The role of the plasma-membrane Ca2+-ATPase in ca2+ homeostasis in Sinapis alba root hairs

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The role of the plasma-membrane Ca2+-ATPase in Ca2+ homeostasis in Sinapis alba

root hairs

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PLANTA · SEPTEMBER 1992

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Planta (1992)188:306-313 P l a n t a 9 Springer-Verlag 1992

The role of the plasma-membrane Ca +-ATPase

in Ca 2§ homeostasis

in

Sinapis alba

root hairs

Hubert H. Felle*, Andrzej Tretyn**, and Gottfried Wagner

Botanisches Institut I der Justus-Liebig-Universiffit, Senckenbergstrasse 17-21, W-6300 Giessen, Federal Republic of Germany Received 10 March; accepted 27 May 1992

Abstract. The regulation o f cytosolic Ca 2+ has been

investigated in growing root-hair cells o f Sinapis alba L. with special emphasis on the role o f the plasma- m e m b r a n e C a 2 + - A T P a s e . F o r this purpose, erythrosin B

was used to inhibit the Ca2+-ATPase, and the C a 2+

ionophore A23187 was applied to manipulate cytosolic free [Ca 2 +] which was then measured with Ca 2 +-selective microelectrodes. (i) At 0.01 ~M, A23187 had no effect on the m e m b r a n e potential but enhanced the C a 2+ per- meability o f the plasma membrane. Higher concentra- tions o f this i o n o p h o r e strongly depolarized the cells, also in the presence o f cyanide. (ii) Unexpectedly, A 23187 first caused a decrease in cytosolic C a 2+ by 0.2 to 0.3 pCa units and a cytosolic acidification by a b o u t 0.5 p H units. (iii) The depletion o f cytosolic free Ca 2 + spontaneously reversed and became an increase, a process which strong- ly depended on the external Ca 2+ concentration. (iv) U p o n removal o f A231s 7, the cytosolic free [Ca 2+] re- turned to its steady-state level, a process which was inhibited by erythrosin B. We suggest that the first reac- tion to the intruding Ca z+ is an activation o f Ca z+ transporters (e.g. ATPases at the endoplasmic reticulum and the plasma membrane) which rapidly remove Ca 2 + from the cytosol. The two observations that after the

addition o f A23187, (i) Ca 2+ gradients as steep as

- 600 mV could be maintained and (ii) the cytosolic p H

rapidly and immediately decreased without recovery in- dicate that the Ca 2 +-exporting plasma-membrane ATPase is physiologically connected to the electrochemi- cal p H gradient, and probably works as an n H + / C a 2+- ATPase. Based on the finding that the Ca2+-ATPase inhibitor erythrosin B had no effect on cytosolic Ca 2+, but caused a strong Ca 2 + increase after the addion o f A23187, w e conclude that these cells, at least in the short Abbreviations and symbols." EB=erythrosin B; Era=membrane potential; pCa = negative logarithm of the Ca 2 + concentration * To whom correspondence should be addressed; FAX: 49 (641) 702 8464

** Permanent address: N. Copernicus University, Institute of Biol- ogy, Gagarina 9, P~87 100 Turun, Poland

term, have enough metabolic energy to balance the loss in transport activity caused by inhibition o f the primary Ca 2 +-pump. We further conclude that this ATPase is a major Ca 2 + regulator in stress situations where the cy- tosolic Ca 2 + has been shifted from its steady-state level, as may be the case during processes of signal transduc- tion.

Key words: ATPase (calcium, plasma membrane) - Cal-

cium regulation - Cytosol (calcium, pH) - I o n o p h o r e

(A23187) - Plasma membrane - Sinapis (calcium homeo-

stasis)

Introduction

F r o m the literature on animal cells, cytosolic free C a 2 + is known to be a link in numerous intracellular processes and responses, and it is generally regarded a second messenger o f signal-transduction pathways (Campbell 1983; Carafoli 1987). In recent years, evidence has accu- mulated that this ion m a y play a similar role in plants (Gilroy and Trewavas 1990), although their sessile lifestyle may require different strategies to cope with the various external stimuli. In order to learn more a b o u t the role o f cytosolic Ca 2 + in plant signal transduction (He- pler and W a y n e 1985; Bush et al. 1989), metabolism (Kauss 1987) or gene expression (Thompson and White 1991), the regulatory components leading to Ca 2+ ho- meostasis have to be investigated. In particular, cytosolic C a 2 + has to be measured under conditions where regula- tion is required. This can be accomplished through ex- perimentally manipulating the resting Ca 2 + level, as well as forcing the cells to react to different stimuli. In doing this, Ca z + ionophores (Pressman 1976; Campbell 1983) and Ca2+-ATPase inhibitors (e.g. erythrosin B; Rasi- Caldogno et al. 1989a, b; Evans et al. 1991) can be indispensible tools.

