Planta

Planta (1989) 178:231-241

9 Springer-Verlag 1989

Calcium influx at the plasmalemma of isolated guard cells of Commelina communis Effects of abscisic acid E.A.C. MacRobbie Botany School, University of Cambridge, Downing Street, Cambridge CB2 3EA,UK

Abstract. The influx of 45Ca into isolated guard cells of C o m m e l i n a c o m m u n i s L. has been measured, using short uptake times, and washing in ice-cold La3+-containing solutions to remove extracellular tracer after the loading period. Over 0.5-4 min the uptake was linear with time, through the origin. Over 20-200 gM external Ca 2 + the influx measured with 10 20 m M external KC1 was in the range 0.3-2.3 pmol-cm 2-s-1 (on the basis of estimated guard-cell area) ; with only 1 m M KC1 externally the 4SCa influx was significantly reduced, in the range 0.3-1.1 p m o l . c m -2 .s -1 for external Ca 2+ of 50-100 gM. The results indicate that the Ca-channel is voltage-sensitive, opening with depolarisation. No consistent effect of the addition of abscisic acid could be found. In different experiments, on the addition of 0.1 m M abscisic acid the Ca 2 § influx was sometimes stimulated by 28-79%, was sometimes unaffected, and was sometimes inhibited by 16-29%. The results rule out a long-lasting stimulation of 45Ca influx by ABA, but they do not rule out a transient stimulation followed by inhibition, perhaps as a consequence of down-regulation of Ca 2 § influx by increasing cytoplasmic Ca 2§ The hypothesis that ABA may act via an action on Ca 2§ influx, increasing cytoplasmic Ca 2 § with consequent effects on voltage-dependent and Ca2+-dependent ion channels in both plasmalemma and tonoplast, is neither proved nor disproved by these results.

Key words: Abscisic acid and

C a 2 + uptake - Calcium uptake - C o m m e l i n a - Guard cell Membrane t r a n s p o r t - Stomata

Introduction The level of cytoplasmic free Ca 2 § plays a vital role in the regulation of many cell processes, and Abbreviations: ABA = abscisic acid; Cao, Ko = external Ca and K concentrations

small increases in its level are responsible for triggering a variety of cell responses. Cytoplasmic Ca 2 + is set by the balanced sum of a number of influxes from various compartments, from outside but also from various internal storage compartments (such as the endoplasmic reticulum, mitochondria and the vacuole), and of a number of different efflux processes, of active transport out of the bulk cytoplasm to the same compartments. The passive influxes involve gated ion channels which can be modulated, and it is likely that the characteristics and sensitivities of the channels in different membranes will differ. Calcium-dependent responses are likely to be initiated by opening of one or more of such gated channels, leading to an increase in cytoplasmic Ca 2 + above the resting level of a few hundred nanomoles per liter as measured in Characean cells (Williamson and Ashley 1982; Miller and Sanders 1987). F u c u s rhizoid cells (Brownlee and Wood 1986), mung-bean roottip protoplasts (Gilroy et al. 1986), and diatoms (Brownlee et al. 1987), and are assumed to be similar in all plant cells. When a response is dependent on external Ca 2+ it is assumed that modulation of the plasmalemma influx is involved, but it is important to confirm this by direct evidence of the flux change. It has long been recognised that control of stomatal aperture is one such Ca-sensitive process, but the mechanism of the sensitivity has not been established. Effects of Ca 2 § on stomatal aperture, and its role in promoting closure or in inhibiting opening were observed by Fujino (1967), Willmer and Mansfield (1969) and Fischer (1972). Schwartz (1985) found that in C o m m e l i n a external Ca 2 § was more effective in promoting stomatal closure than in inhibiting stomatal opening, and argued for a larger effect on ion efflux than on ion influx. Fitzsimons and Weyers (1986) found that Ca 2 § inhibited K-dependent swelling of C o m m e l i n a guard-cell protoplasts, and Inoue and Katoh (1987) showed, again in C o m m e l i n a , that Ca 2 § inhibited both pro-

232

ton extrusion and stomatal opening during ionstimulated opening in the dark, after pre-illumination. De Silva et al. (1985a, b) showed that the inhibition of stomatal opening by abscisic acid (ABA) was a Ca2+-dependent process, and that the inhibition was sensitive both to Ca 2 +-channel blockers (La 3 +, verapamil and nifedipine) and to calmodulin antagonists (trifluoperazine, W7 and compound 48/80). MacRobbie (1986) showed that 86Rb+ influx during the early opening of newly isolated Commelina guard cells was inhibited by Ca 2 +, and that the inhibition of influx in the dark was Ca 2 +-dependent. Investigation of fluxes indicates that the initiation of stomatal opening is likely be regulated by control of the processes of ion uptake, largely dependent on the activity of the proton pump, whereas initiation of stomatal closure, or control of the ability of stomata to stay open, is achieved by regulation of processes of ion efflux, not by control of ion influx. (For discussion of the evidence for this view, see review by MacRobbie 1988a, and references therein.) The work on Ca-effects on apertures indicates that both ion-influx and ion-efflux processes are sensitive to Ca 2+, by separate mechanisms, but there is a need to establish the detailed sequence of events. As far as the closure response is concerned, a working hypothesis (speculation) can be formulated (MacRobbie 1988b) in terms of opening of Ca 2 +-channels in the plasmalemma in response to a closing signal (such as ABA or transfer to the dark), leading to an increase in cytoplasmic Ca 2 + and a depolarisation of the plasmalemma potential. These changes might then be expected to lead to the opening of Ca 2 +-dependent and voltage-dependent ion channels (for both K + and CI-) in the plasmalemma, and to the opening of Ca 2 +-dependent ion channels in the tonoplast. This is similar to the sequence of events involved in the action potential of Characean cells, and the same plasmalemma events are also seen in the hypotonic regulatory responses in the Characean Lamphrothamnium (Okazaki et al. 1984; Okazaki and Tazawa 1987). The sequence is consistent with the electrophysiological properties established for Chara, and also with the properties of ion channels in higher-plant cells, in so far as they have been established by patch-clamp techniques. Thus Schroeder etal. (1987) identified K+-channels, which were opened by depolarisation, in the plasmalemma of Vicia guard cells, and Schauf and Wilson (1987) showed that the kinetic characteristics of the voltage-sensitive K+-channels in the Vicia guard-cell plasmalemma were affected by ABA, which produced long bursts of channel opening.

