ferricyanide or 2,3, 5,6-tetramethyl-p-phenylene diamine. In the presence of ferricyanide, the magnitude of the signal was increased by the addition of ADP (in ...
Plant Physiol. (1973) 51, 1069-1073
The Relation between Photophosphorylation and Delayed Light Emission in Chloroplasts1 Received for publication Novembe: 30, 1972
JOSEPH NEUMANN,2 JIM BARBER, AND PETER GREGORY3 Department of Botany, Imperial College, Lonidon SW7, Entglanid ABSTRACT One millisecond delayed light emission has been studied in isolated coupled lettuce (Lactuca sativa var. romaine) chloroplasts. Delayed light emission was increased upon addition of ferricyanide or 2,3, 5,6-tetramethyl-p-phenylene diamine. In the presence of ferricyanide, the magnitude of the signal was increased by the addition of ADP (in the absence of orthophosphate), ATP, DIO-9, or phlorizin. The signal was also increased by the addition of NaCI and by the addition of NH4Cl in the presence of a high NaCl concentration. The signal of delayed light emission was decreased by the addition of gramicidin, valinomycin, and by the addition of NH4Cl in the presence of a low NaCl concentration. Phosphorylation, whether started by addition of ADP or by addition of glucose plus hexokinase plus ATP, caused a significant decrease in delayed light emission. It was concluded that the magnitude of delayed light emission reflects the size of the proton motive force across the thylakoid membrane. Calibration of delayed light emission by creating KCI gradients indicated that the value for the electrochemical potential gradient for H+ in the presence of ferricyanide was at least 155 millivolts decreasing to 134 millivolts after the onset of phosphorylation.
Goedheer (8) has suggested that DLE' in photosynthetic organisms results from back reactions of electrons and holes in acceptor and donor pools of photosystem II or bacterial reaction centers in a reversal of the light reactions. On the basis of studies involving the effect of uncouplers and phosphorylating agents on DLE in chloroplasts, Mayne (17) suggested that the high energy state of phosphorylation is an additional source of energy for DLE, in direct equilibrium with the photochemical act. Subsequently, Fleischmann (7) suggested that during the photoact charge separation across the thylakoid membrane would provide a store of energy in the form of electrical potential. This so-called membrane potential provides a source of energy in the dark for DLE by presumably lowering the activa'This work was supported by the Science Research Council, the Royal Society, and the Central Research Fund of the University of London. Also supported by a short term EMBO grant to J.N. ' Permanent address: Department of Botany, Tel Aviv University, Tel Aviv, Israel. 'Present address: Department of Biochemistry, Cornell University, Ithaca, N.Y. 14850. 'Abbreviations: DLE: delayed light emission; DAD: 2,3,5, 6-tetramethyl-p-phenylenediamine; p.m.f.: proton motive force.
tion energy for the recombination process. According to a more detailed model (6), the free energy difference between donor and acceptor pools is dependent (in addition to the concentration of Z+ and Q-) both on the proton concentration difference (ApH) and the electrostatic potential difference (AE) across the thylakoid membrane. Since according to the chemiosmotic hypothesis, AE and ApH comprise the driving force for ATP formation (19), DLE should indeed be closely related to photophosphorylation. In the present work, we have further studied this relationship using coupled phosphorylating lettuce chloroplasts. In addition, following the method of Barber (4, 5), the magnitude of the minimal value of the p.m.f. was estimated during electron flow and when electron flow was coupled to phosphorylation.
MATERIALS AND METHODS Chloroplasts were isolated from lettuce leaves Lactuica satii'a var. romaine. One hundred grams of leaves were homogenized for 15 sec in a Waring Blendor in 130 ml of grinding media of 0.4 M sorbitol, 50 mm Tricine (pH 7.8), 10 mm NaCl, 40 mM sodium ascorbate, and 1 mg/ml (final concentration) crystalline bovine serum albumin. The homogenate was transferred through 12 layers of gauze and centrifuged for 7 min at 1000g. The pellet was once washed in the grinding medium which did not contain ascorbate and albumin. The newly obtained pellet was resuspended in the washing medium plus 5 mg/ml (final concentration) bovine serum albumin at a chlorophyll concentration of approximately 1 mg/ml. The concentration of chlorophyll was measured according to Arnon (1). ATP formation was measured according to Nielsen and Lehninger (23) as modified by Avron (2). The reaction mixture for DLE studies included in a volume of 3 ml the following in ,moles (unless specified otherwise): Tricine 50 (pH 8.5), NaCl 50, MgCl 20, KPi 10, and chloroplasts equivalent to approximately 50 prg of chlorophyll. The intensity of delayed light was continuously measured with a rotating sector phosphoroscope as described previously (4). The photomultiplier output current was measured as a voltage developed across a 1 megaohm load resistor and recorded either on a Honeywell recorder or on a storage oscilloscope. The units of the DLE signal are relative. The reaction mixture was illuminated by white light at an intensity of 145 w/m2. During the course of the reaction, various compounds were rapidly injected with a syringe which had its needle inserted through a light-tight rubber diaphragm.
