CIB:3 Eflect on Electron Flow, ApH, pH, and Proton Pump in Chloroplasts a modification .... (from H,O io NADP+) us a,function OfpH ( A ) and offatty acid concen-.
Eur. J. Biochern. 61, 573-580 (1976)
Influence of Unsaturated Fatty Acids in Chloroplasts Shift of the pH Optimum of Electron Flow and Relations to ApH, Thylakoid Internal pH and Proton Uptake Paul-Andre SIEGENTHALER and Franqoise DEPERY Laboratoire de Physiologie Vegetate et Biochimie, Institut de Botanique, Universite de Neuchftel (Received July 31 / October 8, 1975)
Linolenic acid (C18: 3 ) is the main endogenous unsaturated fatty acid of thylakoid membrane lipids, and seems in its free form to exert significant effects on the structure and function of photosynthetic membranes. In this investigation the effect of linolenic acid was studied at various pH values on the electron flow rate in isolated spinach chloroplasts and related to dpH, the proton pump and the pH of the inner thylakoid space (pHi). The ApH and pH, were estimated from the extent of the fluorescence quenching of 9-aminoacridine. Linolenic acid caused a shift (approximately one unit) of the pH optimum for electron flow toward acidity in the following systems: (a) photosystems I1 I (from H 2 0 to NADP' or to 2,6dichlorophenolindophenol) coupled or non-coupled; (b) photosystem I1 (from H,O to 2,6-dichlorophenolindophenol in the presence of dibromothymoquinone). In photosystem I conditions (phenazine methosulphate), the ApH of the control increased as a function of external pH, with a maximum around pH 8.8. When linolenic acid was added, the ApH dropped, but its optimum was shifted toward more acidic pH,. The same phenomena were also observed in photosystems I1 + I (from H 2 0 to ferricyanide) and in photosystem I1 conditions (from H,O to ferricyanide in the presence of dibromothymoquinone). However, the d p H was smaller and the sensitivity of the proton gradient toward linolenic acid was eventually higher than for photosystem I electron flow activity. The proton pump which might be considered as a measure of the internal buffering capacity of thylakoids was optimum at pH, 6.7 in the controls. An addition of linolenic acid diminished the proton pump and shifted its optimum toward higher pH,. As a consequence, pH, increased when pH, was raised. At the optimal pH, 8.6 to 9, pH, were 5 to 5.5. Additions of increasing concentrations of linolenic acid displaced the curves toward higher pHi. A decrease of pH, was therefore required to maintain the pH, in the range of 5 - 5.5 for maximum electron flow. In conclusion, the electron flow activity seems to be delicately controlled by the proton pump (buffer capacity), dpH, pH, and pH,. Fatty acids damage the membrane integrity in such a way that the subtle equilibrium between the factors is disturbed.
+
Unsaturated fatty acids can influence several structural parameters and photochemical functions of chloroplasts in vitro. For instance, it was experimentally verified that fatty acids modified the physicochemical properties of the membrane, namely the osmotic properties, in such a way that the thylakoid membranes swelled [l].These structural changes induced by fatty acids were accompanied by an inhibition of electron flow [2-41 and energy-linked reactions [1,2] in chloroplasts, and also by a dissociation of electron flow activity from photophosphorylation [2]. Abbreviations. pH,, pH of the medium; pHi, pH of inner thylakoid space.
The most intriguing feature of the inhibition of the photochemical reactions was that it was pH dependent. Indeed, unsaturated fatty acids caused a shift of the p H optimum for electron flow toward acidity in both photosystem I (from reduced tetramethyl-p-phenylenediamine to methylviologen) and photosystem I1 photosytem I (from H 2 0 to Fe(CN)Z-). Contrary to the suggestion of several investigators [5-91, such a phenomenon was demonstrated not to be related to the uncoupling event [2]. As a working hypothesis, we assumed that the acid shift of the pH optimum of electron flow and its inhibition by fatty acids are due to a deterioration or
+
CIB:3 Eflect on Electron Flow, ApH, pH, and Proton Pump in Chloroplasts
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a modification of the thylakoid membrane, combined with an inhibition of the light-induced proton uptake mechanism and an increase in the permeability of the membrane to water and protons [2,10]. This approach was based upon the assumption that the rate of electron flow is controlled not only by the degree of energy coupling but also by the internal pH of the thylakoid compartment, as proposed by Avron's group [ l l ] . In this investigation, the acid shift of the pH optimum of electron transport caused by unsaturated fatty acids has been verified in other electron flow systems and related to the proton pump mechanism, the ApH and internal pH (pHi) of the thylakoid, in agreement with the above hypotheses. A preliminary report of some of these findings has been published [12].
