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Correlation between monovalent cation-induced decreases in chlorophyll a fluorescence and chloroplast structural changes
ARCHIVES
OF BIOCHEMISTRY
Correlation Chlorophyll
AND
164, 460-468 (1974)
BIOPHYSICS
Between
Monovalent
a Fluorescence
Cation-induced
and
Chloroplast
Decreases
Structural
in
Changes
E. L. GROSS AND S. H. PRASHER Department
of Biochemistry,
The Ohio State University, Received February
Columbus,
Ohio 43210
12, 1974
Low concentrations (- 3 mM) of salts of monovalent cations such as Na+, K+, and tetraethylammonium were found to decrease the turbidity of chloroplast suspensions. The turbidity changes (AA,,,) had the same kinetics, salt concentration dependence, and pH dependence as the monovalent cation-induced decreases in chlorophyll a fluorescence (9), suggesting that structural changes are the cause of the associated increases in spillover. Electron microscopy revealed that the grana are stacked when spillover is inhibited (in the absence of salts or the presence of divalent cations) and that monovalent cations cause the grana to unstack, thereby promoting spillover.
Murata (l-3) and Homann (4) showed that salts of divalent cations such as Mg2+ or Ca*+ inhibit the spillover of excitation energy from Photosystem II to Photosystern I. The decrease in spillover can be determined by monitoring changes in chlorophyll a fluorescence at room temperature or 77°K under conditions in which photoby 3-(3,4chemistry is inhibited dichlorophenyl)-1, 1-dimethylurea. Murata (3), Murakami and Packer (5), and Mohanty et al. (6) demonstrated that divalent cation-induced structural changes (7, 8) accompany the increases in chlorophyll a fluorescence. In most of these experiments, low concentrations of tricine buffer or salts of monovalent cations were present. Gross and Hess (9) showed that the divalent cation-induced fluorescence increases did not occur when these salts were deleted from the medium. Moreover, addition of low concentrations (2-10 mM) of salts of monovalent cations such as K+, Na+, or tetraethylammonium promoted a decrease in chlorophyll a fluorescence at room temperature. Fluorescence emission spectra obtained at 77°K showed that the decrease in fluorescence at 685 nm emitted by Photosystem II was accompanied by an increase in fluorescence emitted at 735 nm
from Photosystem I, indicating that monovalent cations at low concentrations promote spillaver. Addition of divalent cations, under these conditions, reversed the effects of the monovalent cations (i.e., they inhibited spillover). Since divalent cation-induced increases in chlorophyll a fluorescence are accompanied by structural changes, it was of interest to determine whether structural changes also accompany the monovalent cation-induced decreases in fluorescence. Possible monovalent cation-induced changes in chloroplast membrane structure are of particular interest when considering the mechanism of regulation of energy transfer between the two photosystems since Seely (10, 11) has shown that the change in orientation of a few molecules in a computer model of a photosynthetic unit can dramatically affect the pattern of energy transfer between the two photosysterns.
460 C:opyrigbt All rights
Q 1071 by Academic Press. of reprorlucti<,n in any form
Inc. reserved.
MATERIALS
AND
METHODS
Chloroplast isolation. Spinach chloroplasts were isolated in 350 mM sucrose + 50 mM Tris-Cl buffer (pH 7.5) as previously described (12) after which they were washed once in 100 mM unbuffered sucrose and resuspended in the same medium.
MONOVALENT
CATION-INDUCED
Chlorophyll concentrations. Chlorophyll concentrations were determined according to the method of Arnon (13). Chlorophyll a fluorescence. Changes in chlorophyll a fluorescence were monitored as previously described (9) using an Aminco-Bowman Spectrofluorometer. The excitation and emission wavelengths were 470 and 680 nm, respectively. The reaction mixtures contained 100 mM sucrose + 0.2 mM Tris base (to titrate the chloroplast suspensions to pH 8) + 10 PM 3-(3,4-dichlorophenyl)-l,l-dimethylurea + other additions as indicated. The chlorophyll concentration was 6.7 &ml. Chloroplast
absorption
and
turbidity
changes.