E.A.C. MacRobbie: Effects of ABA on Ca 2 + influx into guard cells

Further, Hedrich and Neher (1987) observed a Cai+-sensitive non-selective channel in the tonoplast in sugar beet, and it may well be that this is a common feature in all plant cells, including guard cells. Thus the controlled operation, in guard cells, of channels of the kind identified in some plant cells (and in some instances in guard cells) could account for the tracer flux responses observed during the closing response in Commelina, both the transient increase in plasmalemma efflux of anions (CI-, B r - ) and cations (Rb + as analogue for K+), and the ABA-induced increase in the flux of anions from vacuole to cytoplasm (MacRobbie 1981, 1983, 1984). A role for increased cytoplasmic Ca 2+ as the trigger for such channel openings is a reasonable hypothesis requiring testing. This discussion establishes a clear need to measure the C a 2+ influx in guard cells, and to look for stimulation of such influx in response to a closing signal, as one part of the test of the hypothetical sequence of events. The present work was undertaken with these aims, using ABA as the closing signal, but with only partial success. It has been possible to measure influx, but not to show consistent effects of ABA on the influx. A major problem in the measurement of 4SCa2+ influx in the past has been the very high tracer Ca 2 + in the Donnan phase in the cell wall, and the difficulty of distinguishing the extracellular tracer from that which has truly entered the cell. The problem can be overcome by the use of a wash in ice-cold La 3 +-containing solution after the labelling period, to remove such extracellular 4 5 C a , while losing negligible intracellular tracer. The method was first used in Characean cells (MacRobbie 1988c), and is here applied to guard cells. Material and methods Plant material. 'Isolated' guard cells of Commelina communis L. were prepared as previously (MaeRobbie 1981a, b, 1983, 1984). Epidermal strips were treated at low pH to kill all cells other than guard cells, with a 10-rain treatment in 150 mM KC1, 10raM 2-(N-morpholino)ethanesulfonic acid (Mes), pH 3.9, followed by 10min in 50 mM KC1, 10raM Mes, pH 3.9. Strips were then floated on stirred 20 m M KC1, l0 m M Mes, pH 3.9 for several hours. Strips were examined for absence of crystals in epidermal cells, a sign of effective isolation. Strips were then incubated overnight, to allow them to reach a steady state, in the solution to be used for the influx (but unlabelled), containing 1, 10 or 20 m M KC1, usually 20 ~M CaSO4, but 10-200 gM in other experiments, and 10 mM 1,4-piperazinediethanesulfonic acid (Pipes), pH 6, in a thermostatted cabinet at 25 ~ C, in light (140 Ixmol. m - z. s- 1 in the range 400-700 mm, from a bank of fluorescent tubes). Influxes were then measured over short labelling periods at various times during the following day.

E.A.C. MacRobbie: Effects of ABA on Ca 2+ influx into guard cells In general, where comparison of influxes under different conditions was to be made, care was taken to prepare replicate sets of strips from the same leaves; where possible, comparison was made between replicate sets of strips from a single leaf, but where too few strips could be obtained for this, sets of strips from a pair of leaves from the same plant were compared. The variability in influx from different strips from the same leaf was much less than that between strips from different leaves and different plants, as might be expected. In 19 out of 31 influx determinations (on batches of 8-20 strips) the standard error of the mean was < 6% of the mean value; of the remaining 12 determinations, 9 had standard errors of 7 10% of the mean, and the other 3 were between 13 and 18%. Differences between batches from different plants in the same experiment could be much larger than this, in one case up to 40%, though in other instances between 1% and 9%. Thus comparison of influx under different conditions, such as with and without ABA, required matched samples of strips, preferably from the same leaf, but at least from the same plant.

233

1,0

0.5

i

E '-8 E

0

t 4

2

i 6

(..)

Influx measurements. Strips were incubated in labelling solution, composition as above~ containing usually 40 7 0 k B q - m 1 - 1 *SCa, in 3.5-ml portions of solution in the wells of plastic culture dishes, on a vibrating shaker. The shaking turned out to be necessary, and in one 8-rain influx period when the tissue was not shaken the influx was reduced by 53%. After the labelling period, strips were rinsed twice in inactive bathing solution, then transferred to a large volume of ice-cold bathing sohit i o n + 2 m M LaC13 (La-sohition), again on a shaker, to remove extracellular 4SCa. The wash-time was 20 min, chosen with reference to a measured time course of efflux to ice-cold Lasolution. After the wash the strip area was measured, and strips were extracted in 0.5 ml of 0.1 M H2SO4, 10 m M CaSO4 in scintillation-vial inserts; 3 ml of Optiphase Safe scintillation cocktail (LKB, Bromma, Sweden) was added, before counting in a Beckman (High Wycombe, Bucks., U K ) LS 7500 scintillation counter. Standard aliquots of loading solution were also counted to allow conversion of disintegrations per minute to moles of Ca. Influxes are expressed on the basis of epidermalstrip area, in pmol. m m - 2 . rain-1. Approximate conversion to the basis of guard-cell area, to real membrane flux units, is considered in the Discussion, using average figures for guardcell area from previous work.

Results

Time course of influx. For the proper measurement of influx it is necessary that the uptake period is short enough for back-flux of tracer during the uptake to be negligible, and that the wash treatment allows complete removal of free-space Ca 2 + but negligible loss of intracellular Ca 2+. The use of an ice-cold La-wash was designed to achieve this, but it is necessary to establish that uptake of tracer over the time periods used is linear with time, through the origin. Figure 1 shows two such time courses, and Table I shows the calculated regressions of tracer content against time for six batches of tissue. On this basis, 2 to 4-min uptake periods were used for most subsequent influx measurements, as being safely within the linear uptake region.

