Proc. Natl. Acad. Sci. USAVol. 89, pp. 3591-3595, April 1992Plant Biology

Gibberellic acid and abscisic acid coordinately regulate cytoplasmiccalcium and secretory activity in barley aleurone protoplasts

(confocal sanning optical microscopy/plant hormones/ratio imaging/secretion)

SIMON GILROY AND RUSSELL L. JONES*Department of Plant Biology, University of California, Berkeley, CA 94720

Communicated by Winslow R. Briggs, December 16, 1991

ABSTRACT The effects of gibberellic acid (GA3) andabscisic acid (ABA) on the temporal and spatial dynamics ofcytosolic calcium concentration ([Ca2+]o) in aleurone proto-plasts of barley (Hordeum vulgare L. cv. Himalaya) weremeasured by using fluorescence ratio analysis and confocalmicroscopy. After 4-6 h of treatment, GA3 induced a sustainedincrease in [Ca2+]1 from 50 to 150 nM in aleurone protoplasts.The increase in [Ca2+]1 preceded the GA3-induced increase ina-amylase synthesis and secretion by 4 h. The elevation of[Ca2+]1 was highest in the peripheral cytoplasm and may playa role in coordinating the secretory events there. ReducingCa2+ levels in the incubation medium to below 500 FMinhibited the increase in [Ca2+]1 and the GA3-induced stimu-lation of a-amylase synthesis and secretion. These data suggestthat GA3 may increase [Ca2+]1 by increasing the influx of Ca2+at the plasma membrane. ABA reversed the effect of GA3 on[Ca21]J within 3 h, 2 h before its effect on a-amylase productioncould be detected. Thus changes in [Ca2+] may play a role inmediating the effects of GA3 and ABA on the synthesis andsecretion of a-amylase in the aleurone cell.

The aleurone layer of barley grain secretes a spectrum ofhydrolytic enzymes, principally a-amylases, which degradethe endosperm during germination. Synthesis and secretionof a-amylase is enhanced by gibberellic acid (GA3) in thepresence of 10 mM extracellular Ca2+, and this stimulation isreversed by abscisic acid (ABA) (1). The effects of thesegrowth substances are visible as changes in the rate oftranscription of a-amylase genes and in the rates of proteinsynthesis and secretion. However, the steps between hor-mone perception and stimulation and coordination of subse-quent secretory events are unknown.Changes in the concentration of cytosolic calcium ([Ca2+]i)

regulate the response of many animal cell types to chemicaland electrical stimuli (2). In particular, increases in [Ca2+]iare known to mediate the stimulation of secretory activity inanimal cells by a wide range of hormones (2). In plants therole of [Ca2+]i in the coupling of stimulus and response is lessclear. Changes in [Ca2+]i have been reported to follow auxinand ABA treatment of coleoptile and root cells (3, 4) but werenot evident in root hairs (5). In stomatal guard cells ABAinduces an increase in [Ca2+]j (6, 7), probably through rapidactivation of Ca2' channels in the plasma membrane (8)coupled to Ca2' release from organelles (7). Artificiallyinducing these increases in [Ca2+]j can mimic the effect ofABA and trigger stomatal closure (9). The link between GA3perception, changes in [Ca2+]i, and modulation of secretoryactivity in the aleurone cell is less well defined. Bush et al.showed that GA3 treatment of aleurone protoplasts increased[Ca2+]i (10) and stimulated Ca2' transport into the endoplas-mic reticulum (11). However, these measurements were

made after 36 h of GA3 treatment, whereas a 5- to 50-foldincrease in a-amylase synthesis and secretion occurs afteronly 8-12 h (reviewed in ref. 1).

In this paper we show that GA3 treatment increases [Ca2+]i3-fold and that this increase precedes the effects of thehormone on a-amylase synthesis and secretion. We alsoshow that the increased (Ca2+]i is localized to the peripheralcytoplasm of the cell and that the stimulatory effects of GA3on [Ca2+]i are reversed by treatment with ABA.

