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The effect of the ionophore A23187 on Mg2+ and Ca2+ movements and internal pH of isolated intact chloroplasts
Plant Science Letters, 9 (1977) 7--15 © Elsevier/North-Holland Scientific Publishers, Ltd.
7
THE E F F E C T O F THE IONOPHORE A23187 ON Mg:+ AND Ca :+ M O V E M E N T S AND INTERNAL pH OF ISOLATED INTACT CHLOROPLASTS
M. M I G I N I A C - M A S L O W
and A. H O A R A U
Laboratoire de Physiologie Cellulaire V$~gJtale,Assoei~ au C.N.R.S., Universit~ de Paris-Sud, Centre d'Orsay, 91405 Orsay Cedex (France)
(Received September 29th, 1976) (Revision received and accepted November 20th, 1976)
S UMMARY
The kinetics of the Mg and Ca efflux from intact spinach chloroplasts were measured in the presence of the ionophore A23187 and/or EDTA. The results suggest that the Mg concentration in the stroma was about 16 mM in the dark and 23 mM in the light. No free Ca was present within the chloroplasts, but it could be artifically introduced in the presence of the ionophore. The results are discussed with respect to the hypothesis of the light-activation of the Calvin cycle through the Mg efflux.
INTRODUCTION
Many reports proposed the increase of the Mg ion concentration in the stroma of the chloroplasts to be at least partially responsible for the lightactivation of the photosynthetic CO2 fixation [see ref.1 ]. This widespread hypothesis was based on observation of magnesium efflux from the isolated thylakoids at the onset of illumination [ 2,3 ]. A light-dependent Ca efflux was also observed when isolated thylakoids were preloaded with Ca [3]. However, a light-dependent divalent-cation efflux could not be measured in the stroma of intact chloroplasts as the chloroplast envelope is relatively impermeable to ions [4,5]. Nevertheless, studies with divalent cation-specific ionophores [6,7] on cation-induced chlorophyll fluorescence quenching and on electron transfer of intact chloroplasts led to the suggestion that Mg ions moved into the chloroplast stroma in exchange for protons taken up by the thylakoids in the light. We have investigated the effect of A23187, an Mg and Ca specific ionophore [8], in an attempt to quantitate the Mg and Ca movements of intact spinach chloroplasts. Abbreviations: Chl, chlorophyll;HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulphonate; Tris,tris(hydroxymethyl)aminomethane.
MATERIAL .~ID METHODS Intact chloroplasts were prepared from field-grown spinach (Spinacia oleracea L., vat. Monstrueux de Viroflay) by a m e t h o d adapted from Cockburn et al. [9]. The grinding medium consisted of 0.33 M sorbitol, 10 mM sodium pyrophosphate pH 6.5, 20 mM NaC1, 1 mM MgC12, 1 mM MnC12 and 2 mM EDTA. The first chloroplast pellet was washed twice and resuspended in a metal-cation free medium consisting o f 0.33 M sorbitol and 10 mM HEPES buffer brought to pH 7.6 with 1 M Tris base. The chloroplasts were 75 to 98% intact, as determined by the ferricyanide method [10]. When suspended in a conventional carboxylation medium [ 11 ], they fixed 40 to 80 /~moles CO2.mg chl-~.h -1. Chlorophyll was determined by the m e t h o d of Arnon [12]. Unless otherwise indicated, the chloroplasts were incubated in the cationfree suspension medium, at 20°C, either in the dark or in the light (incandescent bulbs providing 27 mW. cm-2). Chlorophyll concentration was 150 pg. ml -~. Samples of 0.5 ml were periodically withdrawn and centrifuged 2 min in an E p p e n d o r f microcentrifuge. Mg and Ca were determined on the clear supernatant by atomic absorption s p e c t r o p h o t o m e t r y . The ion c o n t e n t of chloroplast pellets was determined on supernatants after the pellets were digested with 10% HC1 during 24 h, then centrifuged. In some cases, the separation of chloroplasts from the incubation medium was done either by filtration through a 0.45 ~m Millipore filter, or by centrifugation-filtration through a silicone layer [ 13 ]. In these cases, the incubation was made directly on the filter or in the centrifuge tube. The stroma and thylakoid pH and the volume of the chloroplast non-osmotic and osmotic spaces were determined by the m e t h o d of Heldt et al. [ 14 ]. A23187 was a kind gift of Eli Lilly France. It was used as ethanol solution. Each experiment was repeated at least three times, except for the experiments presented in Figs. 1 and 3 and Table I, which were performed twice. RESULTS
Magnesium movements When suspended in a cation-free buffer, isolated intact chloroplasts show a slow loss of magnesium (Fig. 1). This loss m a y be prevented by the addition of MgCl: to the medium, the optimal concentration being 0.4 mM. At higher concentrations, Mg is b o u n d b y the chloroplasts and partially inhibits their CO~ fixation activity. The magnesium leakage is n o t due to disruption of chloroplasts, as only 10 to 15% of the chloroplasts are broken after a 1 h incubation at r o o m temperature, and is not significantly affected by light (Fig.2 control). When the ionophore A23187 was added to the chloroplasts, a fast initial Mg efflux occurred and a plateau was attained after a 20 min incubation. Here again, light had practically no influence on the ionophore-induced magnesium efflux. The initial velocity of Mg efflux in the presence of A23187 was
9
J.tg M g / mg chl
0
~g
Mg / mg chl
~ ~ t -
:L _t
•
+5 - ~ " " ~ 5 ]
"
/
.1×mM .