8,

n

9

0.1

0

/"

I

1 . 1

M

2

TIME (min)

Fig. 1 A, B. Time course of uptake of 45Ca into 'isolated' guard cells of Commelina. Cells were loaded for the time shown, then washed in ice-cold La-containing solution for 20 rain. Each point represents uptake into a single epidermal strip. A 50 gM CaSO4, 20 m M KC1, pH 6; B 50 gM CaSO4, 1 m M KC1, pH 6

The time course of efflux of 45Ca to successive 0.75-ml portions of ice-cold La-wash solution was measured, and the results indicated that the use of a 20-min wash period to remove extracellular tracer was satisfactory. In one experiment, 2 m M LaC13 was added to the uptake solution (containing 20 gM CaC12); the uptake in the presence of La 3+ was reduced to 0.010_+0.005 pmol-mm -2. min ~, compared with 0.078 _+0.009 pmol- m m - 2. min- ~, in the control. The apparent influx declined with longer periods of uptake, as would be expected if the cytoplasmic specific activity rose, and a back-flux of tracer developed. Figure 2 shows the time course over 35 rain of uptake, initially linear with time, but with a significantly lower apparent influx over 35 min of uptake. In the early stages of filling a multi-compartment cell the tracer content will rise with time, approximated by the relation Q * = Papp(l --e kt), where Qavv is the apparent pool size, and k a rate constant for exchange. The interpretation of Qappand k depends on the model assumed

234

E.A.C. MacRobbie: Effects of ABA on Ca 2 + influx into guard cells

Table 1. Regressions of 45Ca content of Commelina guard cells on time of uptake. Q* is the 45Ca content of 'isolated' epidermal strips after tracer loading for varying time periods within the range shown, expressed on the basis of area of epidermal strips. Loading was done by floating 'isolated' epidermal strips, with only guard cells alive, on 4SCa-containing solutions of Ca 2+ and K + concentrations as shown. Cao, Ko = external concentrations Cao (gM)

20 20 20 50 50 50

Ko (mM)

20 10 10 20 1 1

Uptake time (min)

Number of strips

2-8 2~4 2-4.25 2 8 0.5-2 2~4

Regression Q * = a + bt

16 18 13 23 16 12

a (pmol- mm 2)

b (pmol. m m - 2 m i n - 1)

0.056 __+0.026 - 0.072 __+0.060 0.031 __+0.033 -0.053 +0.040 -0.002__+ 0.007 0.008 __+0.028

0.068 _+0.005 0.156 __0.019 0.045 -t-0.009 0.141 +__0.009 0.064-t- 0.005 0.050-t- 0.009

Table 2. Influx of 45Ca into 'isolated' guard cells of Commelina from a solution containing 20 gM Cao. 'Isolated' epidermal strips were labelled by floating on 45Ca-containing solution. Influx is expressed on the basis of the area of epidermal strips

1.5

External Range of ~SCa influx" KC1 (pmol-mm- 2 . m i n 1) (mM) Minimum Maximum

E E 1.O

Mean b

"5 E

10 0.077• (16) 0.129+0.005 (18)0.106+0.005 (10) 20 0.043-t-0.008 (12) 0.144_+0.012 (12)0.093_+0.015 (7) Mean of 17 experiments combined: 0.101 _+0.007 (17)

O.

0.5

0

Mean_+ SE (number of strips) b Mean of different batches (number of sets of strips)

I

10

I

20 TIME (min)

I

30

Fig. 2. Extended time course of uptake of 45Ca into 'isolated' guard cells of Commelina, showing the decline in apparent influx as tracer back-flux develops during uptake. Each datum point represents the uptake from a single epidermal strip. The bathing solution contained 20 ~tM CaSO4, 20 m M KC1, pH 6

0.3

A

7

.E

E '~.

for the cell, and will be considered later. For the time course of Fig. 2 the apparent pool size is about 1 . 3 7 p m o l - m m 2, and the apparent rate constant for exchange is about 3.4. h-1, half-time about 12 min. Most experiments were done at 20 gM external Ca, a concentration which allows wide stomatal opening. Table 2 shows the combined results of these experiments, for external KC1 concentrations of 10 and 20 m M (pH 6). There is no difference between the results at these two concentrations of KC1, and the two sets may be combined in a single mean of 0.101 ___0.007 p m o l . m m - 2 . m i n -1 (17 experiments). A few measurements were made at different concentrations of Ca 2+, and these are

0.2

E E

i//I

"S E C3. X

_=

0.1

C

o

2'o

~

L

~o

~o

Cao (NM)

Fig. 3. Influx of 45Ca into guard cells of Commelina from different concentrations of external Ca (Cao). Each point is the mean influx measured on 5-20 strips, with S E = 5 13% of mean values, e, 20 mM KC1; zx, 1 m M KC1. Solid lines join points for replicate batches of strips measured on the same day; dotted line joins two points measured with 2-d time interval between them

E.A.C. MacRobbie: Effects of ABA

o n C a 2+

influx into guard cells

235

Table 3A-C. Effects of 0.1 mM ABA on 4SCa influx into isolated guard cells of Commelina. After overnight incubation in nonradioactive solution, influx was measured over 2- or 4-min periods at various times during the following day. The uptake time in ABA is shown from the start of the ABA treatment. For 0-2 or 0 4 rain, ABA and tracer were added simultaneously for uptake of 2 or 4 rain; for the 2-4 rain uptake, strips were pre-treated in ABA in the absence of tracer for 2 rain, then labelled for 2 rain in the presence of ABA Date

Cao (gM)

Time (a.m./p.m.)

Uptake time in ABA (min)

Flux (pmol. ram- 2. min 1)

p.m. p.m. p.m. p.m. p.m.

0-4 2-4 0-4 0-4 2-4

0.078 _+0.003 0.24 _+0.02 0.144_+0.012 0.098 _+0.005 0.34 _+0.01

(6) (10) (12) (9) (7)

0.100_+ 0.008 0.43 _+0.05 0.184_+0.008 0.138 _+0.007 0.45 _+0.02

(5) (7) (16) (10) (7)

a.m. a.m. a.m. p.m. p.m. a.m. p.m. p.m. a.m. p.m. a.m./p.m.