Changes in light-scattering (turbidity) were monitored at 540 nm using an Aminco-Chance scanning/ dual wavelength spectrophotometer in its split beam mode of operation. Difference spectra were determined as previously described (14). Other conditions were the same as described above for the fluorescence measurements except that 3-(3,4-dichlorophenyl)-1, 1-dimethylurea was omitted for some experiments-its presence had no effect on the structural changes. Electron microscopy. The procedure used for fixation and embedding for electron microscopy was modified as follows from that of Stoner and Sirak (15). Chloroplasts (at 16.7 &ml chlorophyll) were incubated for 10 min at 4°C in the presence of 100 mM sucrose, 0.3 mM Tris base, and some combination of the salts, 3 mM NaCl and 1 mM CaCl,. Glutaraldehyde was added to a concentration of 1% and allowed to stand 10 min at 4’C. Hepes buffer (pH 8.6) and osmium tetroxide were added (final concentrations 3 mM and 0.25%, respectively) and allowed to stand 1 hr at 4°C. The chloroplast lamellae were pelleted by centrifugation in a Beckman 152 Microfuge, dehydrated with ethanol and propylene oxide, and embedded in Dow Epoxy Resin. The electron microscope used was a JEM 7.
STRUCTURAL
CHANGES
461
Several hundred chloroplasts from 5 different preparations were examined. The only criteria for selection used was that the chloroplasts were sectioned perpendicular to the plane of the membranes since tangential sections gave little information. RESULTS
Time course of monovalent cationinduced structural changes. Addition of 3.3
mM NaCl to chloroplasts suspended in 100 mM sucrose + 0.2 mM Tris base caused a parallel decrease in turbidity and chlorophyll a fluorescence (Fig. 1). Addition of an equal volume of water (not shown) had no effect. When 0.67 mM CaCl, was added after the NaCl, an increase in both chlorophyll a fluorescence and turbidity was observed. These changes are similar to those observed by other authors (3, 5). However, there are differences between the fluorescence and turbidity traces. For example, the turbidity changes are biphasic with one phase too fast to measure and a second slow increase which requires up to 2 min for completion (14). The fluorescence trace, on the other hand, shows only the slow phase. Thus, it may be that only the slow phase of the structural changes is involved in control of spillover. When 0.67 mM CaCl, was added first, a slight decrease in chlorophyll a fluorescence was observed. Similar results have been observed by Wydrinski et al. (16). This slight decrease in fluorescence is accompanied by an increase in turbidity. When 3.3 mM NaCl was added after the CaCl,, no further change in either chlorophyll a fluorescence or turbidity was ob-
FIG. 1. Timecourse of cation-induced changes in chlorophyll a fluorescence and turbidity. The effect of addition of either 3.3 mt.r NaCl or 0.67 mM CaCl, on the chlorophyll a fluorescence level and turbidity (AA,,,) were examined. Other conditions were as described in Materials and Methods.