MATERIALS AND METHODSFluorescent Dyes. Fluorescent dyes were obtained from

Molecular Probes. Calcium green linked to a dextran of Mr10,000 has Ca2+-dependent fluorescence characteristics sim-ilar to fluo-3 but is approximately 5 times brighter.

Isolation and Incubation of Aleurone Protoplasts. Proto-plasts were prepared from aleurone layers isolated fromdeembryonated grain of Hordeum vulgare L. cv. Himalaya(1985 harvest) and purified on a discontinuous Nycodenzdensity gradient (10). Protoplasts were incubated in modifiedGambourg's B-5 medium (10) supplemented with 10 mMCaCl2 and, where specified, 5 ,uM GA3 or 5 ,uM GA3 followedby 5 uM ABA. a-Amylase was assayed according to Jonesand Varner (12).

Radiolabeling. Protein synthesis was monitored by radio-labeling with [35S]methionine (13). Protoplasts (5 X 10' perml) were incubated with 100 uCi (1 Ci = 37 GBq) ofTran-35S-label reagent (ICN) for 1 h, then homogenized inbuffer supplemented with 10% (wt/vol) trichloroacetic acidand 10 mM methionine. [35S]Methionine incorporated intotrichloroacetic acid-precipitable material was measured byliquid scintillation counting (13).

Acid Loading of Protoplasts with Indo-1 and Fluo-3. Proto-plasts were loaded with indo-1 and fluo-3 essentially asdescribed by Bush and Jones (10). Carry-over of extracellulardye contributed <5% of the signal from loaded protoplasts asestimated by quenching extracellular dye with 100 ,uM MnCl2(14). Leakage of indo-1 and fluo-3 from protoplasts wasapproximately 5% and 8% h-1, respectively. For experimentswhere time courses were studied protoplasts were incubatedunder appropriate conditions and samples were removed atthe indicated times for dye loading and [Ca2+], measurement.

Microinjection of Protoplasts. Micropipettes (10- to 20-Mflresistance) were pulled from filament electrode glass (WorldPrecision Instruments, New Haven, CT) by using a ListMedical model 5A electrode puller (List Electronics, Darm-

Abbreviations: ABA, abscisic acid; calcium green-dextran, calciumgreen linked to dextran of Mr 10,000; [Ca2+]I, extracellular Ca2+concentration; [Ca2]ij, cytosolic Ca2' concentration; ER, endoplas-mic reticulum; GA3, gibberellic acid; indo-1, 1-[2-amino-5-(6-carboxyindol-2-yl)phenoxyl]-2-(2'-amino-5'-methylphenoxyl)ethane-N,N,N',N'-tetraacetic acid; rhodamine-dextran, tetramethyl-rhodamine linked to dextran of Mr 10,000.*To whom reprint requests should be addressed.

359.1

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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stadt, F.R.G.). Micropipettes contained 100 AuM indo-1,fluo-3, or Lucifer yellow-CH or 1 mM calcium green-dextranand 1 mM tetramethylrhodamine-dextran (rhodamine-dextran, Mr 10,000). All fluorescent dyes were dissolved indeionized water (resistivity 18 Mfl-cm; Millipore). Proto-plasts were allowed to settle for 10 min onto an acid-washedno. 1 coverslip forming the bottom of a microscope samplechamber (1.5 cm diameter, 1 ml total volume). Dextran-linked fluorescent dyes were pressure injected into impaledprotoplasts by using a SB2 syringe microburet system (MicroMetric Instrument, Cleveland, OH). Indo-1, fluo-3, and Lu-cifer yellow CH were iontophoretically microinjected at <0.5nA for 5 min (model 5-A microelectrode amplifier, Gelting,Iowa City, IA). Injected cells were allowed to rest for 15 minto ensure even diffusion of the dye throughout the cytoplasmand maintenance of viability after the micropipette waswithdrawn.