0-2 0-4 2-4 0-4 0-2 0-4 0-4 2-4 0-4 0-4 2-4

0.086-+0.005 0.114_+ 0.006 0.077 _+0.007 0.043 _+0.002 0.054 _+0.003 0.093 _+0.004 0.123 _+0.007 0.129_+0.005 0.119_+0.007 0.108_+0.006 0.23 _+0.02

(11) (13) (16) (12) (15) (13) (14) (18) (10) (9) (11)

0.091 _+0.003 0.107_+ 0.005 0.078 • 0.003 0.041 _+0.002 0.050 _+0.002 0.094 _+0.006 0.104_+0.007 0.125_+0.006 0.101 _+0.006 0.119_+0.005 0.223_+0.009

(11) (12) (l 5) (14) (16) (13) (14) (9) (10) (10) (11)

2-4 0-4 0-4 0-4 0-4

0.24 __0.02 0.080 -+ 0.004 0.077-+0.007 0.079 +0.005 0.055 -+ 0.002

(10) (14) (6) (9) (13)

0.17 _+0.01 (8) 0.067 -+ 0.002 (16) 0.063 • (9) 0.058-!-_0.004(6) 0.042 -+ 0.004 (11)

--ABA

p

+ABA

A Significant stimulation by ABA 5/5 18/6I a II 29/7 IIb 21/8 II c

20 20 20 20 200

0.025 0.01 0.01 0.001 0.001

B No significant effect of ABA 12/6 I II III 24/6 II d III 29/7 I b 3/8 I e IIe 5/8 II I 21/8 I c

20 20 20 20 20 20 20 20 20 20 100

C Significant inhibition by ABA 18/6I a 24/6 1d 22/4 1 II III

20 20 20 20 20

a.m. a.m. a.m. p.m. p.m.

0.02 0.02 0.05 0.02 0.02

a 18/6: BatehII, p.m., gave significant stimulation by ABA. Batch I was split between a.m. and p.m. The control cells did not differ between morning and afternoon, and a combined mean has been used for comparison with ABA-treated cells. (a.m. control 0.23_+0.04 (4), p.m. control 0.25_+0.025 (6); combined mean for control 0.24_+0.02 (10)). The values for ABA-treated cells in the afternoon were significantly higher than this, and those for ABA-treated cells in the morning were significantly lower b 29/7: Batch I, a.m., gave no effect of ABA. Batch II, p.m., gave significant stimulation by ABA c 21/8: Batch I, a.m., gave no effect of ABA. Batch II, p.m., gave significant stimulation by ABA d 24/6 : Batch I, a.m., gave significant inhibition by ABA. Batch II, p.m. gave no effect of ABA ~ 3/8 : Freshly isolated tissue, used within a few hours of isolation of guard cells

shown in Fig. 3. Influx increases with concentration, but no detailed study of the form of the concentration dependence was made on replicate batches of tissue over several concentrations. Fig. 3 indicates that 4 5 C a influx increases with concentration but not proportionately over the whole range from 20-200 gM; it also indicates that the flux is significantly less with 1 m M external potassium (Ko) than with either 10 or 20 m M Ko, as might be expected if the Ca-channel is voltage-sensitive (as is the channel in Chara), and opens with depolarisation at high Ko.

Effect of 100 BM ABA on 4SCa influx. A primary aim of this work was to test the hypothesis that

ABA stimulated C a 2 + influx to guard cells, a hypothesis indicated by the sensitivity of stomatal closing, or of inhibition of stomatal opening, to external Ca 2 +. In most experiments the influx was measured over 0-2, 0 4, or 2 4 min of A B A treatment, although it is quite possible that there could be a real ABA effect which is not measurable over this time course - for example, a short period of stimulated influx which is over too quickly to show up with this protocol. The results are unsatisfactory in that there was, in some experiments, a significant stimulation of 4SCa influx in the presence of ABA (albeit a relatively small one), but in other experiments there was either no significant effect of ABA, or indeed a small inhibition in the pres-

236

E.A.C. MacRobbie: Effects of ABA on Ca 2 + influx into guard cells

0.4

~.,~,ATT

0.2 E

~

?

E

~

I

o

Dn7

DIT

DI

QbOe~(~

I

I

I

I

I

i

0

E 0.~ x U.

z

cI 0.2

I~ 0

I

1.00

I

12.00

BT i

V~v

I

13 O0 14.00 TIME

Brr

l

I

!

I

15.00

16.00

17.00

18.00

Fig. 4. Effect of time of day on 45Ca influx into Comrnelina guard cells with and without 0.1 mM ABA, in four different experiments ( A D ) . After isolation, all batches of strips were incubated overnight in non-radioactive bathing solution, to reach a steady state. Influxes were measured over 2 or 4 min at the times shown during the following day, in the absence (open symbols) or presence (filled symbols) of ABA. Batches from different plants in the same experiment, on the same day, are indicated by the same letters (A-D). Lines join measurements at different times of day in replicate batches of strips from the same leaf (or occasionally from two leaves from the same plant). Each point represents the mean of four to six strips (except last points _+ABA in CII in which there were only three), and bars show calculated SE. The bathing solutions contained: A, D, 20 mM KC1, 20 gM CaSO4; B, 10 m M KCI, 20 gM CaSO4; CI, 20 mM KC1, 100 gM CaSO4; CII, 20 mM KC1, 200 gM CaSO4. In A, B, C a non-significant effect of ABA is replaced, later in the day, by a significant stimulation. In D significant inhibition by ABA is replaced, later in the day, by no significant effect

ence of ABA. In these experiments great care was taken to match strips in the two treatments; as strips were removed from a single leaf (or occasionally from two leaves from the same plant), they were allocated alternately to the control and to the ABA-treatment set. Different batches of strips in the same experiment, from different leaves (or from different plants), were kept separate. An attempt was made to identify solutions in which an effect could be seen consistently, by varying external Ca 2 § and K +, but without success. The results are collected in Table 3, separated into three groups, showing significant stimulations, no significant effect, and significant inhibition. There is some evidence in Table 3 that 4 5 C a influx is more likely to show stimulation by ABA in the afternoon than in the morning, since all the positive stimulations were observed when the influx was run in the afternoon. In two experiments in Table 3 (18/6 and 29/7) there was stimulation by ABA in the batches of cells run in the afternoon, but not in the morning; in a third experiment (21/

8) there was stimulation in batch II run in the afternoon, but none in batch I, split between morning and afternoon. The effect is seen most strongly in the experiment 18/6, when batch II was run in the afternoon, and batch I split between morning and afternoon. In batch-I cells the control influx did not differ between morning and afternoon, although the flux was inhibited by ABA in the morning but stimulated in the afternoon, both by significant amounts ( P < 0 . 0 2 and 0.01, respectively). In the batch-II cells, run in the afternoon, there was stimulation by ABA. This difference between morning and afternoon is shown in Fig. 4 which plots the 4SCa influx, with and without ABA, against time of day in various experiments (A-D), and for different batches of cells (I, II, and in one case also III) utilized on the same day. It is clear that a stimulation by ABA can occur later in the day, when earlier cells show no effect (experiments A, B, C), or that in experiment D an early inhibition by ABA (batch I) is replaced by no significant effect (batches II and III). In these experiments it

E.A.C. MacRobbie: Effects of ABA

on Ca 2 +

influx into guard ceils

237

Table 4. Effect of ABA on 4SCa influx over short times into guard cells of Commelina. The external solution contained 1 mM KC1, 50 pM CaSO4, pH 6. Each batch of strips came from two to three leaves from the same plant, but all three batches were run on the same day, with the same isolation procedure and pre-treatment.