462
GROSS AND PRASHER
served, indicating that divalent cations do something to the chloroplast membrane which prevents the action of monovalent cations. Furthermore, the Ca’+-induced structural changes alone are not sufficient to induce the fluorescence increases. The monovalent cations are also required. Concentration dependence of the structural changes. If the turbidity decreases
shown in Fig. 1 are related to the decreases in chlorophyll a fluorescence, both phenomena should have the same salt concentration dependence. This prediction is confirmed by the results presented in Fig. 2. Other cations (Table I) such as K+ and tetraethylammonium also cause turbidity decreases in the same concentration range. Thus both the turbidity decreases and the chlorophyll a fluorescence decreases (9) show no selectivity for one monovalent cation over another. pH dependence of Na+-induced decreases in turbidity and chlorophyll a fluorescence. If the turbidity decreases are related to the chlorophyll a fluorescence
decreases, they should show the same dependence on pH. When the pH is lowered (Fig. 3), the chlorophyll a fluorescence level decreases both in the presence and absence of 3.3 mM NaCl. These results agree with those obtained by Wraight et al. (17) obtained for uncoupled chloroplasts or chloroplasts in which the internal pH was lowered by illumination (18-20). However, our results are the opposite of those obtained by Mohanty et al. (5) in which lowering the pH to 3.8 caused an increase in fluorescence from Photosystem II compared to Photosystem I at 77°K. The reason for this difference is not clear. An increase in turbidity accompanied the decrease in fluorescence observed as the pH was lowered. Similar increases in turbidity have previously been observed by Packer et al. (21) and Gross and Libbey (14). Between pH 8 and 6, the fluorescence level obtained in the absence of NaCl decreased with decreasing pH whereas that obtained in the presence of 3.3 mM NaCl remained essentially constant. Therefore, the effect of NaCl on the fluorescence level (which is represented by the difference between the value obtained in the absence of NaCl and
[NaCl](mM)
FIG. 2. The NaCl
concentration dependences changes in turibidity and chlorophyll a fluorescence chloroplast suspensions. Various concentrations
of of
of NaCl were added to chloroplast suspensions and the effects on turbidity (AA,,,) and chlorophyll a fluorescence were recorded. The final fluorescence or turbidity levels obtained after salt addition were calculated as percent of the initial control level obtained prior to salt addition. Other conditions were as described in Materials and Methods. (0) Fluorescence, (0) AA,,,. TABLE I CATION
SPECIFICITY OF MONOVALENT CATION-INDUCED STRUCTURAL CHANCES
Cation NaCl KC1 Na-tricine (pH 8) TEA+ Cl
Lx (o/o) decrease 0.6 i 0.2 0.8 zt 0.3 0.7 f 0.3 0.5 f 0.2
12 11 7 11
aThe fluorescence yield was determined as a function of salt concentration after which the concentrations required for half-maximal response (C, ,..) and the maximal per cent decrease (Ii..) were calculated. Each value represents the average of five separate determinations. Other conditions were as described for Fig. 2 except that 3-(3,4-dichlorophenyl)-I,ldimethylurea was omitted from the reaction mixture.
that obtained in its presence) decreased with decreasing pH over this pH range. A similar result was obtained for the turbidity changes, confirming that the two effects are related. Wavelength dependence of Na+ ioninduced structural changes in chloroplasts.
To obtain more information concerning the nature of the Na+ ion-induced structural changes, effects on the total visible absorption spectra were examined. The results (Fig. 4) are presented as difference spectra in which the chloroplasts in the reference cuvette were suspended in 100 mM sucrose
MONOVALENT
CATION-INDUCED
STRUCTURAL
463
CHANGES
absorption changes of the same sign. Addition of CaCl, first (Fig. 4B) caused an increase in turbidity accompanied by a decrease in pigment absorption, which agrees with previous results (14). Addition of NaCl after the CaCl, caused no change in the turbidity @A,,,), which agrees with the results presented in Fig. 1, but did cause a further decrease in pigment absorption at 435, 470, and 680 nm. Our absorption spectra (Fig. 4) show that addition of NaCl caused a decrease both in turbidity and pigment absorption. However, the question arises as to whether the two effects are related. If they are, they should show the same kinetics upon NaCl 6.0 70 so 9.0 addition. In order to test this hypothesis, PH kinetics of the salt-induced absorption FIG. 3. pH dependence of monovalent cationchanges were determined at three waveinduced changes in turbidity and chlorophyll a fluo- lengths (435,470, and 540 nm). When NaCl rescence. Chloroplasts were incubated in 100 mM was added before the CaCl, (Fig. 5), it sucrose + 10 FM 3-(3,4-dichlorophenyl)-l,l-dimethylcaused decreases in absorbance at all three urea in the presence or absence of 3.3 mM NaCl. The wavelengths. The kinetics are similar original pH was about 6.2. The chloroplasts were then showing an initial rapid decrease followed titrated to the pH values indicated with either HCl or
-I050
Tris base. Then, the levels of turbidity @A,,,) and chlorophyll a fluorescence were determined as described in Materials and Methods.