Fluorescence Microscopy and Photometry. Fluorescencefrom single protoplasts was monitored by using a NikonDiaphot inverted microscope fitted with a 75-W xenon epi-fluorescence light source and optics transmissive to 340-nmlight. A Nikon CF Fluor 40x, 1.30 numerical aperture,oil-immersion objective was used throughout. Excitationwavelengths were determined with a 485-nm, 10-nm half-bandwidth (hbw) interference filter for fluo-3 or 340-nm(10-nm hbw) for indo-1. Excitation intensity was reducedwith a 1/8 transmittance neutral density filter to reduce spec-imen photodamage and photobleaching of the dye. Emissionfiltration consisted of a 400-nm dichroic mirror and a 485-nm(10-nm hbw) or 405-nm (10-nm hbw) interference filter forindo-1 or 510-nm dichroic mirror and 520-nm, long-pass filterfor fluo-3. A Nikon PFX shutter assembly and circularemission aperture were used to define a single protoplast forstudy. Thus, results presented from the photometric systemrepresent an average of [Ca2"] over a whole protoplast.Fluorescence was measured by using a C716-01 photomulti-plier coupled to a C1230 photon counter (Hammatsu, Bridge-water, NJ) with 6-decibel filtering. Autofluorescence repre-sented <15% of the total signal from dye-loaded protoplastsand did not change with time or experimental treatment.

Ratio Imaging. Conditions, equipment, and image process-ing used for ratio imaging were as described by Gilroy et al.(7). Data for ratio images were recorded every 20 min. Themean of 20 autofluorescence measurements from individualprotoplasts (8% ± 6% of the signal from dye-loaded proto-plasts) was subtracted from images prior to ratio calculation.

Confocal Microscopy. Protoplasts were allowed to settleonto an acid-washed glass slide and viewed with a NikonOptiphot microscope and Lasersharp MRC-600 confocal im-aging system (Bio-Rad) or a Phoibos 1000 confocal laser-scanning microscopy system (Sarastro, Philadelphia). Fluo-rescence excitation utilized the 514-nm or 488-nm emissionlines of an argon-ion laser passed through an ND 2 neutraldensity filter. Fluorescence from fluo-3 was visualized byusing a BHS (fluorescein) filter block on the MRC-60 con-focal microscope. Calcium green-dextran and rhodamine-dextran were simultaneously visualized with the Phoibosconfocal microscope using a 520 DRLP dichroic mirror forexcitation. The emitted light passed through a 530 EF long-pass filter to a 565-nm dichroic mirror. Wavelengths above 565nm (rhodamine) passed directly to a detector, whereas wave-lengths below 565 nm were further filtered through a 530 DF30band-pass filter prior to confocal detection. Images werecollected as the average of 8-16 scans with a Nikon PlanApoFluor 60x, 1.40 numerical aperture, oil-immersion objective.

Estimation of Intracellular Indo-1 and Fluo-3 Concentra-tions. Intracellular dye concentration was assayed (7), as-suming the cytosolic volume of the aleurone protoplast to be15 pl. In the case of fluo-3 and calcium green-dextran,Ca2'-saturated fluorescence was measured from loaded pro-

toplasts treated with 10 ,M calcium ionophore BrA23187 and10 mM CaCI2 and compared with Ca2+-saturated fluo-3fluorescence (250 ALM free Ca2+) in the calibration droplets.

Calibration ofFluorescence Versus Ca2l Concentration. Forindo-1 an in vitro and in vivo calibration was performed asdescribed by Gilroy et al. (7). Since fluo-3 does not undergoa spectral shift upon binding, Ca2+ calibration was performedby determining the Ca2+-saturated and Mn2+-quenched flu-orescence from the dye in situ (9, 15). However, due to thedifficulty of ensuring the intracellular Ca2+ level during sucha calibration, fluo-3 measurements should be regarded asapproximations.