Table 5. Estimates of 45Ca influx into guard cells of Commelina on the basis of guard-cell area. Calculations are based on 120 guard cells, m m - z of epidermal strip and a guard-cell area of 20.i0 -6 cm 2. Results from Table 2 and Fig. 3. Figures in 0 are the number of experiments

The combined regressions of tracer content (Q*) against time (t) for batches II and III are: - A B A : Q* =(0.011 +_0.008)+(0.050+0.0035) t 26 d.f. + ABA: Q * = (0.013 __.0.007) + (0.046 +_0.003) t 26 d.f.

Cao (gM)

Batch

Uptake time (min)

45Cainflux (pmol-mm-2-min 1) -ABA

I II

fII

1 0.5 1 2 2 4

0.053_+0.004 0.058 _+0.004 0.066_+0.005 0.062+0.005 0.054_+0.004 0.052_ 0.004

+ A B A (0.1 mM) (6) (6) (5) (6) (6) (6)

0.054_+0.005 0.061 -t-0.005 0.062• 0.053_+0.004 0.058+0.004 0.049 _+0.003

(6) (6) (5) (5) (6) (6)

is not clear whether this represents an endogenous rhythm in response to ABA, or an effect of total time since isolation, but there is clearly some longterm time dependence of the effect, reflecting some internal change in the guard cells which alters the response of their Ca 2 § influx to ABA. Since it is possible that a very fast short-lived response of Ca 2 § influx would not be seen in these experiments a final attempt was made to look at effects over as short a time course as possible. The influx from 50 gM Cao, I m M Ko, was measured over 0.5 to 4 min, with and without 0.1 mM ABA, to test whether an initial stimulation could be picked up, followed by a return to normal influx, or by an inhibition. There was no evidence that this was so, as is seen in Table 4. The time course was linear through the origin for both sets, and there was no significant difference between the ABA-treated cells and the controls, whether considered as mean influxes at various time points in the replicate sets of cells ( + A B A ) within each batch of cells, or as the combined regression against time, combining different batches of cells. Discussion For comparison with other cells it is necessary to convert the measured Ca 2 + influxes, on the basis of area of epidermal strip, to influxes on the basis of guard-cell area. For this purpose a guard-cell area of 20.10-6cm 2 may be used, as being a reasonable estimate for guard cells of the average aperture in these experiments; for wide-open guard cells a figure of 25.10-6 cm 2, and for nearly closed guard cells a lower figure, may be more appropriate, but the area is in any case not very accurate-

20 50 50 100 100 200

Ko (mM)

10-20 20 1 20 1 20

Estimated 4SCa influx (pmol. cm-2. s- 1) Range

Mean

0.3 -1.0

0.69 0.84 0.37 1.6 0.99 1.9

0.3

0.43

0.93 1.1 1.4 -2.3

(17) (1) (3) (1) (3) (3)

ly known, and a middle-range estimate may be used. (Area estimations are based on measurements of guard-cell dimensions at different apertures from earlier work, MacRobbie 1980.) With 120 guard cells per mm z of epidermal strip this comes to 2.4.10-3 c m 2 of guard-cell area per mm 2 of epidermal strip, and thus a flux of 1 pmolmm - 2. min - 1 is equivalent to a plasmalemma flux of 6.9 p m o l . c m - 2 . s -1. The estimates of plasmalemma influx thus obtained from the results in Table 2 and Fig. 3 are shown in Table 5. These figures are comparable to the Ca 2 + influxes measured in Chara (MacRobbie 1988c). At 100 gM external Ca 2§ and low external K +, the basal influx in Chara was about 0 . 2 5 - 0 . 5 p m o l . c m - 2 - s -~, but was up to 1 . 6 p m o l . c m - 2 . s -~ in one batch of cells; in high external K +, in depolarised cells the Ca 2+ influx from 100 gM Ca 2+ was in the range 0 . 4 4 - 2 . 2 p m o l . c m - 2 . s -1. Thus the guard-cell fluxes for Commelina at 100 gM Cao, at either 1 mM Ko or 20 mM Ko, are within the range found for Chara . The guard-cell fluxes at 20 gM Ca~ are probably somewhat higher than would be expected for Chara at that concentration, but there is rough comparability between the two cells. It is clear that guard cells, like Chara, have Ca z+ influxes which are much higher than the earlier accepted estimates for Chara (Spanswick and Williams 1965) of about 0 . 0 4 p m o l . c m - 2 . s - < It should be noted that the influxes measured are much higher than rates of Ca 2 + uptake measured in membrane vesicles of various kinds; such uptake corresponds to active transport of Ca 2+ out of the bulk cytoplasm (to outside, to endoplasmic reticulum or to vacuole), but in the steady state the rates of active transport out of the cytoplasm must be capable of matching the passive inflow of Ca 2 § (See Table 6 in MacRobbie (1988c) for the figures for rates of transport in vesicles, collected from the literature.) Such figures show that the discrep-

238

E.A.C. MacRobbie: Effects of ABA on Ca z+ influx into guard cells

Table 6. Effect of bepridil on Ca influx in Chara pb

Ko Bepridil PreUptake 45Ca influx a (mM) (gM) treatment time in bepridil (rain) (rain) Expt. I 0.4 0.4 0.4

0 10 50

30 J0

10 10 10

0.236_+0.016 (8) 0.32 +0.03 (8) 0.16 _+0.01 (7)

0.001 0.01

20 20 20

0 10 50

25 10

I0 10 10

0.39 _+0.02 (8) 0.51 +0.02 (8) 0.43 +0.08 (8)

0.001 N.S. r

0 10 50 50

20 0 10

10 10 10 10

0.20 0.27 0.31 0.23

0.001 0.01 N.S.