+ 0.2 mM Tris base and those in the sample compartment were suspended in same medium with the addition of the salts in question. Absorption changes in region between 500 and 650 nm are attributable primarily to light-scattering changes whereas those in the absorption bands are due to “absorption flattening” or “seiving” effects (22, 23), resulting from a change in the spatial location of pigments. Addition of 3.3 mM NaCl caused a decrease both in turbidity (at 540 nm) and in pigment absorption (at 430, 470, and 680 nm). This result is surprising since a decrease in turbidity is usually accompanied by an increase in pigment absorption and vice-versa (see Fig. 4B and Refs. 14, 23, and 24). Addition of 0.67 mM CaCl, after the NaCl caused an increase in turbidity (confirming the results of Fig. 1) and a further decrease in pigment absorption. Thus the effects on Na+ and Ca2+ ions on turbidity and pigment absorption changes are quite different. They cause light-scattering changes of opposite sign but pigment
+10r
0
,/--
-10 '\
CO.-Me
r___-----
,-'.I
8
-20 P
9’ 2 +Kl
r
I 400
500
600
700
x h-d
FIG. 4. Difference spectra for cation-induced structural changes in chloroplasts. Chloroplasts (at 10 &ml chlorophyll)were suspended in 100 mM sucrose + 0.2 mM Tris base. Salts as indicated were added to the sample cuvette and the resulting difference spectra were determined. (A) 3.3 mM NaCl (final concentration) was added first (dashed line) followed by 0.67 mM CaCl, (solid line). (B) 0.67 mM CaCl, was added first (dashed line) followed by 3.3 mM NaCl (solid line). Other conditions were as described in Materials and Methods.
464
GROSS AND PRASHER
I
AA=002
~,,,“, :?c FIG. 5. Comparison of the kinetics of the cationinduced absorption changes at 435, 470, and 540 nm.
The absorbance changes at 435,470, and 540 nm were monitored upon addition of either 3.3 mM NaCl or 0.67 mM CaCl,. Other conditions were as for the Materials and Methods section except that 3-(3,4dichlorophenyl)-1 , I-dimethylurea was omitted from the reaction mixture.
concentrations of NaCl caused a decrease in turbidity (AA,,,) but higher concentrations reversed this effect causing an increase in turbidity once more. These results parallel those obtained for chlorophyll a fluorescence (9). The increases in turbidity observed at higher NaCl concentrations also confirm previous results (5, 14). On the other hand, the absorbance at 435 nm showed a steady decrease with increasing NaCl concentration. Thus, pigment absorption at 430 nm decreases with increasing NaCl concentration regardless of the direction of the Na+-induced changes in turbidity and chlorophyll a fluorescence. These results suggest that both chlorophyll a fluorescence and turbidity changes reflect one aspect of the structural changes whereas the pigment absorption changes (monitored at 430 nm) reflect another aspect of the structural changes. It is of interest that Govindjee and Vander Muellen (25) observed that Na+ and Ca2+ both cause an increase in a-anilinonapthalene-1-sulfonic acid fluorescence. Thus, these fluorescence increases may be more closely correlated with the pigment absorption decreases than with changes in turbidity or chlorophyll a fluorescence.
by a slower phase which required 30 set for completion. Addition of CaCl, after the NaCl caused an increase in turbidity (AA,,,) together with a decrease in absorbance at 435 nm. The absorbance changes observed at both wavelengths were biphasic. No change was observed at 470 nm. Ultrastructure of chloroplasts These results are consistent with those presented in Fig. 4 for the corresponding with mono- and divalent cations. steady-state absorption spectra. When the nature of the Na-induced CaCl, was added before the NaCl, it caused an increase in absorbance at 540 nm accompanied by decreases at 470 and 435 nm. These results are consistent with those previously obtained (see Fig. 4 and Ref. 14). Addition of NaCl after CaCl, caused a further decrease in absorbance at 435 and 470 nm similar to that shown in Fig. 4. The similarity of the kinetics observed at 435, 470, and 540 nm for the NaCl-induced structural changes suggests that they re-0.1 flect the same underlying process. The effect of NaCl concentration on 2o ~aCl]~M,*O absorbance
changes at 435 and 540 nm.