RESULTSDye Loading Does Not Impair the Response of Barley

Aleurone Protoplasts to GA3 or ABA. We have loaded barleyaleurone protoplasts with the Ca2+-sensitive fluorescent dyes

UUI -700

-400

-200[Ca2j1nMW

-100

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FIG. 1. Ratio and confocal images of the [Ca2+]1 in protoplaststreated with GA3 and ABA. Protoplasts were loaded with indo-1 orfluo-3 by low-pH incubation (ratio images, a, and confocal images,b, respectively) or with calcium green-dextran by microinjection(confocal images, c). Samples were taken at the time indicated (h)after GA3 treatment, and protoplasts were loaded with the appro-priate dye for estimation of [Ca2+]i. In b, after 16 h of incubation withGA3 the protoplasts were treated with 5 ,uM ABA and [Ca2+]j wasvisualized after 2 h (2+A) and 4 h (4+A). In experiments usingprotoplasts microinjected with calcium green-dextran [Ca2+]j wasmonitored at 0 and 8 h after treatment with 5 AuM GA3. These cellswere simultaneously microinjected with rhodamine-dextran. Thefluorescence from rhodamine-dextran is not Ca2+-sensitive andtherefore reflects the distribution of dextran-linked dye in the cyto-sol. In c, 0 (r) and 8 (r) show the distribution of rhodamine-dextranvisualized simultaneously with the calcium green-dextran images.The [Ca2+]i has been pseudocolor coded in all the images accordingto the color bar on the right. The [Ca2+] scale does not apply to theimages of rhodamine-dextran fluorescence, which have been colorcoded on an arbitrary scale, where blue represents minimum and redmaximum fluorescence intensity, to clarify the distribution of the dyewithin the cell. Protoplasts incubated without GA3 showed noincrease in [Ca2+],. Images are representative of at least 15 cellsanalyzed from three separate experiments. (Scale bar represents 5Jim.)

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FIG. 2. Distribution of fluorescent dyes loaded into barley aleurone protoplasts. (a) Confocal image of protoplasts loaded with fluo-3 bymicroinjection, showing cytosolic localization of the dye. (b) Protoplast loaded with fluo-3 by incubation at pH 4.5, showing vacuolaraccumulation of dye in a protoplast stressed by incubation in 5 mM EGTA. (c) Protoplast microinjected with Lucifer yellow. (d) Protoplastincubated with 10 AtM fluorescein diacetate for 10 min. Images are of the median plane of the protoplast and have been individually pseudocolorcoded to enhance the visualization of dye distribution according to the color scale, where blue is minimum and red is maximum fluorescence.(Scale bar represents 5 Aum.)indo-1 and fluo-3 by two methods: incubation at low pH (Fig.1 d-j and refs. 10 and 16) and microinjection (Fig. 2). Theintracellular concentrations of indo-1 and fluo-3 loaded byeither method were estimated to be 5-20 ,uM. At theseconcentrations the dyes would not be expected to contributesignificantly to the Ca2l-buffering capacity of the cytoplasm(7) since, for example, 100 ,uM buffer concentrations wererequired to interfere with the functioning of Pelvetia zygotes(16). The dyes .vere excluded from the vacuole and evenlydistributed throughout the cytoplasm (Figs. 1 and 2 and refs.10 and 17). Autofluorescence from granular inclusions in thevacuoles (phytic acid crystals) was evident with both fluo-3and calcium green (Fig. 2), but it did not change with time orexperimental treatment and did not interfere with Ca2+measurements. Poor injections or loading of stressed proto-plasts led to vacuolar accumulation of the dye within 5 min(Fig. 2), and these protoplasts were not studied further.