o :O 20 /
_51_
+
5
0.6 m M
~÷
0
10
20
40
60 min
Fig. 1. Time course of the loss or fixation of magnesium by intact chloroplasts suspended in media containing different MgCl 2 concentrations (as indicated on each curve). Positive values: magnesium efflux from chloroplasts. Negative values: Mg binding by chloroplasts. Numbers in brackets indicate the percentage of intact chloroplasts present in the medium. 0 time is the m o m e n t when chloroplasts are suspended in the different media. Fig. 2. The effect of A23187 on magnesium efflux from intact chloroplasts susp.ended in a metal-cation free buffer. L(q~--e), light control; D(o--o), dark control; AL(A--A), 5 ~M A23187, light; A D ( a - - a ) , 5 uM A23187, dark.
proportional to the ionophore concentration in the m e d i u m (Fig. 3) but the absolute a m o u n t exported was the same for all concentrations. To check if the lack of illumination effect was not due to too slow a separation of chloroplasts from their incubation m e d i u m we tried separation by Millipore filtration and by centrifugation through a silicone layer (Table I). However, light-induced Mg efflux was still very limited or absent. The A23187induced Mg efflux was a characteristic of intact chloroplasts: osmotically shocked chloroplasts did not exhibit it (Fig. 4). The measured efflux could therefore be entirely ascribed to intact chloroplasts. The absence of light effect in the presence of the ionophore was surprising, since a higher Mg concentration would be expected in the stroma in the light [6,7]. Two explanations might therefore be proposed: (1) a complete export of all chloroplast Mg by the ionophore; (2) an interaction of the ionophore with the proton gradient collapsing the light-dependent Mg efflux to the stroma. The first hypothesis was tested by adding EDTA to the medium to trap all the exported Mg. Fig. 5 shows that EDTA (at concentrations >f 0.5 mM) in the presence of A23187 induced a rapid Mg loss from the chloroplasts (complete within 3 rain). The maximal level was identical in the dark and in the light, but higher than in the absence of EDTA. Thus, not all of the Mg was exported by the ionophore in the absence of the chelator. EDTA when added alone, enhanced Mg leakage from the chloroplasts. Under this condition a
10
TABLE I
Mg EFFLUX METHODS
FROM
INTACT CHLOROPLASTS,
STUDIED BY RAPID SEPARATION
The chloroplasts were separated from the supernatant after a 3 rain incubation at room temperature. The data for Millipore filtrationand for centrifugation filtrationwere obtained from two separate experiments. The M g content of the pellet is corrected for the M g content of chlorophyll. The given values are the average of 5 replicates. Incubation conditions
Mg Distribution a Millipore Centri_fugation filtrate Pellet
Supernatant
9.2 8.6 13.9 13.7
6.4 5.6 9.4 10.2
Light Dark Light + A23187 (10 #M) Dark + A23187 (10 #M) a
11.4 12.1 6 7.1
#g Mg/mg chlorophyll
12 ~g Mg / mg
chl
B Mg Mg / m g
~x ~ '
chl
B+A
8'
/-
0 i
i
10
20
i
40 rnin
5
10
/
EA] MM I
0
1'0
20
i
30
4'5
6'0 rain
Fig. 3. The effect of different concentrations of A23187 on the time-course of Mg efflux (left) and on the initial velocity of Mg efflux (right). o---o, No addition; x-x, 2 #M A23187; a--o, 5 #M A23187; A--a, 7.5 #M A 2 3 1 8 7 ; m - a , 10 #M A23187. Fig. 4. The effect of A23187 on Mg movements of intact and broken chloroplasts. I, intact chloroplasts; I+A, intact chloroplasts + 5 #M A23187. B, chloroplasts broken by suspension in distilled water, followed by readjustement of the osmotic pressure by addition o f double strength suspension medium. B + A, broken chloroplasts + 5 #M A23187. The experiment was done in the light, but experiments in the dark provide very similar results, as no light-dependent Mg effiux could be measured in broken chloroplasts, unless an electron acceptor was present..
11
~g Mg/ mg chl •
lOrl
(EDTA + A)L B~EDTA
/|
•
chl EDTA + A
+A')D./'"
EDTAL//
I# ."
pg Ca / m g
--III
A
~
4
a/,,,~a.~/ I u 40
/~
s [3p
/ EDTA
sliq ,," i'" ~ /e,..,a
~
D
CL
2£ f ~ O
~
.~_~
A
L 0
10
20
40
60 min
10
20
30
45
Fig. 5. The effect of A 2 3 1 8 7 and/or EDTA on the Mg efflux of intact chloroplasts CL ( i e), light control; C D ( o - - o ) , dark control; EDTA D (~ .... ~), 1 mM EDTA, dark; EDTA L (m.... m), 1 mM EDTA, light; A L (A--A), 10 uM A23187, light; A D ( ~ - - ~ ) , 10uM. A23187, dark; (EDTA + A)L (m--m), 1 mM EDTA + 10 ~M A23187, light; (EDTA + A) D ( c - - u ) , 1 mM EDTA + 10 uM A23187, dark. Fig. 6. The effect of EDTA and/or A 2 3 1 8 7 on Ca loss by intact chloroplasts. Additions: C, no addition; A, 2.5, 5 or 7.5 uM A23187; EDTA, 1 mM EDTA. The experiment was done in the dark, but experiments in the light provide very similar results.