Previously (14), we have observed that NaCl caused an increase in turbidity associated with a decrease in pigment absorption albeit at higher NaCl concentrations. A complete concentration curve for the NaCl-induced changes in absorbancy at 435 and 540 nm (Fig. 6) showed that low
treated
To clarify structural
+5
-20
FIG. 6. NaCl concentration dependence of absorbance changes at 435 and 540 nm. Chloroplasts were incubated in the presence of the NaCl concentrations indicated and the absorbances at 435 and 540 nm were determined. Conditions were as described in Materials and Methods except that 3-(3,4-dichlorophenyl)-1, 1-dimethylurea was omitted from the reaction medium. (0) AA,,,, (0) AA,,,.
MONOVALENT
CATION-INDUCED
change, we used electron microscopy. Figures 7 and 8 show chloroplasts which were incubated in 100 mM sucrose + 0.2 mM Tris base and either no additions (Fig. 7A), 3 mM NaCl (Fig. 7B), 1 mM CaCl, (Fig. 8A), and/or both added together (Fig. 8B). Approximately 95% of the chloroplasts observed had lost their outer membranes. However, the inner membranes are still connected together. Chloroplasts fixed in the absence of either NaCl or CaCl, show grana stacking’ but are somewhat swollen. The observation of the existence of grana stacks under extremely low ionic strength at first glance disagrees with the results of Izawa and Good (26), Okhi et al. (27), and Murakami and Packer (4), who observed no grana stacking under what they called “low ionic strength” conditions. However, the difference can probably be accounted for by the difference in conditions employed. Murakami and Packer (4) suspended their chloroplasts in distilled water, thereby adding osmotic stress to low ionic strength. Since we had 100 mM sucrose present, we were only looking at ionic effects. Both Good and Izawa (26) and Okhi et al. (27) had 50 mM tricine present. Therefore, their conditions did not correspond to our very low ionic strength conditions but may correspond to our chloroplasts incubated in 3 mM NaCl (see below). Also, we can rule out the hypothesis (28) that unstacking of the thylakoids is the result of repulsion of negative charges unmasked by the low ionic strength conditions since our chloroplasts remain stacked at low ionic strength. Addition of 3 mM NaCl to the low ionic strength chloroplasts causes unstacking of the thylakoids (Fig. 7B). This corresponds to the case in which spillover is promoted. It is these chloroplasts that resemble those incubated in the presence of tricine buffer (26, 27, 29) which has also been shown to promote spillover (9). Thus, it is not low ionic strength per se but the presence of low concentrations of NaCl or tricine buffer which causes unstacking. 1In the absence of salts, there sometimes appear to be fewer grana stacks than observed in the presence of CaCl,. However, there is no evidence that this is statistically significant.