MATERIALS AND METHODS Spinach and Preparation
of Chloroplasts
Spinach (Spinacia oleracea var. Nobel) was purchased commercially or grown in a growth chamber under controlled conditions. After being germinated and grown for 3 weeks in vermiculite, the plants were grown in water culture for 8 weeks, using a synthetic nutrient solution. During the first week in vermiculite, the seedlings were supplied with water only, then for 2 weeks with the nutrient solution. The solution was prepared by adding, in 1 1 water, the following (ml): 1 M K N 0 3 (9,1 M Ca(N03)2 (lS),1 M MgSO, (l), 1 M KH2P04 (l), 1 M NH4C1 (l), a micronutrient solution (1) and Fe-EDTA solution (1). The micronutrient solution contained, in 1 1 of water, the following (g): H3B03 (2.863), MnS04 . H 2 0 (1.515), ZnSO, . 7H20 (0.222), CuSO, 5 H 2 0 (0.079), MOO, 8 5 % (0.098) and NH,VO, (0.029). The Fe-EDTA solution was prepared according to Arnon et al. [13]. The other growth conditions were: (a) 10 h lighti14 h dark cycles; the light was turned on and off step-wise and symmetrically in the following way: from 0 to 4000 lux (4000 to 0) within the first (last) half hour and from 4000 to 6600 lux (from 6600 to 4000) after the 4th (6th h), leaving 2 h at maximum light intensity; (b) temperature approximately 22 "C in the light and 16 "C in the dark; (c) humidity 70-90%. After 8 weeks in water culture, the leaves were harvested. Intact chloroplasts were first prepared according to Kalberer et al. [14]in 25 mM Hepes (pH 7.6) and 0.35 M sucrose (centrifugation: 30 s at 1935 x g) and then shocked osmotically in the same medium diluted to 1:10. After centrifuging 5 min at 17300 x g , the resulting pellet was resuspended in the initial medium and adjusted to give 2 mg chlorophyll/ml. Chlorophyll was measured by the method of Bruinsma ~ 1 .
Measurement of Electron Transport
+
The HzO/NADP+ (photosystems I1 I) electron transport was observed spectrophotometrically in a 0.2-mm cuvette by recording the absorbance changes of NADP+/NADPH at 340 nm, in a Zeiss spectrophotometer modified as described by McSwain and Arnon [16]. The actinic light was supplied by a 1000 W iodine lamp passed through interference filters (Balzers Calflex and Balzers DT-red). NADP photoreduction was carried out over a 1-min period at 20 "C at a light intensity of approximately I x lo5 ergs . cmP2 s-' at the cuvette [4]. The results are expressed as the initial rates of the reaction. The Hz0/2,6-dichloroindophenolelectron transport was measured spectrophotometrically at 590 nm by the photoreduction of dichloroindophenol as the oxidant. The activity was determined after 1 min at 20 "C in the presence of white light of approximately 5 x 105ergs . cmP2 . s p l intensity. The reaction mixtures are given in the explanations of the figures. Evaluation of ApH and Internal p H ApH in chloroplast membrane systems was estimated from the extent of the fluorescence quenching of 9-aminoacridine, employing the technique originally proposed by Schuldiner et ul. [17]. The fluorescence was measured with a fluorescence spectrophotometer (Perkin-Elmer type 204) which was adapted for illumination on the side of the cuvette. Actinic light (approximately 5 x lo5 ergs .cm-' . s-') was provided by a halogen lamp passed through a Calflex and a Corning CS-260 filter. The 9-aminoacridine was excited at 370-440 nm with a Klett 42 filter. The emission and the quenching of the fluorescence was measured at 90" angle at 505 nm. The osmotic volume of the chloroplast preparation was estimated from the data provided by Rottenberg et al. [l I] and was found to be 32.5 pl/mg of chlorophyll in our experimental conditions. The basic reaction mixture contained : 30 mM N-tris(hydroxymethyl)methylglycine/maleate or N-tris(hydroxymethyl)methylglycine/glycine