In an attempt to investigate cellular Ca / + homeostasis in plant cells, we tested these agents and focussed our

H.H. Felle et al. : Ca 2+ homeostasis in plant cells

i n t e r e s t o n t h e r e a c t i o n o f the p l a s m a - m e m b r a n e C a 2 +- A T P a s e to p e r t u r b a n c e s o f c y t o s o l i c C a z +. F o r this p u r -

p o s e t h e t i p - g r o w i n g r o o t - h a i r cells o f Sinapis alba

p r o v e d a f a v o u r a b l e q u a s i - u n i c e l l u l a r test s y s t e m .

M a t e r i a l and m e t h o d s

General conditions. Seedlings of Sinapis alba L. (Fa. Hesemann, Giessen, FRG) were grown for 2-3 d on filter paper which was soaked with tap water. The excised roots were mounted in a Plexi- glas chamber which was constantly perfused with the basic test medium. This comprised 0.1 mM KC1, 0.I mM NaCI, 0.1 mM CaClz and a mixture of 5 mM of 2-(N-morpholino)ethanesulfonic acid/Tris, adjusted to pH 6.1. After 2-4 h the roots were equili- brated and had grown new root hairs. Only these were used for the experiments. AU deviations from these conditions are given in the figure legends.

The Ca 2+ ionophore A23187 w a s obtained from Calbiochem

(Frankfurt, FRG) and erythrosin B (EB) from Sigma (Deisenhofen, FRG).

Electrophysiology and ion-selective microelectrodes. The electrical setup for the impalement of root hairs and for measurements of membrane potential (Era) has been described previously (Felle 1982, 1987). The test chamber is open on both sides and allows the simultaneous horizontal approach of two separate electrodes. The fabrication and intracellular use of the pH- and CaZ+-sensitive microelectrodes has been described in detail recently (Felle and Bertl 1986; Felle 1989). These electrodes were connected to high- impedance amplifiers (FD 223 or Duo 773; WP-Instruments, Sar- asota, Fla., USA).

Presentation of the data and stat&tics. Since the ion-selective mi- croelectrodes primarily record the voltage sum of the free-ion con- centration plus Era, the signal of the voltage electrode was simul- taneously subtracted (differential amplifier) from the signal of the

307 ion-selective electrode to obtain the net free-ion concentration (for a detailed description, see Felle and Bertl 1986). For the sake of clarity in presentation, only the traces of the free-ion concentrations are shown or, wherever interesting to the argument, the net trace and the Em are given in the figures. The number of experiments is stated in the figure legends. Typical chemical time resolutions (T1/2) of the ion-selective electrodes were 2-5 s for the pH-electrode and 5-8 s for the Ca 2 +-electrode.

Since cytosolic changes of pH and Ca 2 + were recorded, the sensitivities of these electrodes were cross-checked. All calibrations were carried out in solutions containing 100 mM K + which closely represents the cytosolic [K +] of the root-hair cells. The Ca 2 +-elec- trodes gave a reading of 1-3 mV in response to pH changes from 6 to 8; the pH-electrode did not react at all to changes in pCa from 5 t o 7.

R e s u l t s

Effect o f the Ca 2+ ionophore A23187 on the plasma- membrane potential

of Sinapis

alba root hairs. F i g u r e 1A s h o w s t h e effect o f d i f f e r e n t Az3187 c o n c e n t r a t i o n s o n t h e

Em o f f r e s h l y g r o w n S. alba r o o t h a i r s . I n the p r e s e n c e o f