STRUCTURAL
CHANGES
465
Addition of 1 mM CaCl, to chloroplasts suspended in low ionic strength (Fig. 8A) causes shrinkage in a direction perpendicular to the plane of the lamellae. These results are consistent with the turbidity and absorption flattening changes described above. Addition of 1 mM CaCl, after incubation of the chloroplasts in 3 mM NaCl (not shown) caused the majority of the grana to restack. Thus Caz+ ions can reverse as well as prevent the Na+ ioninduced unstacking of the lamellae. Addition of 1 mM CaCl, prevents the NaClinduced unstacking of the thylakoids (Fig. 8B) as well as the Na-induced fluorescence changes (Fig. 1). Thus, when the grana appear stacked either under very low ionic strength conditions and/or in the presence of divalent cations, spillover is inhibited. When the thylakoids are unstacked, spillover is promoted. Unstacking the grana probably changes the relative positions of the two photosystems, allowing greater spillover. One might question whether such a mechanism for control of spillover could occur in vivo in the presence of a high concentration of cations (30, 31). However, one could imagine local fluctuations in cation concentration which could produce these effects. Also, there may be endogenous agents which, like tricine buffer, allow unstacked thylakoids to exist at higher salt concentrations. Furthermore, a small amount of unstacking which might not require extreme conditions may serve to control the photosynthetic process. The antagonistic effects of mono- and divalent cations on chloroplast structure and chlorophyll a fluorescence are reminescent of similar antagonistic effects on other processes in plants. These include membrane permeability (32, 33) and stomata1 closure (33). CONCLUSIONS
The close correlation observed between monovalent cation-induced decreases in turbidity and chlorophyll a fluorescence indicates that the structural changes are responsible for the increases in spillover. Ultrastructural studies showed that low concentrations of NaCl caused unstacking
FIG. 7. The effect of NaCZ on the ultrastructure ofchlorophts. Chloroplasts were incubated in 100 rn~ sucrose + 0.2 mM Tris base (A) and in the same medium + 3.0 mM NaCl (B) prior to fixation with glutaraldehyde. Other conditions were as described in Materials and Methods. The magnification was 25,000x. 466
FIG. 8. The effect of C&Y, on the ultrastructure of chloroplasts. Chloroplasts were incubated in the presence of 1 mM CaCI, (A) and 1 mM CaCl, + 3 mM NaCl (B) prior to fixation. Other conditions were as described for Fig. 7. The magnification was 25,000x. 467
468
GROSS AND PRASHER
of the grana lamellae which could be prevented or reversed by addition of salts of divalent cations. Thus, spillover was enhanced when the lamellae were unstacked and inhibited when stacking occurred either in the absence of salts and/or the presence of divalent cations. The unstacking of the lamellae caused by the monovalent cations must either change the mutual orientation of the two photosystems or bring them into closer proximity. Experiments are now in progress to determine the molecular mechanism of this process. ACKNOWLEDGMENTS This work was supported in part by Grant No. GB19156 from the National Science Foundation. We thank Dr. Robert M. Pfister, Dr. Clinton Stoner, and Miss Adele Arar for help with the electron microscopy. We also thank Dr. Stoner for use of the electron microscope. REFERENCES 1. MURATA, N. (1969) Biochim. Biophys. Acta 189, 171-181. 2. MURATA, N., TASHIRO, H., AND TAKAMIYA, A. (1970) Btichim. Biophys. Acta 197, 250-256. 3. MURATA, N. (1971) Biochim. Biophys. Acta 245, 365-372. 4. HOMANN, P. (1969) Plant Physiol. 44,932-936. 5. MURAKAMI, S., AND PACKER, L. (1971) Arch. Biothem. Biophys. 146,4837-347. 6. MOHANTY, P., BRAUN, B. Z., AND GOVINDJIE (1973) Biochim. Biophys. Acta 292,459-476. 7. GROSS, E., AND PACKER, L. (1967) Arch. Biochem. Biophys. 121, 779-789. 8. DILLEY, R. A., AND ROTHSTEIN, A. (1967) Biochim. Biophys. Acta 135, 427-443. 9. GROSS, E. L., AND HESS, S. C. (1973) Arch Biochem. Biophys. 159,832-836. 10. SEELY, G. R. (1973) J. Theor. Biol. 40, 173-187. 11. SEELY, G. R. (1973) J. Z’heor. Biol. 40, 188-198. 12. GROSS, E. L. (1971) Arch. Biochem. Biophys. 147, 77-84.
13. ARNON, D. I. (1949) Plant Physiol. 24, 1-15. 14. GROSS, E. L., AND LIBBEY, J. W. (1972) Arch. Biochem. Biophys. 153,457-467. 15. STONER, C. D., AND SIRAK, H. D. (1973) J. Cell Biol. 56.51-64. 16. WYDRINSKI, T., GROSS, E. L., AND GOVINDJEE, in
preparation. 17. WRAIGHT, C. A., KRAAN, G. P. B., ANDGEFXITS, N. M. (1972). Biochim. Biophys. Acto 283, 259-267.