at various pH, 40 mM NaC1,0.8 pM 9-aminoacridine, 0.5 %, ethanol or linolenic acid, 20 pM phenazine methosulphate or 0.2 mM ferricyanide, as indicated in the figures, and chloroplasts (20 pg chlorophyll/ml). Since the fatty acid was dissolved in ethanol, all reaction mixtures contained 0.5 % ethanol. At this concentration, ethanol had no detectable effect on electron flow, dpH, pHi or proton uptake. The internal pH was calculated by subtracting ApH from pH,. Measurement of Proton Pump Chloroplasts were isolated as indicated previously but resuspended in 175mM NaCl (pH 8) without
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Fig. 1. E ( f h of linolenic acid on photosystems 11 and I electron jloir. (from H,O io N A D P + ) us a,function OfpH ( A ) and offatty acid conceniration ( B ) . The reaction mixture contained: 50 mM of buffer [N-tris(hydroxymethyl)methyl-2-aminoethane sulfonate from pH 6 to 7.7 ( 0 ) and N-tris(hydroxymethyl)methylglycine, from 8 to 8.4 (A)], 35 m M NaCI, 6 m M MgCI,, 1 m M ADP, 1 mM KH,PO,, 2 m M NADP', ferredoxin in excess, 0.57; ethanol or, where indicated, linolenic acid and chloroplasts (60 pg chlorophyll per ml)
buffer. pH changes were measured with a Metrohm pH-meter (type E300B) and recorded continuously [18]. The reaction mixture (5 ml) contained: 35 mM NaCl, 20 pM phenazine methosulphate, 0.5 % ethanol or linolenic acid, and chloroplasts (40 pg chlorophyll/ ml). Light intensity of actinic light was approximately 5 x lo5 ergs . cm-' . s-' and the temperature was maintained constant at 20°C. pequiv. H + taken up per mg chlorophyll were estimated from the data provided by Walz et al. (see Fig. 1A in [19]). According to these authors, the proton binding of chloroplasts in the light is a linear function of pH in the range of 6 to 9, which is the range used in our experiments. Clwmicals
1,5Diphenylcarbazide was dissolved in methanol, while fatty acids and 3(3,4-dichlorophenyl)-l,l-dimethylurea were dissolved in ethanol. Ferredoxin was prepared by Dr P. Schiirmann from spinach leaves according to Buchanan and Arnon [20]. 9-Aminoacridine hydrochloride monohydrate was obtained from Fluka. Dibromothymoquinone was kindly provided by Drs A. Trebst and H. Baltscheffsky. RESULTS Shift in pH Optimum
In Fig. 1A, it is shown that the pH optimum of the electron flow rate (photosystems I1 I : from H 2 0 to NADP'), which was 8.2 under control conditions (coupled reaction and no fatty acids), shifted toward the acidic side in the presence of linolenic acid. At 400 pM, linolenic acid generally caused a shift varying
+
from 0.7 to 1.2 units (from 8.2 to 7.5 in the figure) in the pH optimum of the reaction. In the alkaline range (from pH, 7.7 to 8.4) a fatty acid treatment inhibited whereas in the more acidic range (from pH, 6 to 7.7), it activated the reaction. Fig. 1B shows the effects, at two pH,, of increasing linolenic acid concentrations on the electron transport activity. At pH 8.2, linolenic acid (100-200 pM) first stimulated (possibly due to an uncoupling effect), then inhibited strongly the activity which was completely obliterated at 300 pM. At pH, 7, the activity which was much lower in the control experiment (closed symbols), was accelerated by a factor of 3 in the presence of 300 pM of linolenic acid. Raising the concentrations further inhibited the reaction. Under conditions of no photophosphorylation (absence of ADP, P i and Mg2+),the pH, optimum of the electron flow rate was higher (pH, 8.7-8.9 in Fig. 2A) than under coupled conditions (pH, 8.2 in Fig. 1A). This observation is in agreement with the results reported earlier for the electron flow measured from H,O to ferricyanide [2,21]. In these non-coupled conditions (Fig. 2A), the pH, optimum was also shifted toward