Expt. II 0.4 0.4 0.4 0.4

_+0.01 _+0.0I +0.03 -+0.03

(8) (8) (7) (7)

a Influx measured as in MacRobbie (1988c), using La-wash techniques b Significance level for comparison with control flux in the absence of bepridil c Not significant

ancy is particularly marked for the plasmalemma, where the rates of Ca uptake into vesicles at I ~tM Ca 2 + is only a few fmol. c m - 2. s 1, indicating that the proportion of inside-out vesicles in the plasmalemma preparation is very low, and-or that the vesicles are very leaky. The fall-off in apparent influx with time implies that a back-flux of tracer develops as cytoplasmic stores fill up. What is in fact being measured will depend on the relative fluxes out of the bulk cytoplasm to internal stores (endoplasmic reticulum, mitochondria, chloroplasts, vacuole), and to the outside solution. Provided 45os, the summed fluxes to all internal stores including the vacuole, is greater than ~bco, the efflux at the plasmalemma, then the measured influx will be a good estimate of the true plasmalemma influx. In any case, the measured influx is a minimum estimate of the true influx. The evidence available indicates that ~bo~ is likely to be considerably greater than ~co, the plasmalemma efflux. (See figures collected from the literature for C a 2+ fluxes in various isolated membrane fractions, by Blumwald and Poole (1986), or Table 6 of MacRobbie (1988c).) It seems likely, therefore, that the measured fluxes represent a good approximation to the influx at the plasmalemma. In the single experiment where the fall-off in apparent influx with time was measured (Fig. 2) the apparent pool size was 1.37 pmol. r a m - 2 with an apparent rate constant of about 3.4.h-1. On the basis of guard-cell area the apparent pool size

is about 570 pmol. c m - 2, or about 11 fmol per cell; if the cytoplasmic volume is about 30% of the cell volume (or 50% of the closed cell volume) this will correspond to an apparent concentration of about 5.7 m M averaged over the whole cytoplasmic volume. This is rather higher than the average apparent pool size in Chara, (MacRobbie 1988c), which was only about 1.5 m M averaged over the cytoplasm, as the mean of four values. However, in a further experiment in Chara, in which the initial influx was higher, more like that measured here in guard cells, the apparent pool size was also higher at 3.9 mM. While most of the apparent pool must reflect Ca z + sequestered in the endoplasmic reticulum, and other cytoplasmic organelles, it is also possible that with the smaller size of guard cells a back-flux will develop more quickly from the vacuole, and the apparent pool size will also have a vacuolar contribution. The time course has not been accurately established in guard cells, but is within the range that might be expected, if reasonably similar to Chara. The work with ABA was undertaken to test a speculative hypothesis that ABA had a primary action on a Ca-channel in the plasmalemma, stimulating Ca 2 + influx, leading to depolarisation and an increase in cytoplasmic Ca z +, with consequent effects on other Ca-sensitive and voltage-sensitive ion channels in both plasmalemma and tonoplast. The work has not provided a clear-cut answer to this question. It has ruled out consistent and sustained stimulation of C a 2+ influx by ABA, but does not rule out a short-lived stimulation followed by secondary responses. It is worth considering first the results, then the possible interpretations. The results with ABA are clearly unsatisfactory in that the effect between experiments is so variable, although within a given batch of cells the effect is consistent. Neither external C a 2 + nor external K + seem to determine the response to ABA. While it might be possible to continue the variation in time course, external solutions, pre-treatment of tissue etc. to try to establish a set of conditions in which a reproducible and consistent ABA effect could be obtained (of any kind), it is difficult to identify the hidden variables. The results establish that ABA can sometimes stimulate the Ca 2+ influx, by 28-79%, sometimes has no significant effect, and in other instances can inhibit the Ca 2+ influx, by 16 29%. Thus some internal condition of the tissue, not identified and not controlled, also affects the response. Time of day (or time since isolation) may affect the response, but this effect is not clear-cut. Nevertheless, in spite of the unsatisfactory variation between experiments, it seems

E.A.C. M a c R o b b i e : Effects o f A B A o n C a 2 § influx into g u a r d cells

worth presenting and discussing these results since they rule out a straightforward, long-lived, stimulation of Ca 2 § influx by ABA as the primary response, as the explanation of the Ca-dependence of the sensitivity of the guard cells to ABA, established by De Silva et al. (1985a, b). One possibility is that there is consistently a genuine stimulation of Ca 2 + influx by ABA, but that it is short-lived, and that the time course is variable between experiments. One way in which this might arise is by down-modulation of the Cachannel by an increase in cytoplasmic Ca 2 § resulting from initial stimulation of Ca 2 § influx, perhaps also reinforced by release of Ca 2 § from internal stores. In this situation the period of elevated cytoplasmic Ca 2§ and of Ca-dependent effects on other ion channels, would be much longer than the period of high Ca 2§ influx. Such inhibition of Ca-channels by high cytoplasmic Ca 2 § has not only been observed in animal cells, but also in the plasmalemma of Nitellopsis (Zherelova et al. 1987). There is some evidence that treatment of Chara with low concentrations of bepridil, which binds to Ca-channels, gives a stimulation of Ca 2 § influx, but at higher concentrations an initial stimulation is followed by inhibition, as shown in Table 6. An effect of (presumed) increasing cytoplasmic Ca 2 § is the simplest explanation of such observations. If an explanation of this kind is to be applied to guard cells after ABA treatment then it would be necessary to attribute the observed variability of response to variations in the extent and timecourse of the changes in cytoplasmic Ca 2 § which follow an increase in Ca 2 § influx. Such variability might be a consequence of variability in the fluxes of Ca 2 § out of the cytoplasm to different compartments. It is not argued that this is the case, merely that it is possible to imagine that a consistent initial stimulation of Ca 2§ influx by ABA treatment could nevertheless result in the variability of effects observed. While the results rule out a prolonged stimulation of Ca 2 + influx in the presence of ABA, they do not rule out a short-lived transient stimulation of Ca 2 § influx, with longer lasting Ca-dependent effects on K-channels, and on anion channels. Measurements of cytoplasmic Ca 2 § levels, with a Ca-electrode or by a fluorescent indicator such as Fura-2, are required to establish whether A B A does, or does not, result in an increase in cytoplasmic Ca 2§ Clarkson et al. (1988) have made such measurements in root-hair cells, and found no effect of ABA, but this does not rule out an effect of ABA on cytoplasmic Ca 2 + levels in guard cells; only measurements on guard cells could settle the question, and these have not yet been made.