difference between light and dark incubation appeared after 10 min: the Mg efflux in the dark EDTA assay reached progressively the level of the Mg exported by A 2 3 1 8 7 , while the light EDTA assay attained the ( A 2 3 1 8 7 + EDTA) Mg value. Addition of EDTA to chloroplasts suspended in a cation-free medium seems to cause a progressive rise in permeability of the chloroplast envelope, allowing a quantitative measurement of the Mg fluxes out of the thylakoids. The final light minus dark level was the same, whatever the experiment was done with no added acceptor other than the CO2 in the medium, or with the addition of 5raM CO3HNa + 0.15 mM PO4HK2, to insure an active electron transfer. In intact chloroplasts the slow electron transport associated with the fixation of atmospheric CO; seemed to be sufficient therefore to allow maximal lightdependent Mg efflux. The comparison of the results obtained with A 2 3 1 8 7 or EDTA alone and with A 2 3 1 8 7 + EDTA suggest that the A23187-induced Mg efflux, equal in absolute amount to the EDTA induced Mg loss in the dark, represents the dark Mg level in the stroma, while the A 2 3 1 8 7 + E D T A induced Mg efflux, represents the total free and loosely b o u n d Mg content of the chloroplasts. The latter is probably located entirely in the stroma in the light, as it is accessible to E D T A only during illumination. The conclusion that A 2 3 1 8 7 , in the light, depletes the chloroplasts only of the a m o u n t of magnesium which is normally present in the stroma in the dark,
12 TABLE II THE EFFECT OF A23187 ON THE STROMA AND THYLAKOID pH OF INTACT CHLOROPLASTS 3 rain incubations. The given values are the mean of 5 replicates ± SE. For stroma pH values, the differences between "light" treatment and each of the three other conditions are significant at the 95% level. For thylakoid pH values, the differences between "light" treatment and each of the three other conditions are highly significant at the 99% level. Incubation conditions
Stroma pH
Thylakoid pH
Light Dark Light + A23187 (10 uM) Dark + A23187 (10 uM)
7.7 -+0.15 7.1 -+0.15 7.2 ± 0.06 7.15-+ 0.16
5.45 5.89 5.85 6.00
± 0.01 ± 0.03 ± 0.02 -+0.07
m i g h t be easily u n d e r s t o o d if t h e i o n o p h o r e were able t o i n t e r f e r e w i t h t h e p r o t o n p u m p o f t h e t h y l a k o i d s . Evidence is already available in t h e literature t o s u p p o r t this idea: A 2 3 1 8 7 is a c a r b o x y l i c acid w h i c h m e d i a t e s t h e e x c h a n g e o f Mg 2+ or Ca 2+ for H + [ 8 ] . It reverses the M g - d e p e n d e n t slow f l u o r e s c e n c e q u e n c h i n g o f w h o l e and b r o k e n c h l o r o p l a s t s [6] and inhibits p h o t o p h o s p h o r y lation and l i g h t - d e p e n d e n t p r o t o n t r a n s p o r t in b r o k e n chloroplasts [ 1 5 ] . It u n c o u p l e s t h e o x a l o a c e t a t e - d e p e n d e n t o x y g e n e v o l u t i o n in i n t a c t chloroplasts, a l t h o u g h this u n c o u p l i n g is p r e v e n t e d b y a d d i t i o n o f E D T A [ 7 ] . We were able to r e p r o d u c e the latter results u n d e r o u r c o n d i t i o n s , but, t o validate o u r interp r e t a t i o n we also m e a s u r e d the i n f l u e n c e o f A 2 3 1 8 7 on t h e s t r o m a and thylakoid p H o f o u r c h l o r o p l a s t p r e p a r a t i o n s . Table II clearly d e m o n s t r a t e s t h a t the i o n o p h o r e inhibits light-induced p r o t o n u p t a k e . T h u s t h e Mg efflux, which is c o n s i d e r e d to be a c o u n t e r - e x c h a n g e f o r H + influx, m a y be e x p e c t e d t o be i n h i b i t e d t o o . Calcium m o v e m e n t s T h e slow calcium loss o f i n t a c t c h l o r o p l a s t s was n o t e n h a n c e d , b u t slightly l o w e r e d in t h e p r e s e n c e o f A 2 3 1 8 7 (Fig. 6). T h e a d d i t i o n o f E D T A resulted in a c c e l e r a t e d calcium e f f l u x , b u t this e f f l u x was already very i m p o r t a n t at the start o f t h e a d d i t i o n and t h e p r e s e n c e o f A 2 3 1 8 7 had a l m o s t n o accelerating e f f e c t o n it. T h e r e s p o n s e t o E D T A and t h e i n e f f i c i e n c y o f the i o n o p h o r e in p r o m o t i n g a Ca e f f l u x suggests t h a t t h e r e was n o free Ca within the chloroplast and t h a t a great p a r t o f the liberated Ca was loosely b o u n d t o t h e surface o f the chloroplasts. When Ca was a d d e d t o chloroplasts, the i o n o p h o r e e n h a n c e d its u p t a k e (Fig. 7). This f e a t u r e was never observed with Mg, w h e r e the a d d i t i o n o f external m a g n e s i u m simply l o w e r e d t h e Mg e f f l u x i n d u c e d b y A 2 3 1 8 7 (results n o t shown), T h e Ca u p t a k e p r o m o t e d an a c c e l e r a t i o n o f Mg e f f l u x (Fig. 8) " and a rise in t h e t o t a l a m o u n t o f Mg e x p o r t e d , b u t o n l y w h e n A 2 3 1 8 7 was present.
13 ~g Nlg/ mg chE
I [
Cco÷ AbD B
~
A
)
L
u
}J.g Ca / mg chl CD
130
1oo
sll
~.=~,_-~, - ~ - - - ~ L "
7C o
I
I
20
30
60 rnin
0
10
20
40
60 rain
Fig. 7. Effect of A 23187 on Ca absorption by intact chloroplasts suspended in a medium containing 0.5 mM CaCI~. L, light; D, dark; C, control; A, 5 uM A23187. Fig. 8. Effect of Ca on A23187 induced Mg efflux of intact chloroplasts. L, light; D, dark; C, control (no additions). Ca, 0.5 mM CaCl2added; A, 5 ,M A23187 added.