0.1 m M e x t e r n a l C a 2+, 0.01 ~tM A23187 h a d h a r d l y a n y effect o n Era, b u t it m a r k e d l y s t e p p e d - u p t h e s e n s i t i v i t y o f t h e p l a s m a m e m b r a n e t o C a z +, T h i s is s h o w n in F i g . 18 w h e r e , a f t e r t h e a d d i t i o n o f 0.01 ~tM Az3~87, the cells

r e a c t e d to a n i n c r e a s e in e x t e r n a l Ca z§ f r o m 0.1 t o 10 m M b y a t w o t o t h r e e f o l d e n h a n c e d d e p o l a r i z a t i o n , in spite o f t h e p r o t o n - p u m p a c t i v i t y w h i c h t e n d s t o re- p o l a r i z e t h e cells. I n o r d e r t o s h o w t h e i m p a c t o f this i o n o p h o r e o n p a s s i v e m e m b r a n e p a r a m e t e r s o n l y , t h e p r o t o n p u m p w a s d e a c t i v a t e d b y c y a n i d e ( F i g . 1C). I t is k n o w n t h a t c y a n i d e d e p r e s s e s t h e Em to t h e s o - c a l l e d d i f f u s i o n p o t e n t i a l w h i c h b a s i c a l l y d e p e n d s o n the i o n g r a d i e n t s p r e s e n t o n e a c h side o f t h e p l a s m a m e m b r a n e

- oo[

|

~_>E -t50[

-6

_4-

200 ~

_{i q}

c

0.01

0.1

1 ~Jl~ A23187

E -50

o

-6

C3L

(~

~ -I00

'- -100r-

2 rain

( ~ )

~

E

-150[ ~ A 2 3 1 8 7

~=~'-

wEP

-150

-200

,

J

E -200

I I I I | I 9 I ,I -I

0.1 10

0.1

10

0.1

externa[ Ca2* / rnH

0

9

6 r a i n _i

NaCN ~

/

2L

Fig. 1A-C. Effect of the Ca 2 + ionophore A 23 t 87 on the E~ of freshly

grown root hairs ofSinapis alba. A Different A23187 concentrations,

as indicated, were added in the presence of 0.1 mM external Ca 2 +. B Effect of 10 mM Ca 2+ on the E m before and after the addition

0.1

I I I I I I I I I I I I I ,I

10

~ 0.1

of 0.01 ~tM A231s 7. C Action of 0.5 pM A23ts 7 o n the diffusion

potential in the presence of 1 mM NaCN and at the indicated external Ca 2 + concentrations. Representative curves of six to ten equivalent measurements

308 5 @ ~ 10 0.5

pM

y

m M

Co

2+

A 23187

- - 0.1

~ - -

0.01

7

()

5

10 min

Fig. 2. A Effect of 0.5 gM A23187 on cytosolic free Ca 2+ (pCac), measured with a CaZ+-selective microelectrode. The curves are

individual reactions of different Sinapis alba root hairs to the ap-

plication of the ionophore in the presence of different external Ca 2 + concentrations, as indicated. All curves are difference traces ob- tained by measuring and simultaneously subtracting the voltage

trace from the trace of the Ca z +-selective microelectrode (see Ma-

,--,6

o U ,-'a >

100

50 - 5 0

H.H. Felle et al. : Ca z + homeostasis in plant cells

I I I I I

8 7 6 5 4

pCa

terial and methods). Each curve is representative of three to five experiments, carried out under equivalent conditions. B Calibration of the Ca z + selective microelectrode used for the tests given in A. Eca is the net potential difference obtained by subtraction of the voltage electrode signal from the Ca z + electrode signal. Precalibra- tion (e), recalibration (o). The reference buffer filling the Ca z+- electrode had a pCa of 7

pF-o c

~ 7

T

I

'

|

0.5 pM

j

0.5

pH

f

A23187 9 ._ . , E m

X q5o

P ~55 min ; = -250 u I QJ ~1 :D

E

0.01

0.1

externQ[ Ca2"/mH

pCo c

|

0,1 I

Fig. 3A, B. Cytosolic free Ca 2 § (pCac), measured in the presence of

0.5 MM A23187 and different external Ca 2+ concentrations (hor-

izontal bars). The recordings demonstrate the ability of the Sinapis alba root-hair cells to remove from the cytosol Ca 2+ which had first leaked in through the action of A23187. Simultaneously Em was

measured. Initial external Ca 2+ concentrations were 0.01 mM (A) or 0.1 mM (B), added together with the ionophore, followed by 0.1 mM (A) or 1 mM Ca 2 + (B). Representative recordings from three (A) or four (B) equivalent experiments