239

Alternatively, if the effect of ABA is not mediated via Ca-channels, then the dependence of the ABA effect on external Ca must be explained in some other way. Schauf and Wilson (1987) showed that ABA altered the kinetics of the outwardly rectifying K+-channels in isolated patches of guardcell plasmalemma, producing long bursts of opening. This implies either a direct effect of ABA on the K § or an indirect effect on K-channel gating, mediated by some other membrane protein. An analysis of whole-cell current-voltage relationships of the plasmalemma of Vicia guard cells shows that ABA reduces current through the inward-rectifying K+-channel (open at negative membrane potentials), but stimulates current through the outward-rectifying K +-channels which open on depolarisation. (personal communication from M.R. Blatt, Botany School, Cambridge). In whole cells this could reflect either direct effects on the K-channels, or indirect effects involving either soluble proteins or other membrane proteins. Sensitivity of K-channel kinetics to external Ca 2+ should be investigated, as one possible way of conferring Ca-sensitivity to the ABA response. Schroeder (1988) argues that the outward-rectifying K+-channels are responsible for K § release during stomatal closure, but points out that it is still necessary to establish a mechanism for producing the necessary depolarisation to activate the K § gating. He argues that opening of stretch-activated channels might play this role, leading to release of anions from the cell and consequent depolarisation. Edwards and Pickard (1987) give evidence for the occurrence of such channels in plant cells, and Schroeder (1988) also discusses their existence. However, if the ABA response is mediated by a primary effect on stretchactivated channels, its Ca-dependence would suggest that such channels should be Ca-dependent. This is probably unlikely, in view of the insensitivity to Ca 2 § of stretch-activated channels in animal cells (Brehm et al. 1984; Guharay and Sachs 1984). Thus it remains uncertain how ABA could lead to sufficient depolarisation to activate the outward-rectifying K§ even if ABA did have a direct effect on K§ opening. The merit of a hypothesis involving Ca-channels and changes in cytoplasmic Ca 2 § is that it is plausible to imagine consequent effects on Ca-dependent and voltage-dependent channels in both plasmalemma and tonoplast, producing both the triggering and all the observed effects on fluxes of R b + ( K +) and B r - ( C l - ) . If ABA does not affect the Ca-channel directly then there remains the problem of explaining how

240

there can be effects of ABA o n Ca 2 + influx, either stimulation or inhibition. One possibility is that the small effects observed reflect small changes in membrane potential, the result of opening some other ion channel, or of an effect on the proton pump. There is evidence that ABA partially inhibits H + efflux from epidermal strips (Gepstein et al. 1982), or from guard-cell protoplasts (Shimizaki et al. 1986; Rashke 1987), but it is not clear whether this reflects a direct effect on the proton pump or an indirect effect via an effect on an ion channel. If the Goldman equation is used as a rough approximation for the potential dependence of the influx, then for negative membrane potentials the C a 2+ influx will be roughly proportional to the membrane potential (for Em more negative than even 29 mV the term (l-exp Z F E / R T ) is greater than 0.9, and can be taken as 1.) Thus small changes in membrane potential could produce small changes in C a 2 + influx (increase on hyperpolarisation or decrease on depolarisation), without any dramatic change in Ca-channel gating being involved. A final point may be made about the nature of the ABA effect on ion fluxes in guard cells. In the tracer experiments the stimulation of efflux of anions and cations on adding ABA is strictly transient, and after a period of high ion loss the efflux declines again, with the rate constant for Rb + efflux similar to the value before ABA treatment. Thus ABA does not simply increase the permeability to both anions and cations. Instead it looks rather more like a change in the 'set point' to which the ion content is regulated, and when ion loss has reduced the content to a new set-point the high efflux declines. A similar argument can be advanced to explain the effects of ABA on protoplast volume, as observed by Fitzsimons and Weyers (1987). In that study, ABA induced protoplast shrinkage, but only in guard cells which had previously swelled in KC1; the K concentration was 108 m M in the ABA-treated protoplasts (2.4pl volume), compared with 145 m M after swelling in the absence of ABA (2.8 pl). Thus ABA induced loss of 'excess' salt content but did not induce leakage below some basal level, with other conditions of the cell setting the definition of what is 'excess'. This indicates that ABA acts through interaction with the regulatory systems, in ways not yet understood. In Characean cells the hypoosmotic regulatory response, in which ions are lost to reduce excessive turgor, does involve C a 2 + influx, and transient increase in cytoplasmic C a 2+, with consequent effects on K-channels (Okazaki and Tazawa 1987). The same may still be true of guard cells.

E.A.C. MacRobbie: Effects of ABA on Ca 2+ influx into guard cells

The present work does not establish how ABA acts. It rules out a long-lasting stimulation of C a 2 + influx as the primary action of ABA, but it does not rule out an action mediated by C a 2 +, and increase in cytoplasmic C a 2+ levels, with secondary regulatory effects o n C a 2 + influx obscuring the initial stimulation. Measurements of cytoplasmic C a 2+ levels after A B A treatment are required to test this. The 'Ca-hypothesis' remains an attractive, speculative hypothesis, in need of firm evidence for or against its operation. The support of the Science and Engineering Research Council is gratefully acknowledged. Thanks are due to Mr J. Banfield for technical assistance.