DISCUSSION
Our results indicate that A23187 has an opposite effect on Mg and Ca movements within intact chloroplasts, while chemical studies [8] demonstrated that this ionophore had a very similar efficiency for the transport of both ions. It seems therefore that although free Mg is readily available within the chloroplasts for the action of the ionophore, there is no free Ca present in the chloroplast stroma. The Ca ion can only be artificially introduced into the chloroplasts by the ionophore, probably partially in exchange for Mg. As a consequence, it may be inferred that Ca, contrary to Mg, does n o t significantly contribute to the light-dependent ion efflux from the thylakoids, in agreement with the conclusion of Barber et al. [6]. The studies of Mg movements in the presence of A23187 and/or EDTA suggested that the A23187 induced Mg loss represented the dark stroma Mg level, while the A23187 + EDTA induced loss represented the light stroma Mg level. From these values, knowing the [ ~4C] sucrose impermeable space which was determined during the pH experiments and found to be equal to 18 g l / m g chl, and considering that the stroma volume represents a b o u t 87% of this space [14], the Mg concentration in the stroma can be calculated. After subtraction of the a m o u n t of Mg p~esent in the suspension medium at the start of the experiment, the Mg concentration in the stroma would approach 16 mM in the dark and 23 mM in the light. The light m i n u s dark concentration increase,
14
ca. 7 mM, is of the same order of magnitude as the light-induced variations in the Mg2+ concentration reported for broken chloroplasts [3], when expressed in nEq./mg chl. The f a c t t h a t the Mg concentrations were determined with the aid of A23187, the function of which is to exchange internal Mg ions for external protons, suggests that the measured concentrations represent essentially free Mg. The same may be true for EDTA which induces a rise in the permeability of the chloroplast envelope, thus enabling the free stroma Mg to diffuse into the incubation medium where it is trapped by the chelator. However, in both cases, the depletion of the free stroma magnesium may lead to gradual dissociation and efflux of reversibly bound magnesium leading to an overestimation of the free Mg content. Certainly not all of the bound stroma Mg is depleted under these conditions as more Mg is liberated by disruption of chloroplasts than by addition of A23187 (Fig. 4}. This additional Mg represents a 3 to 4 mM concentration increase on a stroma volume basis. Considering the fast initial efflux of Mg in the presence of A23187 (2/3 of the total Mg efflux occurs during the first 2 min) it may be speculated that this initial efflux (ca. 10 mM Mg) represents the free stroma magnesium content in the dark. If this assumption proved to be true, it might be inferred that the increase of the Mg concentration in the stroma in the light might be especially important for the regulation of fructose biphosphatase and ribulose biphosphate carboxylase. These two enzymes indeed are saturated at fairly high (> 20 mM) Mg concentrations at suboptimal pH values [16,17,18] which may correspond to the stroma pH of illuminated chloroplasts [ 14]. While PGA kinase which is saturated at 2 mM Mg [19], and Ru5P kinase which is saturated at 5 mM Mg [20] would scarcely be affected by the light dependent Mg movements. ACKNOWLEDGEMENTS
We wish to thank professor A. Moyse for reading the manuscript and Dr. M.-L. Champigny for introduction to the pH determination technique. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12
D.A. Walker, N e w Phytol., 72 (1973) 209. R.A. Dilley and L.P. Vernon, Arch. Biochem. Biophys., 111 (1965) 365. G. Hind, H.Y. Nakatani and S. Izawa, Proc. Nat. Acad. Sci. USA, 71 (1974) 1484. R. Pfl~ger, Z. Naturforsch., 28 (1973) 779. H. Gimmler, G. Sch~fer and U. Heber, in M. Avron (Ed.), Proc. 3rd Int. Cong. Photosynth., Elsevier, Amsterdam, 1974, p. 1381. J. Barber, J. Mills and J. Nicolson, F E B S Lett., 49 (1974) 106. A. Telfer, J. Barber and J. Nicolson, Biochim. Biophys. Acta, 396 (1975) 301. P.W. Reed and H.A. Lardy, J. Biol. Chem., 247 (1972) 6970. W. Cockburn, D.A. Walker and C.W. Baldry, Biochem. J., 107 (1968) 89. U. Heber and K. Santarius, Z. Naturforsch., 25b (1970) 718. M. Miginiac-Maslow and M.L. Champigny, Plant Physiol., 53 (1974) 856. D.I. Arnon, Plant Physiol., 24 (1949) 1.
13 K. Werdan, H.W. Heldt and G. Geller, Biochim. Bi0phys. Acta, 283 (1972) 430. 14 H.W. Heldt, K. Werdan, M. Milovancev and G. Geller, Biochim. Biophys. Acta, 314 (1973) 224. 15 C.S. Andreo and R.H. Vallejos, FEBS Lett., 46 (1974) 343. 16 J. Preiss, M.L. Biggs and E. Greenberg, J. Biol. Chem., 242 (1967) 2292. 17 D. Baier and E. Latzko, Biochim. Biophys. Acta, 396 (1975) 141. 18 J.A. Bassham, P. Sharp and I. Morris, Biochim. Biophys. Acta, 153 (1968) 898. 19 B. Axelrod and R. Bandurski, J. Biol. Chem., 204 (1953) 939. 20 J. Hurwitz, A. Weissbach, B.L. Horecker and P.Z. Smyrniotis, J. Biol. Chem. 218 (1956) 769.