a n d the r e s p e c t i v e i o n - p e r m e a b i l i t y r a t i o s ( S l a y m a n 1965; F e l l e 1981). A f t e r t h e c y a n i d e i n d u c e d d e p o l a r i z a - t i o n to - 115 m V , 0.5 g M A z 3 i s 7 f u r t h e r d e p o l a r i z e d the cells to a b o u t - 20 m V . T h e f o l l o w i n g i n c r e a s e in exter- n a l C a z+ f r o m 0.1 to 1 0 m M d r o v e the E m to + 3 0 m V w h i c h s t r o n g l y i n d i c a t e s a shift o f the p e r m e a b i l i t y r a t i o s o f t h e p l a s m a m e m b r a n e in f a v o u r o f C a 2 +.

Cytosolic free

[Ca2+].

I n t h e tips o f Sinapis r o o t h a i r s t h e c y t o s o l i c free [Ca 2+] rests b e t w e e n 378 a n d 831 n M ( n = 2 3 ; see a l s o T r e t y n et al. 1991b). I n the b a s a l p a r t s o f t h e s e cells t o w a r d s the c o r t e x we f o u n d s i g n i f i c a n t l y

l o w e r free

[Ca2+],

viz. 98

to

253 n M ( n = 9 ; d a t a n o t

s h o w n in figures). Since

A23187

is k n o w n as a C a 2+

i o n o p h o r e ( P r e s s m a n 1976), its e x t e r n a l a p p l i c a t i o n w o u l d b e e x p e c t e d to c a u s e C a 2+ i n t r u s i o n i n t o the

c y t o p l a s m , r e s u l t i n g in a n i n c r e a s e o f c y t o s o l i c [Ca2+]. W e f o u n d this i n c r e a s e b u t , m o s t s u r p r i s i n g l y , it o c c u r r e d a f t e r a n initial d e c r a s e w h i c h s t r o n g l y d e p e n d e d o n e x t e r - n a l [Ca2+]. A s s h o w n in F i g . 2, this C a 2+ d e p l e t i o n was o n l y v e r y s h o r t t e r m e d in the p r e s e n c e o f 10 m M C a 2§ b u t l a s t e d a b o u t 4 - 5 m i n in the p r e s e n c e o f 0.1 m M C a 2 +, b e f o r e a n i n c r e a s e o f c y t o s o l i c C a 2 § was o b s e r v e d .

W i t h 0.01 m M C a 2+ in the e x t e r n a l m e d i u m

no A23187-

induced

i n c r e a s e in c y t o s o l i c free [Ca 2+] o c c u r r e d o v e r the t i m e p e r i o d o f the m e a s u r e m e n t s . I n the p r e s e n c e o f 10 m M e x t e r n a l C a 2+ the c y t o s o l i c [Ca 2+] r o s e r a p i d l y to a b o u t p C a 5. F i g u r e s 3 a n d 4 give r e p r e s e n t a t i v e e x a m p l e s o f the cells r e g u l a t i v e p o w e r , as t e s t e d in t h e p r e s e n c e o f 0.5 g M

A23t87

a n d a t different e x t e r n a l C a 2+ c o n c e n t r a t i o n s . T h e i n i t i a l d e c r e a s e in c y t o s o l i c free [Ca 2 +], m e a s u r e d in

H.H. Felle et al.: Ca z + homeostasis in plant cells 309 t = :

o

> I=

-'" -50

, m

400

O

-150

r . -

-200

E GJ

E -250

6

pea c

7t t

T

:

7

t

/

i \

I/ T

',\

;

I/

',

II '

!

'1

;

;

~

, 5 min

,

'

/

I I

I

I

0.1

10

0.1

I

0.5 ~M A23187

Fig. 4. Changes in cytosolic free [Ca 2+] (pCac) of

Sinapis alba

root

hairs before and after increasing the external Ca 2§ from 0.1 mM to

10 mM Ca 2+ in the presence and absence 0.5 laM A23187. After the

first addition of 10 mM Ca 2+, the increase in cytosolic [Ca 2+] was interrupted intentionally to prevent a Ca z +-overload of the cytosol. The intruded Ca 2 § is exported again after removal of the ionophore

i t

9 1 . 4 ,

I0 mM

Coil +

0.I

1

0.5 ~M A23187

a n d reduction of external Ca 2 + to 0.1 raM. As a consequence o f the following addition of A z 318 v ( + 10 m M Ca z +), the cytosolic Ca 2 + again massively increases to concentrations higher t h a n p C a 5, but n o t into equilibrium with the external Ca z § Representative for four equivalent experiments -200