Successfully acid-loaded or microinjected protoplastswere morphologically indistinguishable from untreated con-trols (Fig. 3). Protoplasts that had been acid-loaded withindo-1 and fluo-3 at 10 ,uM exhibited only a 10% inhibition inamylase synthesis and secretion (Fig. 4). Also, after 8 h oftreatment with GA3, these protoplasts showed the usualincrease in amylase synthesis and secretion (Fig. 5B and ref.17), which was reversible within 1-2 h ofABA treatment (Fig.6B). Dye-loaded protoplasts showed no loss of viability asassessed by fluorescein diacetate staining (76 ± 12%6 viabilitybefore loading, 81 ± 8% after loading). We interpret theseobservations as indications that our loading and measure-ment protocols did not disrupt protoplast function.Modulation of Cytosolic Ca2+ and Secretory Activity by GA3

and ABA. Aleurone protoplasts showed an increase in [Ca2+],after 4-6 h of GA3 treatment from approximately 50 nM to150 nM [monitored with fluo-3 (Fig. SA, Table 1)] or 120-500nM [monitored with indo-1 (Table 1)]. Protoplasts microin-jected with fluo-3, indo-1, or calcium green-dextran showedresponses of similar magnitude and timing (Table 1, Fig. 1).This increase in [Ca2+]i preceded the stimulation of amylasesynthesis and secretion by GA3 (Fig. SB).

:a

FIG. 3. Light micrographs of barley aleurone protoplasts loadedwith fluo-3 by incubation at low pH (a) and microinjection (b). (c)Unloaded control. Micrographs were taken using Hoffman differen-tial interference contrast optics. V, vacuole; P, phytin globoid;arrow, point of microinjection. (Scale bar represents 10 ,Lm.)

The increase in [Ca2+] was not uniform across the cyto-plasm. Ratio imaging of indo-1-loaded protoplasts stimulatedwith 5 AuM GA3 showed that the increase in [Ca2f]i wasinitially homogeneous throughout the cytoplasm but after 8 hbecame maximal in the periphery of the cell (Fig. la). Theratio calculation used in the production of the images of Ca2"distribution from indo-1 fluorescence (Fig. 1) should correctfor uneven dye distribution (2, 14). However, to fully assessthe contribution of an edge effect or out-of-focus blur in thispicture of [Ca2+]i distribution we optically sectioned thefluo-3-loaded protoplasts by using confocal microscopy.Confocal imaging (Fig. lb) revealed the same peripheral ringof high calcium subtending the plasma membrane as seen inthe ratio images from indo-1. Protoplasts microinjected withLucifer yellow or loaded with fluorescein diacetate (Fig. 2 cand d) showed no evidence of dye exclusion from any regionof the cytosol or appreciable signal attenuation throughdifferential absorption across the cell. It is therefore unlikelythat redistribution of the dye or internal absorption in thecenter of the cell could explain the high peripheral calciumlevels seen in the confocal imaging of fluo-3.Endoplasmic reticulum (ER) is known to accumulate in the

cytoplasm below the plasma membrane in GA3-treated barleyaleurone protoplasts (18), and ER has been reported to havea high lumenal Ca2+ concentration and to take up indo-1 invivo (11). Thus, it is possible that uptake of Ca2' indicatorinto cortical ER could account for the apparently high [Ca2+]in the periphery of the GA3-treated protoplasts. To test thispossibility protoplasts were microinjected with the fluores-cent Ca2+ indicator calcium green linked to a 10,000 Mr

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FIG. 4. Effect of loading aleurone protoplasts with fluo-3 orindo-1 on a-amylase secretion. Protoplasts were incubated with theappropriate concentration of indo-1 (e) or fluo-3 (o) for 2 h at pH 4.5in dye-loading buffer. Protoplasts were then washed free of extra-cellular dye and incubated with 5 IM GA3 and 10 mM CaCl2 inGambourg's B-5 medium supplemented with 300 mM mannitol.Amylase secretion into the medium was assayed from 5 X 105protoplasts per ml after 16 h and is expressed as percent secretion inunloaded cells (control). Results represent mean ± SEM, n 45individual cells from three separate experiments. Curves were fittedby using a smoothed curve-fitting algorithm (Kaleidagraph, Albel-beck software; Reading, PA).

