trigger the signaling cascade leading to long-term K+ efflux from guard cells and a resulting decrease in stomatal aperture (MacRobbie, 1990). Furthermore, the initial rates of stomatal opening were not measurably affected by ABA microinjection (Figs. 3 and 6). Therefore, the possibility that rapid degrada- tion of A13A resulted in the complete lack of effecíiveness of microinjected ABA in this study is unlikely. Since guard cells remained viable throughout the duration of the experiments reported here, significant leakage of ABA from guard cells, within severa1 minutes, was unlikely. In addition, any leakage of injected ABA may contribute to an inhibition of stomatal opening that was never observed in this study.

In conclusion, this report provides strong eviderice for the requirement of an ABA receptor at the extracellular side of the plasma membrane of guard cells which functions in the regulatiori of stomatal aperture. Furthermore, data show that intracellular ABA alone does not suffice to inhibit stomatal opening under the imposed conditions. The identification of ABA receptors and components of ABA-induced signaling pathways using cell biological, molecular biological, and genetic approaches should provide further insigh t into the mechanism of ABA reception.

ACKNOWLEDCMENTS

Jennifer Sheaffer is gratefully acknowledged for performing sev- era1 control experiments. We thank Drs. Karen Schumaker and Douglas Bush for critica1 comments on the manuscript and Dr. Simon Gilroy for ,advice conceming neutral red staining.

Received September 7, 1993; accepted December 14, 19513. Copyright Clearance Center: 0032-0889/94/104/1177/07.

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