Q5HM EB

l

Ern 0.5pM A23187

(0,1 mM Co 2")

§

2 rain

|

(0.01 mM Ca 2.}

5 ~,

1

0.1 mMC(]

U

6 . ~

. ~ / / ~

cont ro~

J7

Fig. 5A, B. Inhibition of the plasma- m e m b r a n e Ca z +-ATPase by 0.5 taM EB. A Action of EB on Em a n d cytosolic free [CaZ+], before and after the addition o f A23t87. In B 0.5 laM A2a187 was added in the presence of 0.01 m M external Ca 2+ a n d

was left working for 8 10 min. After that period the external Ca 2 § was increased to 0.1 mM and two tests were performed, one with EB (+EB) and one without it (con- trol). Comparison of the two curves demon- strates the ability of the cells to reset cyto- solic Ca 2 + and its loss of this ability in the presence of EB. The curves are each repre- sentative of three equivalent experiments

the presence o f s u b m i l l i m o l a r external [ C a 2 + ] , w a s ac-

c o m p a n i e d b y a rapid d e p o l a r i z a t i o n . A d d i t i o n s o f 0.1 a n d 1 m M external C a 2+, resprectively, resulted in an i m m e d i a t e increase in cytosolic C a z + w h i c h slowly (with- in minutes) recovered. It m a y be interesting to p o i n t o u t t h a t in Fig. 3B the Em r a p i d l y repolarized in spite o f the increased C a 2+ c o n c e n t r a t i o n .

I n Fig. 4 the limits o f the cells ability to restore p e r t u r - b a n c e s in cytosolic free [Ca z+] are d e m o n s t r a t e d . A n increase in external [Ca 2+] f r o m 0.1 to 10 m M (in the

presence o f A 2 3 1 8 7 ) rapidly shifted cytosolic C a 2 + to p C a

values a r o u n d 5. F r o m this l o a d the cells o n l y r e c o v e r e d after a r e d u c t i o n in external [Ca z +] a n d after the r e m o v a l o f the i o n o p h o r e f r o m the m e d i u m .

The effect of erythrosin B.

I n s u b m i c r o m o l a r c o n c e n t r a - tions e r y t h r o s i n B (EB) is k n o w n to specifically inhibit the p l a s m a - m e m b r a n e C a 2 + - A T P a s e , w h e r e a s the H § A T P a s e is inhibited at c o n c e n t r a t i o n s a b o u t 100 times higher ( R a s i - C a l d o g n o et al. 1989a, b). Since the p l a s m a -

m e m b r a n e C a 2 § is p r e s u m e d to e x p o r t C a 2 +, an

310 H.H. Felle et al.: Ca 2+ homeostasis in plant cells E o Q _ o o ; -5o Pl-lc 6.5 -I 50 , ] ~ ~ . ~ E m 7.5 W -25( 5 rain -350 0.5 JM A23187

Fig. 6. A Cytosolic pH (pile) and E~, measured before and after the external addition of 0.5 l.tM A23187. Note that the initial pH change is rapid and does not return to control, although E., completely recovers after removal of A23187 from the medium (W). For back- ground information the trace of the pH-electrode (pH-el) also is

EpHlmV -50 -150 -250 -350

|

/"

/~

0 /

. /

shown. Representative of five equivalent measurements. B Calibra- tion of the pH-sensitive microelectrode used for the tests in A. Epn represents the net potential difference obtained by subtraction of the voltage electrode signal from the pH electrode signal. The reference buffer filling of the pH-electode had a pH of 5

homeostasis. As shown in Fig. 5A, 0.5 ~tM EB slightly hyperpolarized the cells, but it had no appreciable effect on cytosolic free [Ca 2+] o f the Sinapis r o o t hairs. How- ever, as soon as A23187 was added (in the presence o f EB), the cytosolic free [Ca 2+] rose massively after an appar- ently short period o f depletion.