Plant Biology: Gilroy and Jones

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Protoplasts were loaded with fluo-3 by incubation at low pH. [Ca2+I](A) and rate of amylase production (B) were measured at the

indicated times with (o) or without (s) 5 ,uM GA3 added at zero time.

At the indicated times samples of protoplasts were removed from a

bulk incubation and the mean [Ca2+I1 over a whole protoplast was

measured photometrically. Results represent mean SEM, n 45

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FIG. 6. Effect ofABA on the GA3-induced increase in [Ca2+]j (A)and amylase synthesis (B). Protoplasts were treated with 5 ,.M GA3and 10 mM CaCl2 for 16 h; [Ca2+]i was then assayed after no furtheradditions (A) or after addition of 5 MM ABA (e). Control protoplastswere incubated for 16 h with 10mM CaC12 and 5,uM ABA was addedat time zero (o). Protein synthesis was monitored by liquid scintil-lation counting of [35S]methionine incorporated into newly synthe-sized proteins, principally a-amylase.

Table 1. Effect of GA on [Ca2+]i as measured bydifferent methods

[Ca2+], nM

Loading Incubation in 5 ,JM GA3 Controlmethod Dye 0 h 6 h 12 h 12 h

Low pH Fluo-3 64 ± 34 115 ± 39 207 ± 31 78 ± 40Indo-1 143 + 29 179 ± 19 426 ± 45

Microinjection Fluo-3 81 + 39 140 ± 42 231 ± 40 53 ± 37Indo-1 131 ± 30 185 ± 41 509 ± 35

Protoplasts were incubated with 10 mM CaC12 and 5 ,uM GA3 wasadded at 0 h. Controls were treated identically but without GA3.Results represent mean ± SEM, n > 35 (low-pH loading) or >11(microinjection) from at least three separate experiments. Figuresrepresent average [Ca2+]i measured over a whole protoplast.

dextran. The large size of dextran-linked dyes is thought toprevent their uptake by organelles. Confocal imaging ofprotoplasts microinjected with calcium green-dextran re-vealed a peripheral ring of increased Ca2+ concentration inGA3-treated protoplasts (Fig. lc). Imaging of the distributionof rhodamine-dextran that had been microinjected simulta-neously with the calcium green-dextran showed no evidenceof localization of dextran-linked dyes within the cell (Fig. lc).The fluorescence from rhodamine-dextran was not affectedby Ca2+ (data not shown). These results suggest that theincreased Ca2+ observed in the cortical cytoplasm of GA3-treated protoplasts was due to an increase in [Ca2+]j.

After 1- to 3-h treatment with 5 ,uM ABA, the [Ca2 ]i inGA3-treated protoplasts was reduced (Figs. lb and 6A). Thisreduction in [Ca2W]i preceded the first measurable effects ofABA on a-amylase synthesis and secretion by about 2 h (Fig.6B).The GA3-Induced Increase in [Ca2+] Depends upon Extra-

cellular Ca2+. The localized increase in (Ca2+]i induced byGA3 could have arisen from increased Ca2` influx at theplasma membrane and/or by release of Ca2" from intracel-lular stores. Influx at the plasma membrane can be reducedby lowering the extracellular Ca2` concentration ([Ca2+]k).Addition of 5 mM EGTA to the protoplasts to chelateextracellular Ca2` led to a rapid loss in protoplast viability(data not shown) and relocalization of indo-1 or fluo-3 to thevacuoles within 5 min (Fig. 2b). Lowering [Ca2W+ to 10-4 M

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did not affect protoplast viability but did affect [Ca2+]i anda-amylase secretion. At [Ca2+]e below 1 mM the GA3-stimulated increase in [Ca2+], was inhibited (Fig. 7A), and thiseffect directly paralleled the inhibition of a-amylase produc-tion (Fig. 7B). Confocal imaging of fluo-3-loaded protoplastsindicated that lowering the [Ca2'le lowered the amplitude ofthe changes in [Ca2+], throughout the cytoplasm (data notshown).