7
THE E F F E C T O F THE IONOPHORE A23187 ON Mg:+ AND Ca :+ M O V E M E N T S AND INTERNAL pH OF ISOLATED INTACT CHLOROPLASTS
M. M I G I N I A C - M A S L O W
and A. H O A R A U
Laboratoire de Physiologie Cellulaire V$~gJtale,Assoei~ au C.N.R.S., Universit~ de Paris-Sud, Centre d'Orsay, 91405 Orsay Cedex (France)
(Received September 29th, 1976) (Revision received and accepted November 20th, 1976)
S UMMARY
The kinetics of the Mg and Ca efflux from intact spinach chloroplasts were measured in the presence of the ionophore A23187 and/or EDTA. The results suggest that the Mg concentration in the stroma was about 16 mM in the dark and 23 mM in the light. No free Ca was present within the chloroplasts, but it could be artifically introduced in the presence of the ionophore. The results are discussed with respect to the hypothesis of the light-activation of the Calvin cycle through the Mg efflux.
INTRODUCTION
Many reports proposed the increase of the Mg ion concentration in the stroma of the chloroplasts to be at least partially responsible for the lightactivation of the photosynthetic CO2 fixation [see ref.1 ]. This widespread hypothesis was based on observation of magnesium efflux from the isolated thylakoids at the onset of illumination [ 2,3 ]. A light-dependent Ca efflux was also observed when isolated thylakoids were preloaded with Ca [3]. However, a light-dependent divalent-cation efflux could not be measured in the stroma of intact chloroplasts as the chloroplast envelope is relatively impermeable to ions [4,5]. Nevertheless, studies with divalent cation-specific ionophores [6,7] on cation-induced chlorophyll fluorescence quenching and on electron transfer of intact chloroplasts led to the suggestion that Mg ions moved into the chloroplast stroma in exchange for protons taken up by the thylakoids in the light. We have investigated the effect of A23187, an Mg and Ca specific ionophore [8], in an attempt to quantitate the Mg and Ca movements of intact spinach chloroplasts. Abbreviations: Chl, chlorophyll;HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulphonate; Tris,tris(hydroxymethyl)aminomethane.
MATERIAL .~ID METHODS Intact chloroplasts were prepared from field-grown spinach (Spinacia oleracea L., vat. Monstrueux de Viroflay) by a m e t h o d adapted from Cockburn et al. [9]. The grinding medium consisted of 0.33 M sorbitol, 10 mM sodium pyrophosphate pH 6.5, 20 mM NaC1, 1 mM MgC12, 1 mM MnC12 and 2 mM EDTA. The first chloroplast pellet was washed twice and resuspended in a metal-cation free medium consisting o f 0.33 M sorbitol and 10 mM HEPES buffer brought to pH 7.6 with 1 M Tris base. The chloroplasts were 75 to 98% intact, as determined by the ferricyanide method [10]. When suspended in a conventional carboxylation medium [ 11 ], they fixed 40 to 80 /~moles CO2.mg chl-~.h -1. Chlorophyll was determined by the m e t h o d of Arnon [12]. Unless otherwise indicated, the chloroplasts were incubated in the cationfree suspension medium, at 20°C, either in the dark or in the light (incandescent bulbs providing 27 mW. cm-2). Chlorophyll concentration was 150 pg. ml -~. Samples of 0.5 ml were periodically withdrawn and centrifuged 2 min in an E p p e n d o r f microcentrifuge. Mg and Ca were determined on the clear supernatant by atomic absorption s p e c t r o p h o t o m e t r y . The ion c o n t e n t of chloroplast pellets was determined on supernatants after the pellets were digested with 10% HC1 during 24 h, then centrifuged. In some cases, the separation of chloroplasts from the incubation medium was done either by filtration through a 0.45 ~m Millipore filter, or by centrifugation-filtration through a silicone layer [ 13 ]. In these cases, the incubation was made directly on the filter or in the centrifuge tube. The stroma and thylakoid pH and the volume of the chloroplast non-osmotic and osmotic spaces were determined by the m e t h o d of Heldt et al. [ 14 ]. A23187 was a kind gift of Eli Lilly France. It was used as ethanol solution. Each experiment was repeated at least three times, except for the experiments presented in Figs. 1 and 3 and Table I, which were performed twice. RESULTS
Magnesium movements When suspended in a cation-free buffer, isolated intact chloroplasts show a slow loss of magnesium (Fig. 1). This loss m a y be prevented by the addition of MgCl: to the medium, the optimal concentration being 0.4 mM. At higher concentrations, Mg is b o u n d b y the chloroplasts and partially inhibits their CO~ fixation activity. The magnesium leakage is n o t due to disruption of chloroplasts, as only 10 to 15% of the chloroplasts are broken after a 1 h incubation at r o o m temperature, and is not significantly affected by light (Fig.2 control). When the ionophore A23187 was added to the chloroplasts, a fast initial Mg efflux occurred and a plateau was attained after a 20 min incubation. Here again, light had practically no influence on the ionophore-induced magnesium efflux. The initial velocity of Mg efflux in the presence of A23187 was
9
J.tg M g / mg chl
0
~g
Mg / mg chl
~ ~ t -
:L _t
•
+5 - ~ " " ~ 5 ]
"
/
.1×mM .