In Fig. 5B it is demonstrated that after the typical A231sT-induced decrease in cytosolic [Ca2+], a tenfold increase o f external [Ca 2+] from 0.01 to 0.1 m M only caused a transient increase in cytosolic free [Ca 2 +] (con- trol). However, when EB was added together with the Ca 2 +, there was a massive increase in cytosolic [Ca z +] to a b o u t pCa 5 without any sign o f spontaneous recovery. Also, a tenfold reduction o f external [Ca 2+] to the origi- nal concentration only partly reduced the cytosolic [Ca 2 + ].

CytosolicpH.

A231s 7 had an immediate effect on cytosol- ic pH. As shown in Fig. 6, cytosolic p H rapidly decreased from 7.5 to 6.6 following the addition o f 0.5 gM A23~87. The cytosolic p H remained at 6.8 after a transient in- crease to a b o u t 7.1, although the Er, completely re- covered after the withdrawal o f the i o n o p h o r e from the medium.

Discussion

The cytosolic free [Ca2+], recorded from S. alba root hairs, is in agreement with the values reported by other authors for plant cells (see Evans et al. 1991). Especially interesting was the finding o f an intracellular Ca 2 + "gra- dient" with elevated free [Ca 2+] in the growing tip (Tretyn et al. 1991b). Such differences in cytosolic free [Ca 2 +] are not unusual and have been reported for Lilium pollen tubes (Jaffe et al. 1975; Reiss and H e r t h 1978; Nobiling and Reiss 1987), for the Fucus rhizoid (Brown- lee and W o o d 1986), and for Chara rhizoids (Hodick et al. 199l). Since it is u n d e r s t o o d that in tips o f longitudi-

nally growing cells an elevated [Ca 2+ ] as well as an intracellular Ca 2 + gradient m a y be linked to an increased metabolic activity (Speksnijder et al. 1989), we assumed that for investigations o f cytosolic Ca 2 + regulation these root-hair tips should be well suited.

The meanin 9 o f the initial Ca 2 + depletion. Puzzling, but most interesting from the regulatory point o f view, was the observation of the A231 s 7-induced initial depletion o f free cytosolic Ca 2+ which was especially evident in the presence o f low external [Ca2+]. At first sight one could have assumed this to be an artifact caused by the different time resolutions o f the pH- and voltage electrodes, re- spectively (Felle and Bertl 1986). This is not the case: (i) the changes are Ca 2+-dependent (Fig. 2); (ii) the Ca 2 + depletion is also present in cases where no or very little voltage change occurs; (iii) the Ca 2 + depletions remain for minutes (Figs. 2-5); if they were just temporal arti- facts caused by fast voltage changes across the plasma membrane, the difference traces would return to their starting point after the voltage changes (Felle and Bertl 1986), but they do not. On the other hand, the sharp initial peaks o f the Ca 2 + trace, observed during the first seconds after the addition o f the ionophore (Figs. 4, 5), should be treated as such temporal artifacts.

In fact, the initial Ca 2 + decreases depend on the exter- nal [Ca 2+] (Fig. 2), i.e. on the a m o u n t o f Ca 2+ forming complexes with the i o n o p h o r e ; they could indicate that the intruding Ca 2 + triggers regulatory forces which ini- tially remove more Ca 2 + from the cytosol than was origi- nally imported. This is by no means paradoxial, and can be interpreted in different ways. (i) This effect may be caused by the A231s 7 acting as a 2H+/Ca2+-antiporter, as proposed by Pressman (1976). Unfortunately, at present we do not have enough information to follow up and discuss this possibility in a satisfactory manner, but it is under investigation in our laboratory. (ii) Calcium ions move rapidly into the cytosol where they are sensed by Ca z + transporters, such as the Ca 2 +-ATPases at the

H.H. Fel|e et al. : Ca 2 § homeostasis in plant cells 311

ER and the plasma membrane, and are rapidly removed from the cytosol. Apart from the intruding Ca z +, other possible signals to trigger such a reaction could be the rapid depolarization, or the observed fast and strong acidification (Fig. 6).