DISCUSSIONOur data show that the plant hormones GA3 and ABA resetthe steady-state [Ca2+], in barley aleurone protoplasts. GA3induces a sustained 3-fold elevation in [Ca2+], and ABAreduces this increase to near non-GA3-stimulated levels.These changes in [Ca2+], precede the effects ofGA3 and ABAon a-amylase synthesis and secretion (Figs. 5 and 6) and maybe associated with the early events whereby GA3 and ABAregulate protein synthesis and secretion in the aleurone cell.We have used three Ca2+-indicating dyes (fluo-3, indo-1,

and calcium green-dextran) and two methods of loading pro-toplasts with these dyes (acid loading and microinjection;Table 1). This approach has allowed us to ensure that theresponses we observed were not artifacts of the Ca2+ indicatoror its loading protocol and that dye-loaded protoplasts werefunctioning similarly to unloaded controls (Fig. 4). The 5-25,uM intracellular dye concentrations used in this study are wellbelow the 100,tM levels required to interfere with develop-ment of other plant cells (16) and should not contributesignificantly to the buffering capacity of the cytoplasm (7).The increase in [Ca2+] induced by GA3 was not uniform

throughout the cell. The highest [Ca2+], was localized to thecytoplasmjust inside the plasma membrane (Fig. 1). The datain Fig. 7 also show that the GA3-induced increases in [Ca2+],and stimulation of a-amylase secretion have nearly identicaldependencies on [Ca2+]e. These results suggest that theGA3-induced rise in [Ca2+]i and increase in a-amylase secre-tion are supported by enhanced Ca2+ influx at the plasmamembrane. The lowering of the [Ca2+]e would inhibit thisincreased influx and hence lower [Ca2+] below the levelrequired to sustain a-amylase synthesis and secretion.The mechanism whereby GA3 would lead to an enhance-

ment of Ca2+ influx at the plasma membrane is unknown. Arecent report indicates that the GA3 receptor may be on theexternal face of the plasma membrane in the aleurone cell(19). Such a receptor could indirectly modulate the activity ofCa2+ channels or pumps in the plasma membrane. Receptorsfor animal hormones are known to act in this manner (2). Inplants, ABA has already been shown to activate Ca2` chan-nels in the plasma membrane of stomatal guard cells (8). Butit is interesting to note that although ABA seems to open Ca2+channels in the guard cell plasma membrane, it may have theopposite effect in the aleurone cell, where ABA reverses theelevation in [Ca2+] induced by GA3. However, hormonalregulation of Ca2+-pump activities at the plasma and organel-lar membranes may also occur.The localization of the GA3-induced increase in [Ca2+], to

the peripheral cytoplasm of the cell leads to the intriguingpossibility that this may reflect differential localization offunction within the cortical cytoplasm. Hepler et al. (20) havenoted that many plant and animal cells possess a fenestratedmatrix of cortical ER that is tightly associated with theplasma membrane. This cortical ER is sometimes structurallydistinct from ER further into the cell and may facilitatesecretion (20). GA3-treated aleurone protoplasts also containcortical ER (18), and elevated ER Ca2+ has been implicatedin the enhancement of secretory activity induced by GA3

treatment of these cells (1). a-Amylase is the major proteinsecreted from the ER of aleurone cells and is a Ca2+_containing metalloprotein that requires Ca2+ for its stabilityand activity (21). Thus, an elevated concentration ofER Ca2+may be required to support the increased synthesis of a-amy-lase. GA3 treatment is known to lead to enhanced activity ofthe Ca2+ uptake system in barley aleurone ER (11), and thiseffect is reversed by ABA (22), paralleling the effect ona-amylase synthesis and secretion. The level of the [Ca2+]we observe in the peripheral cytoplasm of GA3-stimulatedcells (600 nM; Fig. 1) is within the range found to activate thisCa2+-uptake system of barley aleurone ER in vitro [Km forCa2+ = 0.5 AM (11)] and could facilitate a localized inductionof secretion as is seen in some animal cells (23).Thus the changes in [Ca2+], that we have measured precede

the effects of GA3 and ABA on a-amylase synthesis andsecretion and may be acting to regulate Ca2'-dependentprocesses, such as a-amylase formation, in the ER. Increased[Ca2+1] may also have regulatory effects at other points in thesecretory apparatus, such as vesicle transport and fusion(24). Therefore these changes in [Ca2+], may contribute to thecoordination and integration of the complex series of eventsthat lead to the activation of secretion in the aleurone cell.