18. MURATA, N., AND SUGAHARA, K. (1969) Biochim. Biophys. Acta 189, 182-192. 19. COHEN, W. S., AND SHERMAN, L. A. (1971), FEBS Lett. 16, 319-323. 20. WRAIGHT, C. A., AND CROFTS, A. R. (1970) Eur. J. Biochem.
17,319-327.
21. PACKER, L., DEAMER, D. W., AND CROFTS, A. R. (1966) In Energy Conversion by the Photosynthetic Apparatus, pp. 281-302, Brookhaven Symposium No. 19. 22. DUYSENS, L. M. N. (1956) Biochim. Biophys. Acta 19, 1-12. 23. ITOH, M., IZAWA, S., AND SHIBATA, K. (1963) Biochem. Biophys. Acta’69, 130-140. 24. SHAVIT, N., AND AVRON, M. (1967) Biochim. Biophys. Acta 131, 516-525. 25. GOVINDJEE AND VANDER MUELLEN, D., in prepara-
tion. 26. IZAWA, S., AND GOOD, N. E. (1966) Plant Physiol. 41,544-552. 27. OHKI, R., KUNIEDA, R., AND TAKAMIYA, A. (1971) Biochim. Biophys. Acta 226, 144-153. 28. KIRK, J. T. 0. (1971). in Annual Review of
Biochemistry, Vol. 40, pp. 161-196, Annual Reviews Inc., Palo Alto. 29. ANDERSON, J. M., AND VERNON, L. R. (1967) Biochim. Biophys. Acta 143, 363-376. 30. NOBEL, P. S. (1969) Biochim. Biophys. Acta 172, 134-143. 31. GROSS. E. L., AND HESS, S. C. (1974) Biochim. Biophys. Acta (in press). 32. BONNEX, J., AND GLASTON, A. W. (1952) in Princi-
ples of Plant Physiology, p. 95, W. H. Freeman and Co., San Francisco. 33. KLJIPER, R., J., C., (1972) Annu. Reu. Plant Physiol. 23,157-72.
OF BIOCHEMISTRY
Correlation Chlorophyll
AND
164, 460-468 (1974)
BIOPHYSICS
Between
Monovalent
a Fluorescence
Cation-induced
and
Chloroplast
Decreases
Structural
in
Changes
E. L. GROSS AND S. H. PRASHER Department
of Biochemistry,
The Ohio State University, Received February
Columbus,
Ohio 43210
12, 1974
Low concentrations (- 3 mM) of salts of monovalent cations such as Na+, K+, and tetraethylammonium were found to decrease the turbidity of chloroplast suspensions. The turbidity changes (AA,,,) had the same kinetics, salt concentration dependence, and pH dependence as the monovalent cation-induced decreases in chlorophyll a fluorescence (9), suggesting that structural changes are the cause of the associated increases in spillover. Electron microscopy revealed that the grana are stacked when spillover is inhibited (in the absence of salts or the presence of divalent cations) and that monovalent cations cause the grana to unstack, thereby promoting spillover.
Murata (l-3) and Homann (4) showed that salts of divalent cations such as Mg2+ or Ca*+ inhibit the spillover of excitation energy from Photosystem II to Photosystern I. The decrease in spillover can be determined by monitoring changes in chlorophyll a fluorescence at room temperature or 77°K under conditions in which photoby 3-(3,4chemistry is inhibited dichlorophenyl)-1, 1-dimethylurea. Murata (3), Murakami and Packer (5), and Mohanty et al. (6) demonstrated that divalent cation-induced structural changes (7, 8) accompany the increases in chlorophyll a fluorescence. In most of these experiments, low concentrations of tricine buffer or salts of monovalent cations were present. Gross and Hess (9) showed that the divalent cation-induced fluorescence increases did not occur when these salts were deleted from the medium. Moreover, addition of low concentrations (2-10 mM) of salts of monovalent cations such as K+, Na+, or tetraethylammonium promoted a decrease in chlorophyll a fluorescence at room temperature. Fluorescence emission spectra obtained at 77°K showed that the decrease in fluorescence at 685 nm emitted by Photosystem II was accompanied by an increase in fluorescence emitted at 735 nm
from Photosystem I, indicating that monovalent cations at low concentrations promote spillaver. Addition of divalent cations, under these conditions, reversed the effects of the monovalent cations (i.e., they inhibited spillover). Since divalent cation-induced increases in chlorophyll a fluorescence are accompanied by structural changes, it was of interest to determine whether structural changes also accompany the monovalent cation-induced decreases in fluorescence. Possible monovalent cation-induced changes in chloroplast membrane structure are of particular interest when considering the mechanism of regulation of energy transfer between the two photosystems since Seely (10, 11) has shown that the change in orientation of a few molecules in a computer model of a photosynthetic unit can dramatically affect the pattern of energy transfer between the two photosysterns.