References Blumwald, E., Poole, R.J. (1986) Kinetics of Ca 2+/H § antiport in isolated tonoplast vesicles from storage tissue of Beta vulgaris L. Plant Physiol. 80, 727-731 Brehm, P., Kullbery, R., Moody-Corbett, F. (1984) Properties of non-functional acetylcholine receptor channel on innervated muscle ofXenopus laevis. J. Physiol. 350, 631-648 Brownlee, C., Wood, J.W. (1986) A gradient of cytoplasmic free calcium in growing rhizoid cells of Fucus serratus. Nature 320, 624-626 Brownlee, C., Wood, J.W., Briton, D. (1987) Cytoplasmic free calcium in single cells of centric diatoms. The use of Fura-2.. Protoplasma 140, 118 122 Clarkson, D.T., Brownlee, C., Ayling, S.M. (1988) Cytoplasmic calcium measurements in intact higher plants cells: results from fluorescence ratio imaging of fura-2. J. Cell Sci. 91, 71 80 De Silva, D.L., Cox, R.C., Hetherington, A.M., Mansfield, T.A. (1985 a) Suggested involvement of calcium and calmodulin in the responses of stomata to abscisic acid. New Phytol. 101, 555-563 De Silva, D.L.R., Hetherington, A.M., Mansfield, T.A. (1985b) Synergism between calcium ions and abscisic acid in preventing stomatal opening. New Phytol. 100, 473-482 Edwards, K.L., Pickard, B.G. (1987) Detection and transduction of physical stimuli in plants. In: The cell surface in signal transduction, pp. 41 66, Wagner, E., Greppin, H., Millet, B., eds. (NATO ASI Series, vol. H 12) Springer-Verlag, Heidelberg Fitzsimons, P.J., Weyers, J.D.B. (1986) Volume changes of Commelina communis guard cell protoplasts in response to K +, light and CO2. Physiol. Plant. 66, 463-468 Fitzsimons, P.J., Weyers, J.D.B. (1987) Responses of Commelina eommunis L. guard cell protoplasts to abscisic acid. J. Exp. Bot. 38, 99~1001 Fischer, R.A. (1972) Aspects of potassium accumulation by stomata of Viciafaba Aust. J. Biol. Sci. 25, 1107 1123 Fujino, M. (1967) Role of adenosinetriphosphate and adenosinetriphosphatase in stomatal movement. Sci. Bull. Fac. Educ., Nagasaki Univ. 18, 1-47 Gepstein, S., Jacobs, M., Taiz, L. (1982) Inhibition of stomatal opening in Viciafaba epidermal tissue by vanadate and abscisic acid. Plant Sci. Lett. 28, 63-72 Gilroy, S., Hughes, W.A. Trewavas, A.J. (1986) The measurement of intracellular calcium levels in protoplasts from higher plant cells. FEBS Lett. 199, 217-221 Guharay, F., Sachs, F. (1984) Stretch activated single ion channel currents in tissue-cultured embryonic chick skeletal muscle. J. Physiol. 352, 685-701

E.A.C. MacRobbie: Effects of ABA on Ca z + influx into guard cells Hedrich, R., Neher, E. (1987) Cytoplasmic calcium regulates voltage-dependent ion channels in plant vacuoles. Nature 329, 833 836 lnoue, H., Katoh, Y. (1987) Calcium inhibits ion-stimulated stomatal opening in epidermal strips of Commelina communis L. J. Exp. Bot. 38, 142-149 MacRobbie, E.A.C. (1980) Osmotic measurements on stomatal cells of Commelina communis L. J. Membr. Biol. 53, 189 198 MacRobbie, E.A.C. (1981 a) Ion fluxes in isolated guard cells of Commelina comrnunis L. J. Exp. Bot. 32, 545 562 MacRobbie, E.A.C. (1981 b) Effects of ABA in 'isolated' guard cells of Commelina communis L. J. Exp. Bot. 32, 563-572 MacRobbie E.A.C. (1983) Effects of light/dark on cation fluxes in guard cells of Commelina communis L. J. Exp. Bot. 34, 1695-1710 MacRobbie, E.A.C. (1984) Effects of light/dark on anion fluxes in 'isolated' guard cells of Commelina communis L. J. Exp. Bot. 35, 707-726 MacRobbie, E.A.C. (1986) Calcium effects in stomatal guard cells In: Molecular and cellular aspects of plant development, pp. 383-384, Trewavas, A.J., ed. Plenum Press, New York MacRobbie, E.A.C. (1988a) Stomatal guard cells. In: Solute transport in plant cells and tissues, pp. 453-497, Baker, D.A., Hall, J.L., eds. Longman, Harlow, Essex, UK MacRobbie, E.A.C. (1988b) Control of ion fluxes in stomatal guard cells. Bot. Acta 101, 140-148 MacRobbie, E.A.C. (1988c) Calcium influx at the plasmalemma of Chara coralIina. Planta 176, 98 108 Miller, A.J., Sanders, D. (1987) Depletion of cytosolic free calcium induced by photosynthesis. Nature 326, 397 399 Okazaki, Y., Shimmen, T., Tazawa, M. (1984) Turgor regulation in a brackish charophyte, Lamprothamnium succinctum. II. Changes in K +, Na +, and C1- concentrations, membrane potential and membrane resistance during turgot regulation. Plant Cell Physiol. 25, 573 581

241 Okazaki, Y., Tazawa, M. (1987) increase in cytoplasmic calcium content in internodal cells of Lamprothamnium upon hypotonic treatment. Plant Cell Environ. 10, 619-621 Raschke, K. (1987) Action of abscisic acid on guard cells. In: Stomatal function, pp. 253-279, Zeiger, E., Farquar, G.D., Cowan, I.R., eds. Stanford University Press, Stanford Schauf, C.L., Wilson, K.J. (1987) Effects of abscisic acid on K + channels in Viciafaba guard cell protoplasts. Biochem. Biophys. Res. Commun. 145, 284-290 Schroeder, J.I. (1988) Potassium transport properties of potassium channels in the plasma membrane of Vicia faba guard cells. J. Gen. Physiol. 92, 66%683 Schroeder, J.I., Raschke, K., Neher, E. (1987) Voltage dependence of K + channels in guard cell protoplasts. Proc. Natl. Acad. Sci. USA 84, 41084112 Schwartz, A. (1985) Role of Ca z+ and EGTA on stomatal movements in Commelina communis L. Plant Physiol. 79, 1003-1005 Shimizaki, K., Iino, M., Zeiger, E. (1986) Blue-light-dependent proton extrusion by guard cell protoplasts of Vicia faba. Nature 319, 324-326 Spanswick, R.M., Williams, E.J. (1965) Ca fluxes and membrane potentials in NitelIa translucens. J. Exp. Bot. 16, 463473 Williamson, R.E., Ashley, C.C. (1982) Free C a 2 + and cytoplasmic streaming in the alga Chara. Nature 296, 647-650 Witlmer, C., Mansfield, T.A. (1969) A critical examination of the use of detached epidermis in studies of stomatal physiology. New Phytol. 68, 363-375 Zherelova, O.M., Kataev, A.A., Berestovsky, G.N. (1987) Intracellular calcium control of calcium in Nitellopsis obtusa plasmalemma. Biofizika 32, 348 350

Received 18 August; accepted 30 November 1988