Recently, a similar effect has been reported for pH regulation: Johannes and Felle (1987) demonstrated that during the initial depolarization of H+/amino acid (or

hexose-) symport in the aquatic liverwort

Rieeiafluitans,

cytosolic pH increased just as unexpectedly, although protons as the driving ions obviously must have entered the cells first. Since depolarization and pH increase were found to be related, this effect was interpreted as the result of a voltage-stimulated H +-ATPase which presum- ably led to an overcompensated H + export. At present we cannot rule out such an effect on the Ca 2 +-ATPase, but as long as the problem of electrogenicity of this ATPase is not solved (see below), we favour the Ca 2+- signal hypothesis. It may not be too speculative to as-

sume that as a consequence of the action o'f

A23187,

the

[Ca z+] will first rapidly increase in locations close to the plasma membrane. Since the gap between plasma mem- brane and ER is very small, as shown for instance in etiolated oat coleoptiles (Tretyn et al. 1992), such an increase in [Ca 2 +] will be sensed by both Ca 2 +-ATPases. Also, some Ca 2 +-storage capacity in the mitochondria should be taken into account, since in growing pollen tubes, for example, mitochondria are densely packed in a zone just behind the tip (Steer and Steer 1989). Clearly, both mitochondria and ER as internal compartments have only limited storage capacity: therefore, in stress situations, where Ca 2+ overfloods parts of the cyto- plasm, the plasma-membrane Ca2+-ATPase will take over and export Ca 2+, or the surplus will have to be shifted into the vacuole.

The inhibited plasma-membrane Ca2+-ATPase.

Ever since ATP-dependent Ca z + transport across the plasma membrane of plant cells was reported by Gross and Marm6 (1978), and Dieter and Marm6 (1981), the impor- tance of this pump for Ca z + homeostasis has been em- phasized (Rasi-Caldogno et al. 1987, 1989a, b; Evans 1988; Dupont et al. 1990), and is demonstrated here through the experiments with EB (Fig. 5). Although EB per se does not affect the resting cytosolic

[Ca 2+]

of

Sinapis

root hairs, its impact on cytosolic Ca 2 + regula- tion is massive when it added in situations where Ca 2 + constantly leaks into the cell, i.e. in the presence of

A23187, where the Ca 2 + ATPase is under stress: then the

C a 2 + rapidly enters the cytosol down its electrochemical gradient, and within minutes increases the cytosolic level from 0.66 gM to 5.2 gM.

Is the plasma-membrane

Ca2+-pump an nH+/Ca 2+-

ATPase?

In this study we present evidence that

A23187,

besides its effect on Em and cytosolic free [Ca2+], also induces an immediate decrease of cytosolic pH which seems to be concomitant with the increased activity of the Ca 2 +-pump. Miller and Sanders (1987) have pointed out that under conditions where cells build up EmS of

--200 mV or over,

a Ca 2 +-ATPase

may not have enough

energy from ATP hydrolysis to export C a 2 +, i.e. to main-

tain free Ca z + in the submicromolar range. The inwardly directed Ca 2 + driving force across the plasma membrane can be formulated as:

A I a C a Z + / F = 2 E r a + 5 9 ( p C a o - - p C a c ) ( E q . 1 )

where E m denotes the membrane potential, AgCa z +/F is

the electrochemical Ca 2+ gradient across the plasma membrane, and pCao and pCac are the external and cyto- solic pCa, respectively. From this equation it is evident that the Em is by far the largest fraction of the Ca / +-driv- ing force. Inserting a typical Em of - 200 mV, an external pCa of 4, and a cytosolic pCa of 6.5, a Ca 2 +-driving force of about - 550 mV results before the addition of A z 3 t s 7- In Fig. 3B we demonstrate that in the presence of 0.5 gM

A23187

the cells are well able to regulate cytosolic C a 2+

even at 1 mM external Ca2+; moreover, the cells re- polarize to - 190 mV, i.e. restore and even increase the electrochemical Ca 2 + gradient to about - 590 inV. This provides strong evidence that the C a z + - r e g u l a t i n g system must be connected to some other driving force and, in accordance with Miller and Sanders (1987) and Rasi- Caldogno et al. (1987), we suggest this to be the elec- trochemical proton gradient, built up and maintained by the electrogenic proton pump at the plasma membrane. This means that for every Ca 2 + exported one or more protons should enter the cell driven by the same trans- porter, viz. the C a 2 + - A T P a s e . We give evidence here that this may indeed be the case. As demonstrated in Fig. 6, cytosolic pH decreased immediately after the addition of