the acidic side (from pH, 8.8 to 7.5) but with lower concentrations of linolenic acid (200 pM instead of 400 pM). The same shift phenomenon was found to be also true when the electron flow was measured in the presence of 2,6-dichloroindophenol (photosystem I1 + photosystem I) as the oxidant of the Hill reaction (Fig. 2B). As an example, going from 0 to 300 pM of linolenic acid displaced the pH, optimum from 7 to 6. As noted earlier with Fe(CN):- as the electron acceptor [2], the overall activity of electron flow diminished with increasing fatty acid concentration. In more
C,,:, Erect on Electron Flow, ApH, pHi and Proton Pump in Chloroplasts
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Fig.2. Ej’f;.ct of linolenic acid on phoiosystems I! and I electron flow from H20 to MADP’ ( A ) and from 1,S-diphenycarbazide to 2,6-dichloroindophenol ( B ) as a function of p H . In (A) the reaction mixture contained: 50 mM of buffer [N-tris(hydroxymethy1)methyl-2-aminoethane sulfonate from pH 6.0 to 7.7 (0)and N-tris(hydroxymethyl)methylglycine, from 8 to 9 (A)], 35 mM NaCI, 2 mM NADP, ferredoxin in excess, 0.5 ”/, ethanol or linolenic acid at the indicated concentrations, and chloroplasts (60 1.16chlorophyll per ml). In (B) the reaction mixture contained: 50 mM of Tris-maleate, 35 mM NaCI, 5 mM MgC12, 0.5 mM 1,5-diphenylcarbazide, 0.15 mM 2,6dichloroindophenol, 0.5 ”/, ethanol or linolenic acid at the indicated concentrations and chloroplasts (20 pg chlorophyll per ml). Other conditions are described in Materials and Methods. In (A) the valuc of 10 varied from 80 to 160 pmol NADP’ reduced x mg chlorophyll-’ x h-’ and in (B) from 150-225 pmol 2,6-dichloroindophenol reduced x mg chlorophyll-’ x h-’ at pH 7
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Fig. 3. ApH dependence on p H , and linolenic acid concentrations in photosystem I . (A) In photosystem I (phenazine methosulphate), and (B,C) in photosystem I1 + photosystem 1 [H,O/Fe(CN);-] electron flow systems
specific conditions of photosystem 11 electron flow (i. e. dichloroindophenol + 1 pM dibromothymoquinone), the results (not shown) were essentially the same as in the H,O/dichloroindophenol system, except for the activity and the shift in pH optimum which were smaller. Influence of Fatty Acids on ApH
In order to explain the shft of the pH optimum of the electron flow rate toward acidity, it was first postu-
lated [2,10] that fatty acids deteriorate the membrane so as to impair the light-induced proton uptake. If this postulate is true, one would expect that the lightproduced ApH (ie. the difference between the external and internal pH of the thylakoid compartment) should diminish in the presence of linolenic acid. Fig. 3A shows that in phenazine-methosulphate-catalysedelectron flow (photosystem I), the ApH of the control increased as a function of pH, with a maximum around 8.8. The ApH values of the control are in agreement with those reported by Avron’s group [17,22]. When
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Fig.4. p H , dependence on p H , und linolenic acid concentrations. ( A ) In photosystem I (phenazinc methosulphate), and (B, C) in photosystem 11 electron flow systems
+ photosystem I [H,O/Fe(CN):-]