o :O 20 /
_51_
+
5
0.6 m M
~÷
0
10
20
40
60 min
Fig. 1. Time course of the loss or fixation of magnesium by intact chloroplasts suspended in media containing different MgCl 2 concentrations (as indicated on each curve). Positive values: magnesium efflux from chloroplasts. Negative values: Mg binding by chloroplasts. Numbers in brackets indicate the percentage of intact chloroplasts present in the medium. 0 time is the m o m e n t when chloroplasts are suspended in the different media. Fig. 2. The effect of A23187 on magnesium efflux from intact chloroplasts susp.ended in a metal-cation free buffer. L(q~--e), light control; D(o--o), dark control; AL(A--A), 5 ~M A23187, light; A D ( a - - a ) , 5 uM A23187, dark.
proportional to the ionophore concentration in the m e d i u m (Fig. 3) but the absolute a m o u n t exported was the same for all concentrations. To check if the lack of illumination effect was not due to too slow a separation of chloroplasts from their incubation m e d i u m we tried separation by Millipore filtration and by centrifugation through a silicone layer (Table I). However, light-induced Mg efflux was still very limited or absent. The A23187induced Mg efflux was a characteristic of intact chloroplasts: osmotically shocked chloroplasts did not exhibit it (Fig. 4). The measured efflux could therefore be entirely ascribed to intact chloroplasts. The absence of light effect in the presence of the ionophore was surprising, since a higher Mg concentration would be expected in the stroma in the light [6,7]. Two explanations might therefore be proposed: (1) a complete export of all chloroplast Mg by the ionophore; (2) an interaction of the ionophore with the proton gradient collapsing the light-dependent Mg efflux to the stroma. The first hypothesis was tested by adding EDTA to the medium to trap all the exported Mg. Fig. 5 shows that EDTA (at concentrations >f 0.5 mM) in the presence of A23187 induced a rapid Mg loss from the chloroplasts (complete within 3 rain). The maximal level was identical in the dark and in the light, but higher than in the absence of EDTA. Thus, not all of the Mg was exported by the ionophore in the absence of the chelator. EDTA when added alone, enhanced Mg leakage from the chloroplasts. Under this condition a
10
TABLE I
Mg EFFLUX METHODS
FROM
INTACT CHLOROPLASTS,
STUDIED BY RAPID SEPARATION
The chloroplasts were separated from the supernatant after a 3 rain incubation at room temperature. The data for Millipore filtrationand for centrifugation filtrationwere obtained from two separate experiments. The M g content of the pellet is corrected for the M g content of chlorophyll. The given values are the average of 5 replicates. Incubation conditions
Mg Distribution a Millipore Centri_fugation filtrate Pellet
Supernatant
9.2 8.6 13.9 13.7
6.4 5.6 9.4 10.2
Light Dark Light + A23187 (10 #M) Dark + A23187 (10 #M) a
11.4 12.1 6 7.1
#g Mg/mg chlorophyll
12 ~g Mg / mg
chl
B Mg Mg / m g
~x ~ '
chl
B+A
8'
/-
0 i
i
10
20
i
40 rnin
5
10
/
EA] MM I
0
1'0
20
i
30
4'5
6'0 rain
Fig. 3. The effect of different concentrations of A23187 on the time-course of Mg efflux (left) and on the initial velocity of Mg efflux (right). o---o, No addition; x-x, 2 #M A23187; a--o, 5 #M A23187; A--a, 7.5 #M A 2 3 1 8 7 ; m - a , 10 #M A23187. Fig. 4. The effect of A23187 on Mg movements of intact and broken chloroplasts. I, intact chloroplasts; I+A, intact chloroplasts + 5 #M A23187. B, chloroplasts broken by suspension in distilled water, followed by readjustement of the osmotic pressure by addition o f double strength suspension medium. B + A, broken chloroplasts + 5 #M A23187. The experiment was done in the light, but experiments in the dark provide very similar results, as no light-dependent Mg effiux could be measured in broken chloroplasts, unless an electron acceptor was present..
11
~g Mg/ mg chl •
lOrl
(EDTA + A)L B~EDTA
/|
•
chl EDTA + A
+A')D./'"
EDTAL//
I# ."
pg Ca / m g
--III
A
~
4
a/,,,~a.~/ I u 40
/~
s [3p
/ EDTA
sliq ,," i'" ~ /e,..,a
~
D
CL
2£ f ~ O
~
.~_~
A
L 0
10
20
40
60 min
10
20
30
45
Fig. 5. The effect of A 2 3 1 8 7 and/or EDTA on the Mg efflux of intact chloroplasts CL ( i e), light control; C D ( o - - o ) , dark control; EDTA D (~ .... ~), 1 mM EDTA, dark; EDTA L (m.... m), 1 mM EDTA, light; A L (A--A), 10 uM A23187, light; A D ( ~ - - ~ ) , 10uM. A23187, dark; (EDTA + A)L (m--m), 1 mM EDTA + 10 ~M A23187, light; (EDTA + A) D ( c - - u ) , 1 mM EDTA + 10 uM A23187, dark. Fig. 6. The effect of EDTA and/or A 2 3 1 8 7 on Ca loss by intact chloroplasts. Additions: C, no addition; A, 2.5, 5 or 7.5 uM A23187; EDTA, 1 mM EDTA. The experiment was done in the dark, but experiments in the light provide very similar results.