A23187. Together with the accompanying depletion of cytosolic free C a 2 +, this is exactly what must be expected

of a stimulated ATPase which exchanges nH+ for C a 2 +

So far, we do not have much information about whether this ATPase is electrogenic or electroneutral, but we found that the addition of EB to the cells caused a hyperpolarization by about 5 mV (Fig. 5A). This is not very impressive, but considering that the activity of the Ca 2 +-ATPase may be only a small fraction of what the H+-ATPase exports (Sanders and Slayman 1989), the impact of an electrogenic C a 2 +-ATPase on the total E m

can only be of minor importance. Assuming that this Ca/+-ATPase is electrogenic, then the small hyper- polarization could be indicative of more than 2H + being imported for every C a 2+ exported, a problem which is under current investigation in our laboratory.

Final remarks

We are well aware of the pitfalls in using substances like EB and ionophores. In particular, the in-vivo test situa- tion requires a careful analysis to separate facts from fancies. Still, we are able to present some new insights into how these compounds may work on a living cell. (i) We could demonstrate that the inhibition of the CaZ+-ATPase through EB (without A/31sT) had no im- pact on the cytosolic [Ca 2 +]. This means that, at least for some time, the cells can keep the cytosolic free [Ca 2+] constant without the support of this transporter. On the other hand, cytosolic [Ca z+] rapidly increases when EB

312 H.H. Felle et al.: Ca 2 + homeostasis in plant cells is applied d u r i n g C a z+ stress. W e conclude, therefore,

t h a t the m a i n role o f the p l a s m a - m e m b r a n e Ca 2+- A T P a s e is to act in situations where the cytosolic C a z + gets p e r t u r b e d , as f o r instance d u r i n g cellular signalling ( T r e t y n et al. 1991 b).

(ii) W e d e m o n s t r a t e t h a t because o f the steep elec- t r o c h e m i c a l C a 2+ g r a d i e n t across the p l a s m a m e m b r a n e o f these cells the h y d r o l y s i s o f 1 A T P does n o t p r o v i d e e n o u g h e n e r g y to e x p o r t C a 2 + against this gradient o r to m a i n t a i n it. Since we find t h a t the intruded C a z+ is indeed r e m o v e d f r o m the cytosol, a n d at the same time c y t o s o l i c p H r a p i d l y decreases, we suggest t h a t the plas- m a - m e m b r a n e C a 2 + - A T P a s e m a y be an n H + / C a 2+- A T P a s e with n possibly greater t h a n 2, as indicated by the E B - i n d u c e d h y p e r p o l a r i z a t i o n .

(iii) T h e C a 2 + r e c o v e r y s h o w n in this study a p p e a r s stow if c o m p a r e d with a n i m a l cells where d i s t u r b a n c e s in cytosolic [Ca 2+] are often restored in the s u b s e c o n d range. A p a r t f r o m the fact t h a t the presence o f the Ca 2 + i o n o p h o r e Az3ts 7 within the p l a s m a m e m b r a n e m a y have p r e v e n t e d a faster recovery, p l a n t cells a p p a r e n t l y do need longer. I n g u a r d cells, G i l r o y et al. (1990), M c A i n s h et al. (1990), a n d S c h r o e d e r a n d H a g i w a r a (1990) have s h o w n that the abscisic-acid-induced changes in cytosolic C a 2§ r e c o v e r within minutes, as does the abscisic-acid-induced decrease in cytosolic Ca 2 + reported in barley a l e u r o n e by W a n g et al. (1991). This is in a g r e e m e n t with r e p o r t s o n the liverwort Riccia fluitans, where cytosolic C a 2 + p e r t u r b a t i o n s also recovered with- in a b o u t 1 m i n (Felle 1991), whereas auxin caused oscilla- tion o f C a z+ in Z e a m a y s (Felle 1988a, b), a n d a stable decrease in Sinapis r o o t hairs ( T r e t y n et al. 1991). I n o u r s t u d y we f o u n d t h a t in spite o f the relative slow Ca 2 + r e c o v e r y after the A23187 t r e a t m e n t , the C a 2 +-exporting A T P a s e reacts quite rapidly, which m e a n s t h a t the physi- ological r e c o v e r y times are intrinsic properties o f the u n d e r l y i n g chain o f events.

This work was supported by the Deutche Forschungsgemeinschaft (H.F.) and the Alexander-von-Humboldt-Foundation (A.T.).

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