RESULTS Effects of Phosphorylation on the "Discharge" of the DLE Signal. Addition of ferricyanide in the light causes an increase in the extent of the signal of DLE which decays to a new steady state (Fig. la). Addition of ADP (to a reaction mixture
1069
1070
NEUMANN, BARB] ER, AND GREGORY
0 ADP 1,
ADP I
OFF I
-
l--
-_j!OFF
t
D.-
FeCN
ON
ON
FIG. 1. Effect of addition of cofactor and phosphorylating reagents on DLE. a: The reaction mixture contained in ,umoles the following compounds in a total volume of 3.0 ml: Tricine-50 (pH 8.5), NaCl-50, MgCl2-20, and KPi-10. It also contained chloroplasts equivalent to 40 ,ug of chlorophyll. Where indicated, 1 ,mole of potassium ferricyanide and 2 Amoles of ADP were added. The cuvette was illuminated with white light at an intensity of 145 w/m2. b: Conditions as in a, except that 0.6 ,umole of DAD was added.
ADP
GRAM
FBCN
Z6.
,20seS
Plant Physiol. Vol. 51, 1973
rapidly into ADP.) ATP by itself causes a stimulation of the signal of DLE, resembling the effect of ADP when added in the absence of Pi (see above). The reason for the stimulation of DLE by hexokinase and glucose (in the absence of ATP) is not clear and has not been studied further. Effect of Energy Transfer Inhibitors. If the decrease of the signal upon the addition of ADP is due to phosphorylation, it should be prevented by "energy transfer" inhibitors. In Figure 4, it is shown that DIO-9, a well known inhibitor of the terminal steps of ATP formation, when added by itself, stimulates the signal of DLE (resembling the effect of ADP or ATP). When ADP is added in the presence of a low concentration of DIO-9 (3.3 jig/ml), it causes only a small decrease in DLE (Fig. 4a); at a somewhat higher concentration of DIO-9 (10 ,tg/ml), there is no decrease at all upon addition of ADP (Fig. 4c). The latter indicates that DIO-9 and ADP (in the absence of Pi and Mg) increase DLE by the same mechanism. Furthermore, when DIO-9 is added after the addition of ADP, it abolishes the effect of ADP, and the signal is increased (Fig. 4, b and d). Wraight and Crofts (29) studied in detail the rise time of 1 msec DLE. In the presence of an electron acceptor, they observed a fast phase which they attributed to the formation of a membrane potential (AE), followed by a slow phase due to the formation of ApH. In our preparations, using the same time resolution as Wraight and Crofts, the kinetics of the formation of the signal of DLE did not appear to contain two distinct phases (Fig. 5a). Also, it was of interest to study the effect of phosphorylation and of DIO-9 on the DLE signal using the faster time resolution of the oscilloscope recording. Again, addition of ADP (in the presence of phosphorylating reagents) causes a decrease of the signal of DLE (Fig. 5b). DIO-9, when added after the addition of ADP, causes an increase of the signal above that of the control (Fig. 5c). Valinomycin at 1 /uM inhibits strongly the signal (Fig. Sd). It is interesting that the effects of DIO-9 and valinomycin on DLE are opposite (this difference will be discussed further below). Phlorizin, another energy transfer inhibitor (13), has a similar effect to DIO-9: When added alone, it causes an increase
ON
FIG. 2. Effect of ADP on DLE in the absence of Pi. The conditions were the same as those in Figure la, except that chloroplasts equivalent to 77 ,g of chlorophyll were added to the reaction mixture, and no KPi was present. Gramicidin at 0.33 ,uM was added where indicated.