linolenic acid was added, the ApH dropped. As an example, at high pH, (8.8), the ApH decreased from a value of 4 in the control to 0 in the presence of 140 pM of linolenic acid. At a lower pH, (7.9), the extent of the ApH drop was smaller, i.e. from 3.6 to 2.3. This implies that going from 0 to 140pM of linolenic acid caused a shift of the d p H optimum toward more acidic pH, (Fig. 3A). Fig. 3B shows that the same phenomena were also observed in the H,O/Fe(CN);- electron flow system (photosystem I1 photosystem I). It is particularly evident that increasing concentrations of linolenic acid shifted the d p H optimum toward acidity. However, the ApH values were smaller and the peaks of the curves in treated chloroplasts were sharper than in photosystem I. Also, the sensitivity of the proton concentration gradient toward linolenic acid was eventually higher than in photosystem I electron flow conditions (see for example the effect of 50 pM of linolenic acid in Fig. 3A and B). In Fig. 3C, the ApH was plotted as a function of linolenic acid concentrations. It can be seen that at pH, 9, the d p H was at its maximum in the control and dropped to zero in the presence of 40pM linolenic acid. Around pH, 8, this acid had a much smaller effect on the ApH. These results have been verified in other electron flow conditions (a) without electron acceptors [12], (b) from H,O to methylviologen (photosystems I1 + I), (c) from diaminodurene to methylviologen in the presence of 3(3,4-dichlorophenyl)-l ,l-dimethylurea, azide and ascorbate (photosystem I), (d) from H,O to oxidized p-phenylenediamine (photosystem 11) ;in this system however, the fluorescence of the acceptor interfered with the acridine fluorescence, (e) from H,O to
+
Fe(CN):- in the presence of 1 pM dibromothymoquinone (photosystem 11). Influence of Fatty Acids on the Internal p H
As previously suggested [17,23], the rate of electron transport seems to be controlled not only by the lightinduced ApH but also by the pH of the inner thylakoid space (pHi) with a maximum around 5 [23]. Therefore it was of interest to investigate the dependence of pHi on various pH, in the presence of linolenic acid. Fig. 4A shows in agreement with previous reports [ l l , 171 that in control experiments (conditions of photosystem I electron flow), pHi increased when pH, was raised. At pH, 7.5 and 9.0, pHi were 4.2 and 5.0, respectively. It is interesting to mention that the maximum electron flow in photosystem I was found to be at pH, 8.7-9.0 [2] which correspond to pHi 5. Addition of increasing concentrations of linolenic acid displaced the curves toward higher pHi. For instance, at pH, 9, the pHi were 5.0, 5.8, 6.5 and 9.0 in the presence of 0 (control), 50, 100 and 140pM of linolenic acid, respectively. At pH, 7.5, the pHi were respectively 4.2, 4.8, 5.1 and 5.7. Since pHi 5 was found to be the optimum for the maximum rate of photosystem I electron flow, Fig.4A shows clearly that in the presence of increasing concentrations of linolenic acid, a decrease of pH, would be required to maintain pHi in the range of 5 and hence maximum activity. Fig. 4A shows also that below pH, 8.2, the pHi values were generally constant, indicating the existence of a natural buffering capacity in the acidic pH range as suggested by Schuldiner et al. [17]. This phenomenon is also
C , , : , Effect on Electron Flow, ApH, pH, and Proton Pump in Chloroplasts
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acidic range which might also control pHi. In order to test it, the extent of proton uptake, ApH and pH, were plotted as a function of pH, and compared in the absence and presence of linolenic acid. Fig. 5 shows that in the controls, the proton pump pH, optimum (6.7) was 2 to 3 units lower than the optimum ApH (9.0). When linolenic acid was added, both the proton pump and dpH diminished and the optima were displaced toward one another (see arrows in Fig.5). If the extent of proton pump is basically a measure of the internal buffer capacity [ll], one may conclude that linolenic acid decreases buffering capacity. Fig. 5 shows that above pH, 8.1 and possibly below 6, the buffering capacity in treated chloroplasts was considerably lower and pHi increased. Only around pH, 7.3 were the conditions (proton uptake, ApH, pHi and pH,) optimal for electron flow.