difference between light and dark incubation appeared after 10 min: the Mg efflux in the dark EDTA assay reached progressively the level of the Mg exported by A 2 3 1 8 7 , while the light EDTA assay attained the ( A 2 3 1 8 7 + EDTA) Mg value. Addition of EDTA to chloroplasts suspended in a cation-free medium seems to cause a progressive rise in permeability of the chloroplast envelope, allowing a quantitative measurement of the Mg fluxes out of the thylakoids. The final light minus dark level was the same, whatever the experiment was done with no added acceptor other than the CO2 in the medium, or with the addition of 5raM CO3HNa + 0.15 mM PO4HK2, to insure an active electron transfer. In intact chloroplasts the slow electron transport associated with the fixation of atmospheric CO; seemed to be sufficient therefore to allow maximal lightdependent Mg efflux. The comparison of the results obtained with A 2 3 1 8 7 or EDTA alone and with A 2 3 1 8 7 + EDTA suggest that the A23187-induced Mg efflux, equal in absolute amount to the EDTA induced Mg loss in the dark, represents the dark Mg level in the stroma, while the A 2 3 1 8 7 + E D T A induced Mg efflux, represents the total free and loosely b o u n d Mg content of the chloroplasts. The latter is probably located entirely in the stroma in the light, as it is accessible to E D T A only during illumination. The conclusion that A 2 3 1 8 7 , in the light, depletes the chloroplasts only of the a m o u n t of magnesium which is normally present in the stroma in the dark,
12 TABLE II THE EFFECT OF A23187 ON THE STROMA AND THYLAKOID pH OF INTACT CHLOROPLASTS 3 rain incubations. The given values are the mean of 5 replicates ± SE. For stroma pH values, the differences between "light" treatment and each of the three other conditions are significant at the 95% level. For thylakoid pH values, the differences between "light" treatment and each of the three other conditions are highly significant at the 99% level. Incubation conditions
Stroma pH
Thylakoid pH
Light Dark Light + A23187 (10 uM) Dark + A23187 (10 uM)
7.7 -+0.15 7.1 -+0.15 7.2 ± 0.06 7.15-+ 0.16
5.45 5.89 5.85 6.00
± 0.01 ± 0.03 ± 0.02 -+0.07
m i g h t be easily u n d e r s t o o d if t h e i o n o p h o r e were able t o i n t e r f e r e w i t h t h e p r o t o n p u m p o f t h e t h y l a k o i d s . Evidence is already available in t h e literature t o s u p p o r t this idea: A 2 3 1 8 7 is a c a r b o x y l i c acid w h i c h m e d i a t e s t h e e x c h a n g e o f Mg 2+ or Ca 2+ for H + [ 8 ] . It reverses the M g - d e p e n d e n t slow f l u o r e s c e n c e q u e n c h i n g o f w h o l e and b r o k e n c h l o r o p l a s t s [6] and inhibits p h o t o p h o s p h o r y lation and l i g h t - d e p e n d e n t p r o t o n t r a n s p o r t in b r o k e n chloroplasts [ 1 5 ] . It u n c o u p l e s t h e o x a l o a c e t a t e - d e p e n d e n t o x y g e n e v o l u t i o n in i n t a c t chloroplasts, a l t h o u g h this u n c o u p l i n g is p r e v e n t e d b y a d d i t i o n o f E D T A [ 7 ] . We were able to r e p r o d u c e the latter results u n d e r o u r c o n d i t i o n s , but, t o validate o u r interp r e t a t i o n we also m e a s u r e d the i n f l u e n c e o f A 2 3 1 8 7 on t h e s t r o m a and thylakoid p H o f o u r c h l o r o p l a s t p r e p a r a t i o n s . Table II clearly d e m o n s t r a t e s t h a t the i o n o p h o r e inhibits light-induced p r o t o n u p t a k e . T h u s t h e Mg efflux, which is c o n s i d e r e d to be a c o u n t e r - e x c h a n g e f o r H + influx, m a y be e x p e c t e d t o be i n h i b i t e d t o o . Calcium m o v e m e n t s T h e slow calcium loss o f i n t a c t c h l o r o p l a s t s was n o t e n h a n c e d , b u t slightly l o w e r e d in t h e p r e s e n c e o f A 2 3 1 8 7 (Fig. 6). T h e a d d i t i o n o f E D T A resulted in a c c e l e r a t e d calcium e f f l u x , b u t this e f f l u x was already very i m p o r t a n t at the start o f t h e a d d i t i o n and t h e p r e s e n c e o f A 2 3 1 8 7 had a l m o s t n o accelerating e f f e c t o n it. T h e r e s p o n s e t o E D T A and t h e i n e f f i c i e n c y o f the i o n o p h o r e in p r o m o t i n g a Ca e f f l u x suggests t h a t t h e r e was n o free Ca within the chloroplast and t h a t a great p a r t o f the liberated Ca was loosely b o u n d t o t h e surface o f the chloroplasts. When Ca was a d d e d t o chloroplasts, the i o n o p h o r e e n h a n c e d its u p t a k e (Fig. 7). This f e a t u r e was never observed with Mg, w h e r e the a d d i t i o n o f external m a g n e s i u m simply l o w e r e d t h e Mg e f f l u x i n d u c e d b y A 2 3 1 8 7 (results n o t shown), T h e Ca u p t a k e p r o m o t e d an a c c e l e r a t i o n o f Mg e f f l u x (Fig. 8) " and a rise in t h e t o t a l a m o u n t o f Mg e x p o r t e d , b u t o n l y w h e n A 2 3 1 8 7 was present.
13 ~g Nlg/ mg chE
I [
Cco÷ AbD B
~
A
)
L
u
}J.g Ca / mg chl CD
130
1oo
sll
~.=~,_-~, - ~ - - - ~ L "
7C o
I
I
20
30
60 rnin
0
10
20
40
60 rain
Fig. 7. Effect of A 23187 on Ca absorption by intact chloroplasts suspended in a medium containing 0.5 mM CaCI~. L, light; D, dark; C, control; A, 5 uM A23187. Fig. 8. Effect of Ca on A23187 induced Mg efflux of intact chloroplasts. L, light; D, dark; C, control (no additions). Ca, 0.5 mM CaCl2added; A, 5 ,M A23187 added.