which contained Mg2+ and Pi) causes a rapid decrease of the signal to a lower steady state level which returns to the zero level upon turning the light off. A similar sequence of changes takes place when ferricyanide is replaced by DAD (Fig. lb). DAD can support cyclic phosphorylation (10); however, in lettuce chloroplasts it can act as an autooxidizable electron acceptor (9). Apparently, the free radical of DAD mediates the transfer of electrons from the photosystem to oxygen (9). Injection of water or ethanol (at an equivalent volume to that of the solution of the electron carrier) had only a negligible effect on the signal. The first part of our work was aimed to show that the decrease of the signal in the light, upon addition of ADP, is indeed the result of ATP formation. The decrease of the signal caused by addition of ADP is dependent on the presence of Pi (Fig. 2). ADP by itself actually causes an increase in the signal (see "Discussion" for a possible explanation of this effect). In Figure 3, it is shown that addition of hexokinase-glucose and ATP has the same effect as addition of ADP (both in the presence of Pi) in causing a decrease of the signal. (In the presence of an excess of hexokinase and glucose, ATP is converted very
GLUCOSE HEXOKINASE
A20 sec, ON ON FIG. 3. Effect of glucose-hexokinase and ATP on DLE. The conditions were the same as those of Figure la, except that chloroplasts equivalent to 52 Ag of chlorophyll were added to the reaction mixture. One unit of Sigma hexokinase (grade III) and 10 ,umoles of glucose were added where indicated; 2 ,umoles of ATP were added where indicated.
Plant Physiol. Vol. 51, 1973
Calibration of the Proton Motive Force. Barber (4) has shown that it is possible to establish a diffusion potential across
I
ADP
ADP i i
4
DlO-9
1071
DELAYED LIGHT EMISSION
I;l
I,
OFF 4W
the thylakoid membrane (positive inside) by injecting a KCl solution into the chloroplasts suspension. He calibrated this signal quantitatively and used this calibration for the estimation of the light-induced DLE signal. However, as pointed out
/
OFF
|
a I-
b
i F
IFCN
2
lc~~~~~~~~~~~~~~~~~~~ ON
ON
,20 *sc
ON
d
@) IA
2
II
I
vI
I %
\
I,t Ij
1
A ADII
ON
ON I
I
I
.2
III
1.0
.6
I
I
IA
I I I 1.8 2.2
seconds DIO-9
F*CN
ON
ON , 20 ,
FIG. 4. Effect of DIO-9 on DLE. Experimental conditions as in Figure la. a; b: DIO-9 was added at a final concentration of 3.3 /Ag/ml; c, d: DIO-9 was added at a final concentration of 10 jig/ml.
in the signal, and in the presence of phlorizin, ADP does not decrease in the signal of DLE. Effect of Uncouplers. Several uncouplers were shown previously to inhibit DLE (17). Upon the addition of gramicidin, we have observed that the extent of "discharge" of the signal by subsequent addition of ADP decreased with increasing concentrations of the uncoupler. When gramicidin was added after the addition of ADP, the signal of DLE was further reduced (Fig. 2). The concentration of salt in the reaction mixture has a pronounced effect on the size of the DLE signal. When the concentration of NaCl was increased from 3.5 mm to 166 mm the magnitude of the signal of DLE was very much increased both in the "cofactorless" system and in the presence of ferricyanide (Fig. 6). Addition of NH4Cl to a reaction mixture which contained 3.5 mm NaCl inhibited the signal of DLE; however, the signal was markedly stimulated by NH4Cl in the presence of 166 mm NaCl (Fig. 6). Effect of Valinomycin. Valinomycin at 10 uM inhibits strongly the signal of DLE (Fig. 7). If ADP is added after the addition of valinomycin, there is only a very small additional decrease of the signal. In this experiment, the rate of ATP formation was measured (by following the incorporation of 3P into ATP) for 30 sec (after the addition of ADP) while the signal of DLE has been recorded. It is obvious from this experiment that the extent of the lowering of the signal by ADP is related to the rate of ATP formation. Valinomycin at 10 /tM inhibited both processes approximately 80%.