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Fig.5. The ej’ect qf linolenic acid on the proton uptake (A,A) ApH I@, 0 ) a n d p H i (m, 0)as a,function ofpH,,. The experimcnts were performed in photosystem I conditions (20 pM phenazine rnethosulphate). In fatty-acid-treated chloroplasts, the molar ratio C18:3/ chlorophyll was 3.15. Filled symbols are controls, open symbols with linolenic acid
illustrated in Fig. 4B and C where the pHi were plotted versus pH, and linolenic acid concentrations in the H,O/Fe(CN)i- electron flow system (photosystem I1 + photosystem I). In the presence of 0-30pM of linolenic acid, the pH, remained almost constant over a wide range of low pH,. At higher concentrations of linolenic acid (40- 50 pM), the pH were displaced to higher pH, with an exception at pH, 8 where the buffering capacity seemed to be more effective. In this range of linolenic acid concentration, two pHi were similar to their respective pH, (see arrows in Fig. 4B). With 100 pM linolenic acid, all the pHi were identical to the pH, (see arrows in Fig.4B and C). Upon addition of 1 pM of the inhibitor dibromothymoquinone (specific conditions for photosystem I1 electron flow), the results were essentially the same. Influence ofFatty Acids on Proton Uptake and ApH
In both photosystems, we have observed that in the acidic range of pH,, pH were maintained constant. This was the case for photosystem I1 + photosystem I (Fig. 4B) in the presence of 0- 30 pM linolenic acid and for photosystem I electron flow (Fig.4A) from 0 to about 100 pM of the acid. These results have suggested the existence of a buffering capacity in the
Ejfect of Linolenic Acid on the Electron Flow p H Dependence
It was shown previously [2] that unsaturated fatty acids shifted the pH optimum of electron flow toward lower pH in the following systems: H,O/Fe(CN);(photosystem I1 + photosystem I) and reduced N , N , N‘,N’-tetramethyl-p-phenylene diamine/methylviologen (photosystem I). This investigation extends this observation to new electron flow conditions, i. e. photosystems I1 + I (from H,O to NADP’), photosystem I1 + photosystem I (from H,O to 2,6-dichloroindophenol) and photosystem I1 (from H 2 0 to dichloroindophenol in the presence of dibromothymoquinone). It appears therefore that the shift in pH optimum toward acidity is a general phenomenon which concerns both photosystem activities, measured separately or in series. The shift in pH optimum was observed whether ADP, P i and MgZ+ were present or not in the medium and at concentrations of linolenic acid which did not uncouple but inhibited the electron flow at its optimum pH, (Fig. 1A and 2A). Agents such as NH,Cl and EDTA [5,6,8], detergents [8] and sonication [9] have also been found to induce a shift in the optimum of the electron transport rate which was attributed [7-91 to their uncoupling effect. As shown previously, this interpretation does not seem to be valid for the effect of linolenic acid [2]. At neutral pH (see Fig. 1B), linolenic acid stimulated electron transport, mainly by shifting the internal pH towards the optimum (say from 4.5 to 5 ) . Relations between the Shift in p H Optimum, ApH and p H i
In a recent series of papers, Avron’s group [ll,17, 231 has proposed that the rate of electron transport
P.-A. Siegenthalcr and F. Deptry
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may be controlled not only by energy coupling but also by d p H and pHi. From this, we predicted [2, lo], as did Avron’s group for uncouplers, that any treatment that deteriorates chloroplast membranes would decrease the light-induced proton uptake and dpH, and consequently modify p H i and the electron transport rate. The results of this investigation are in agreement with this hypothesis and show that fatty acids, which are known to damage the chloroplast membrane [l]. caused a decrease in ApH (Fig.3) and proton pump (Fig.5) which in turn, depending on pH,, changed pHi (Fig. 4) and the electron transport activity (Fig. 1 and 2). Similar results have been found in chloroplasts aged in vitro, such a treatment having in many respects the same deteriorating effects as fatty acids [I] (unpublished results). Fig. 6 illustrates our interpretation for the shift of the pH, optimum of electron flow toward acidity under these two conditions. In intact thylakoids, light initiates the proton pump mechanism which creates a ApH between the outer and inner space of the membrane. This results in a lowering of the pHi. In the light, pH, 8.5-9.0