We are grateful to Prof. Tony Trewavas and Dr. Nick Read for useof the ratio imaging equipment, the National Science FoundationCenter for Plant Developmental Biology and the Department ofMolecular and Cellular Biology, University of California at Berkeleyfor use of the confocal microscopes, and Eleanor Crump for assis-tance in preparing this manuscript. This work was supported bygrants to R.L.J. from the National Science Foundation.

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Sci. USA 87, 9645-9649.4. Felle, H. (1988) Planta 174, 495-499.5. Clarkson, D. T., Brownlee, C. & Ayling, S. M. (1988) J. Cell Sci.

91, 71-80.6. McAinsh, M., Brownlee, C. & Hetherington, A. M. (1990) Nature

(London) 343, 186-188.7. Gilroy, S., Fricker, M. D., Read, N. D. & Trewavas, A. J. (1991)

Plant Cell 3, 333-344.8. Schroeder, J. I. & Hagiwara, S. (1990) Proc. Natl. Acad. Sci. USA

87, 9305-9309.9. Gilroy, S., Read, N. D. & Trewavas, A. J. (1990) Nature (London)

346, 769-771.10. Bush, D. S. & Jones, R. L. (1988) Eur. J. Cell Biol. 46, 466-469.11. Bush, D. S., Biswas, A. K. & Jones, R. L. (1989) Planta 178,

411-420.12. Jones, R. L. & Varner, J. E. (1967) Planta 72, 155-161.13. Sticher, L., Biswas, A. K., Bush, D. S. & Jones, R. L. (1990) Plant

Physiol. 92, 506-513.14. Grynkiewicz, G., Poenie, M. & Tsien, R. Y. (1985) J. Biol. Chem.

260, 3440-3450.15. Kao, J. P. Y., Harootunian, A. C. & Tsien, R. Y. (1989) J. Biol.

Chem. 264, 8171-8178.16. Speksnijder, J. E., Miller, A. L., Weisenseel, M. H., Chen, T.-H.

& Jaffe, L. F. (1989) Proc. Natl. Acad. Sci. USA 86, 6607-6611.17. Bush, D. S. & Jones, R. L. (1987) Cell Calcium 8, 455-472.18. Hillmer, S., Bush, D. S., Robinson, D. G., Zingen-Sell, I. & Jones,

R. L. (1990) Eur. J. Cell Biol. 52, 169-173.19. Hooley, R., Beale, M. H. & Smith, S. J. (1991) Planta 183, 274-280.20. Hepler, P. K., Palevitz, B. A., Lancelle, S. A., McCauley, M. M.

& Lichtscheidl, I. (1990) J. Cell Sci. 96, 355-373.21. Bush, D. S., Sticher, L., Van Huystee, R., Wagner, D. & Jones,

R. L. (1989) J. Biol. Chem. 264, 19392-19398.22. Bush, D. S. & Jones, R. L. (1991) in Calcium in Plant Growth and

Development, eds. Leonard, R. T. & Hepler, P. K. (Am. Soc. PlantPhysiol. Symp. Ser., Rockville, MD), Vol. 4, pp. 60-66.

23. Cheek, T. R., Jackson, T. R., O'Sullivan, A. J., Moreton, R. B.,Berridge, M. J. & Burgoyne, R. D. (1989) J. Cell Biol. 109, 1219-1227.

24. Steer, M. J. (1988) Physiol. Plant 72, 213-220.

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