FIG. 5. Fast kinetics of DLE. The four experiments (a, b, c, d) were performed with separate samples of chloroplasts. In each experiment, the numbering indicates consecutive illuminations; thus in experiment a, DLE was measured in the absence of cofactor (1), FeCn was added and the sample was illuminated again (2). In experiment b, the sample was first illuminated in the presence of FeCN (1), then ADP was added (2). Otherwise experimental conditions as in Figure la. a: 1, no electron carrier; 2, FeCN (ferricyanide). b: 1, FeCN; 2, FeCN, ADP. c: 1, FeCN; 2, FeCN, ADP; 3, FeCN, ADP, DIO-9 10 ,ug/ml. d: 1, FeCN; 2, FeCN, 1 ,uM valinomycin. OFF
cause a
th1~~~41 NH4C_
NH4C1
OFF
FeCN ON
i
min
ON
FIG. 6. Effect of NH4C1 and NaCl on DLE. The reaction mixture contained 50 mM Tricine (pH 8.5). NaCl was present in a at 3.5 mm and in b at 166 mM. NH4Cl, where indicated, was added at 1.7 mm. The amount of chloroplasts added to the reaction mixture was equivalent to 50 ,ug of chlorophyll. Otherwise experimental conditions were as in Figure la.
1072
NEUMANN, BARBER, AND GREGORY
Plant
Physiol. Vol. 51,
1973
DISCUSSION Wraight and Crofts (29) studied in detail the intensity of lop M light emitted 1 msec after illumination as a function of the time of illumination. They observed a large increase in DLE ADP upon addition of ferricyanide or DAD. In the presence of an acceptor, they found a two phase kinetics of "formation" of the DLE signal: a rapid phase, complete in less than 0.1 sec which was correlated with the formation of a membrane potenADP tial and a slower phase with a half rise time of about 0.3 sec OFF correlated with formation of ApH. Ito et al. (12) also observed OFF two phases in the build up of the DLE signal; however, in their experiments the completion of the slow phase required approximately 25 sec. The signal of DLE obtained in our chloroplast preparation was also considerably stimulated upon the addition of an electron acceptor (Fig. 1). However, we did not observe two phases in the formation of the signal even in the -J FeCN presence of an acceptor (Fig. 5). Perhaps the "two phase" ON kinetics depends on dark preincubation (12). In addition, it is FIG. 7. Effect of valinomycin on ATP formation and DLE. Ex- possible that the chloroplast preparation from lettuce exhibited perimental conditions as in Figure la, except that chloroplasts a larger noncyclic "endogenous" electron transport which equivalent to 49 ,ug of chlorophyll were added to the reaction mix- supported the formation of both a membrane potential and a ture. ATP formation was measured for 30 sec after addition of ApH, even in the absence of an exogenous electron acceptor. ADP. Valinomycin at 10 /uM was added where indicated. The rate It has been reported previously that in the presence of phosof ATP formation was 77 ,umoles/mg chlorophyll-hr of illuminaphorylating reagents the extent of DLE is diminished (17). In tion in the control and 8 Amoles/mg chlorophyll- hr in the presence the work, we have monitored this effect kinetically. present of valinomycin. Injection of ADP into the reaction mixture in the light caused a decrease in DLE to a new steady state. This decrease of the mV signal is due to phosphorylation since it depends on the presence of phosphate, it does not take place in the presence of 160 ADP energy transfer inhibitors, and it can be obtained also when ADP is replaced by ATP plus glucose plus hexokinase. More155mV over, by measuring ATP formation it was shown that under the present experimental conditions, after addition of ADP, 150ATP is indeed formed (Fig. 7). From studies which involved the effect of various inhibitors, it can be concluded that the rate of ATP formation can be correlated at least semiquantitaOFF 140owith the extent of the DLE signal decrease due to phostively X.-1 34 mV phorylation (Figs. 4 and 7). This aspect of the work requires further investigation. 120Gramicidin, an uncoupler of photophosphorylation (3), was shown to inhibit proton uptake (26) and ApH (24). This comFe CN pound inhibited the signal of DLE whether added in the absence of ADP or after the addition of ADP (Fig. 2). The inhibition of DLE by gramicidin has been reported previously ON
mm.-
100
0
ON
(17).