corresponds to pH, close to 5, both pH values being optimal for electron transport. Under these normal conditions, the uptake of protons is accompanied by an exchange of cations and anions [25], by a shrinkage of the chloroplast and maximum photophosphorylation. Moreover, the extent of proton uptake, which may be a measure of the internal buffering capacity of the thylakoid [ll], is maximal around pH, 6.7. In any appreciation of the situation, one has to remember that the exact sequence of this series of events is difficult to establish: based on our results, the electron flow activity seems to be delicately controlled by the proton pump (buffering capacity), ApH, pHi, pH,, the kind of photosystem taken into consideration and, in treated chloroplasts, by the linolenic acid/chlorophyll ratio. In thylakoids treated by fatty acids or aged in vitro (Fig. 6 ) , the membrane becomes deteriorated in such a way that the proton uptake and ApH diminish, the extent of the decreases depending upon linolenic acid concentrations (Fig. 3) and aging time (unpublished results). The pHi values of course increase and eventually reach pH, (Fig.4); these values are no longer optimal for electron transport activity (Fig. 1 and 2). Since the size of ApH is lowered in treated chloroplasts, the pH, optimum for electron flow is shifted toward the acidic side and photophosphorylation diminishes [2]. A similar photophosphorylation dependence on the size of the d p H has also been reported by Pick et al. [22]. Under these conditions, one has to postulate that the fluxes of cations and anions are altered in such a way that swelling occurs, and lightinduced shrinkage decreases [l,261. Moreover, the overall extent of proton uptake which is supposed to be a measure of the internal buffering capacity [I 11 is diminished in treated chloroplasts and its optimum is shifted from pH, 6.7 to higher pH, as suggested by the results reported in Fig.3B and 4B and observed in Fig. 5. Although the extent of the proton uptake is mainly a function of the internal buffer capacity under normal conditions, it is also dependent on the rate of proton pumping and proton leakage. Linolenic acid may increase the proton leakage which in itself will decrease the extent of proton uptake and therefore no change in buffer capacitypev se will occur. At present, it is difficult to make a decision on this alternative. We have recently postulated [2] that in aged and fatty-acid-treated chloroplasts, lowering of pH, which thereby increased the external proton concentration, would accelerate the passive proton diffusion into the thylakoid compartment. In view of the present results, such a passive transport does not seem to be conclusive since in the presence of fatty acids, the size of light-induced d p H (and pH,) accounted for the observed electron flow rate. However, at very high concentrations of linolenic acid, where ApH is zero, proton diffusion might occur in the light in order to
580
P.-A. Siegenthaler and F. Deptry: ClSr3Effect on Electron Flow, dpH, pH, and Proton Pump in Chloroplasts
get the same pH in both inner and outer space of thylakoid. The present investigation offers an explanation for several observations [2] which were otherwise difficult to understand. The sensitivity of photosystem-I-associated photophosphorylation was greater at pH, 8.5 than at pH, 7.7 (see Fig. 3 in [2]). This difference can now be explained by considering the size of ApH in the two conditions (see Fig.3A); in the presence of appropriate concentrations of linolenic acid (see Fig.3A), ApH was larger at pH, 7.7 than 8.5 as was the photophosphorylation. On the other hand, we have demonstrated (see Fig. 1B and 2B in [2]) that in intact thylakoids, unsaturated fatty acids caused a sequential inhibition of photosystems I1 and I electron transports and of the associated photophosphoryations. This difference in sensitivity toward fatty acids can be directly correlated with the sensitivity of ApH in the two photosystems (compare Fig. 3A and B). Finally, we would like to mention that the meaning of the fluorescence quenching of acridines has been questioned recently by Kraayenhof [27] who assumes that the quenching is related to the energization of the membrane rather than to ApH [17]. Although not incompatible with the energization theory, our results and others [28] bring credit to the theory of Schuldiner et ul. [17]. We thank the Swiss National Foundation for ScientificResearch for its gcnerous support (Grant 3.2470.74 to P.A.S.), Mrs Jarmila Hordkovd for her able technical assistance in various parts of this work and Pierre-Andre Leuba for growing spinach. We are indebted to Dr R. A. Dilley for this critical review of the manuscript, to Mrs A. Robert for correcting the English and to the Geology Institute of the University of Ncuchdtcl for permitting the use of their spectrofluorimetric equipment.
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P.-A. Siegenthaler and F. Depery, Labordtoire de Physiologie VCgetale et Biochimic, Universite de Neuchlitel, Rue de Chantemerle 20, CH-2000 Neuchdtel, Switzerland
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