DISCUSSION
Our results indicate that A23187 has an opposite effect on Mg and Ca movements within intact chloroplasts, while chemical studies [8] demonstrated that this ionophore had a very similar efficiency for the transport of both ions. It seems therefore that although free Mg is readily available within the chloroplasts for the action of the ionophore, there is no free Ca present in the chloroplast stroma. The Ca ion can only be artificially introduced into the chloroplasts by the ionophore, probably partially in exchange for Mg. As a consequence, it may be inferred that Ca, contrary to Mg, does n o t significantly contribute to the light-dependent ion efflux from the thylakoids, in agreement with the conclusion of Barber et al. [6]. The studies of Mg movements in the presence of A23187 and/or EDTA suggested that the A23187 induced Mg loss represented the dark stroma Mg level, while the A23187 + EDTA induced loss represented the light stroma Mg level. From these values, knowing the [ ~4C] sucrose impermeable space which was determined during the pH experiments and found to be equal to 18 g l / m g chl, and considering that the stroma volume represents a b o u t 87% of this space [14], the Mg concentration in the stroma can be calculated. After subtraction of the a m o u n t of Mg p~esent in the suspension medium at the start of the experiment, the Mg concentration in the stroma would approach 16 mM in the dark and 23 mM in the light. The light m i n u s dark concentration increase,
14
ca. 7 mM, is of the same order of magnitude as the light-induced variations in the Mg2+ concentration reported for broken chloroplasts [3], when expressed in nEq./mg chl. The f a c t t h a t the Mg concentrations were determined with the aid of A23187, the function of which is to exchange internal Mg ions for external protons, suggests that the measured concentrations represent essentially free Mg. The same may be true for EDTA which induces a rise in the permeability of the chloroplast envelope, thus enabling the free stroma Mg to diffuse into the incubation medium where it is trapped by the chelator. However, in both cases, the depletion of the free stroma magnesium may lead to gradual dissociation and efflux of reversibly bound magnesium leading to an overestimation of the free Mg content. Certainly not all of the bound stroma Mg is depleted under these conditions as more Mg is liberated by disruption of chloroplasts than by addition of A23187 (Fig. 4}. This additional Mg represents a 3 to 4 mM concentration increase on a stroma volume basis. Considering the fast initial efflux of Mg in the presence of A23187 (2/3 of the total Mg efflux occurs during the first 2 min) it may be speculated that this initial efflux (ca. 10 mM Mg) represents the free stroma magnesium content in the dark. If this assumption proved to be true, it might be inferred that the increase of the Mg concentration in the stroma in the light might be especially important for the regulation of fructose biphosphatase and ribulose biphosphate carboxylase. These two enzymes indeed are saturated at fairly high (> 20 mM) Mg concentrations at suboptimal pH values [16,17,18] which may correspond to the stroma pH of illuminated chloroplasts [ 14]. While PGA kinase which is saturated at 2 mM Mg [19], and Ru5P kinase which is saturated at 5 mM Mg [20] would scarcely be affected by the light dependent Mg movements. ACKNOWLEDGEMENTS
We wish to thank professor A. Moyse for reading the manuscript and Dr. M.-L. Champigny for introduction to the pH determination technique. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12
D.A. Walker, N e w Phytol., 72 (1973) 209. R.A. Dilley and L.P. Vernon, Arch. Biochem. Biophys., 111 (1965) 365. G. Hind, H.Y. Nakatani and S. Izawa, Proc. Nat. Acad. Sci. USA, 71 (1974) 1484. R. Pfl~ger, Z. Naturforsch., 28 (1973) 779. H. Gimmler, G. Sch~fer and U. Heber, in M. Avron (Ed.), Proc. 3rd Int. Cong. Photosynth., Elsevier, Amsterdam, 1974, p. 1381. J. Barber, J. Mills and J. Nicolson, F E B S Lett., 49 (1974) 106. A. Telfer, J. Barber and J. Nicolson, Biochim. Biophys. Acta, 396 (1975) 301. P.W. Reed and H.A. Lardy, J. Biol. Chem., 247 (1972) 6970. W. Cockburn, D.A. Walker and C.W. Baldry, Biochem. J., 107 (1968) 89. U. Heber and K. Santarius, Z. Naturforsch., 25b (1970) 718. M. Miginiac-Maslow and M.L. Champigny, Plant Physiol., 53 (1974) 856. D.I. Arnon, Plant Physiol., 24 (1949) 1.
13 K. Werdan, H.W. Heldt and G. Geller, Biochim. Bi0phys. Acta, 283 (1972) 430. 14 H.W. Heldt, K. Werdan, M. Milovancev and G. Geller, Biochim. Biophys. Acta, 314 (1973) 224. 15 C.S. Andreo and R.H. Vallejos, FEBS Lett., 46 (1974) 343. 16 J. Preiss, M.L. Biggs and E. Greenberg, J. Biol. Chem., 242 (1967) 2292. 17 D. Baier and E. Latzko, Biochim. Biophys. Acta, 396 (1975) 141. 18 J.A. Bassham, P. Sharp and I. Morris, Biochim. Biophys. Acta, 153 (1968) 898. 19 B. Axelrod and R. Bandurski, J. Biol. Chem., 204 (1953) 939. 20 J. Hurwitz, A. Weissbach, B.L. Horecker and P.Z. Smyrniotis, J. Biol. Chem. 218 (1956) 769.