20 Sec
FIG. 8. Calibration of proton motive force. Experimental ditions as in Figure la.
con-
(5) this approach has some drawbacks when applied under conditions where both electrical and pH gradients are contributing to the high energy state. Under these conditions, it is impossible to decide what fraction of the DLE represents the electrical or chemical gradients. Nevertheless, bearing in mind that there seems to be an exponential relationship between the intensity of emission and the p.m.f. (6), then one can at least use the KCI calibration procedure to predict the minimum value of the p.m.f. (that is, if all the p.m.f. was AE). Based on these considerations, the estimated (minimal) value of p.m.f. in the presence of ferricyanide was found to be 155 mv, and this value decreased to 134 mv upon addition of ADP (Fig. 8). The scale in Figure 8 has been calculated by analyzing KCI signals obtained with chloroplasts from the same preparations which were used for obtaining the signals with ferricyanide and with ADP but for calibration the chloroplasts were suspended in the simple buffer medium as described previously (4. 5).
NH4Cl is a well known uncoupler of photophosphorylation. Recently, we have shown that the mode of action of NH4Cl is complex and that under some conditions it can stimulate the rate of ATP formation (20-22). These conditions include the presence of high salt (22). As shown in the results of Figure 6, addition of NaCl at 166 mm causes a large increase in DLE. This effect might be related to the observation of Rottenberg et al. (25) who had shown that by increasing the salt concentration in the reaction mixture there is an increase in ApH.
The effect of NH4Cl on DLE as a function of salt concentration is clearly related to its effect on ATP formation. In low NaCl, NH4Cl inhibits ATP formation and DLE, whereas in high NaCl, NH4Cl stimulates both DLE and the rate of ATP formation (ref. 21 and Fig. 6). Since NH4Cl even in the presence of high salt inhibits proton uptake (21) and (as based on preliminary experiments) also ApH, the increase of DLE by NH4Cl under these conditions is presumably due to an increase in AE. ADP, ATP, and DIO-9 were shown to increase the extent of light-dependent proton uptake apparently by decreasing the rate of the leakage of protons through the thylakoid membrane at the ATP synthesizing site (18, 27). These compounds in-
Plant
Physiol. Vol. 51, 1973
DELAYED LIGHT
crease the magnitude of DLE as shown in Figures 2, 3, 4, and 5c. Phlorizin has a similar effect, indicating that it too may cause an increase of p.m.f. in the steady state. Our result with phlorizin differ in this respect from the results of Wells et al. (28). Valinomycin at low concentrations (1 nM-O. 1 pM) increases specifically the permeability of various membranal systems toward (11). In this concentration range, valinomycin does not affect ATP formation in chloroplasts (3). At higher concentrations valinomycin was found to inhibit the rate of coupled electron flow and ATP formation, while "basal" electron flow was inhibited only at high pH values, showing several properties which are characteristic of "energy transfer" inhibitors (15, 16). However, Keister and Minton (16) noted, that contrary to the action of other energy transfer inhibitors, valinomycin (at 1-33 Mm) when added in the presence of phosphorylating reagents further inhibited the signal of DLE (beyond the inhibition obtained by phosphorylating reagents). We have obtained similar results (Fig. 7). Also, it should be emphasized that the effect of valinomycin when added without phosphorylating reagents is opposite to that of DIO-9 or phlorizin (compare Fig. 4a and 4c with Fig.Sd). The work of Wraight and Crofts (29) and of Ito etal. (12). as well as our data, support the notion that the magnitude of DLE is a function of the total p.m.f. contributed both by ApH and by AE. The decrease of p.m.f. due to phosphorylation would support the chemiosmotic hypothesis. The extent of proton uptake in the light was shown by some workers (14) to be larger under phosphorylating conditions than under nonphosphorylating conditions. However, this result has recently been questioned, since it was found that ATP (which was formed during phosphorylation) increases the extent of proton uptake and consequently could have masked the decrease in the proton gradient which resulted from phosphorylation (18). If both AE and _pH comprise the driving force of phosphorylation in chloroplasts, and if DLE is an indicator of the total p.m.f., our results would strongly support the chemiosmotic hypothesis, by showing that in the steady state in light the p.m.f. is lower under phosphorylating than under nonphosphorylating conditions. Quantitative analyses of the signals in relation to ApR and AE is difficult, since one may be dealing with the product of two exponential functions (6). Nevertheless, the type of analysis presented in this paper may have some meaning, since it gives an estimate of the minimal value of the p.m.f. during the photoreactions.
K+
Acknzowledgment-We would like to thianik Dr. MT. Avron for rea(ling the
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dlelayed
XVRRAIGHT,