460 C:opyrigbt All rights
Q 1071 by Academic Press. of reprorlucti<,n in any form
Inc. reserved.
MATERIALS
AND
METHODS
Chloroplast isolation. Spinach chloroplasts were isolated in 350 mM sucrose + 50 mM Tris-Cl buffer (pH 7.5) as previously described (12) after which they were washed once in 100 mM unbuffered sucrose and resuspended in the same medium.
MONOVALENT
CATION-INDUCED
Chlorophyll concentrations. Chlorophyll concentrations were determined according to the method of Arnon (13). Chlorophyll a fluorescence. Changes in chlorophyll a fluorescence were monitored as previously described (9) using an Aminco-Bowman Spectrofluorometer. The excitation and emission wavelengths were 470 and 680 nm, respectively. The reaction mixtures contained 100 mM sucrose + 0.2 mM Tris base (to titrate the chloroplast suspensions to pH 8) + 10 PM 3-(3,4-dichlorophenyl)-l,l-dimethylurea + other additions as indicated. The chlorophyll concentration was 6.7 &ml. Chloroplast
absorption
and
turbidity
changes.
Changes in light-scattering (turbidity) were monitored at 540 nm using an Aminco-Chance scanning/ dual wavelength spectrophotometer in its split beam mode of operation. Difference spectra were determined as previously described (14). Other conditions were the same as described above for the fluorescence measurements except that 3-(3,4-dichlorophenyl)-1, 1-dimethylurea was omitted for some experiments-its presence had no effect on the structural changes. Electron microscopy. The procedure used for fixation and embedding for electron microscopy was modified as follows from that of Stoner and Sirak (15). Chloroplasts (at 16.7 &ml chlorophyll) were incubated for 10 min at 4°C in the presence of 100 mM sucrose, 0.3 mM Tris base, and some combination of the salts, 3 mM NaCl and 1 mM CaCl,. Glutaraldehyde was added to a concentration of 1% and allowed to stand 10 min at 4’C. Hepes buffer (pH 8.6) and osmium tetroxide were added (final concentrations 3 mM and 0.25%, respectively) and allowed to stand 1 hr at 4°C. The chloroplast lamellae were pelleted by centrifugation in a Beckman 152 Microfuge, dehydrated with ethanol and propylene oxide, and embedded in Dow Epoxy Resin. The electron microscope used was a JEM 7.
STRUCTURAL
CHANGES
461
Several hundred chloroplasts from 5 different preparations were examined. The only criteria for selection used was that the chloroplasts were sectioned perpendicular to the plane of the membranes since tangential sections gave little information. RESULTS
Time course of monovalent cationinduced structural changes. Addition of 3.3
mM NaCl to chloroplasts suspended in 100 mM sucrose + 0.2 mM Tris base caused a parallel decrease in turbidity and chlorophyll a fluorescence (Fig. 1). Addition of an equal volume of water (not shown) had no effect. When 0.67 mM CaCl, was added after the NaCl, an increase in both chlorophyll a fluorescence and turbidity was observed. These changes are similar to those observed by other authors (3, 5). However, there are differences between the fluorescence and turbidity traces. For example, the turbidity changes are biphasic with one phase too fast to measure and a second slow increase which requires up to 2 min for completion (14). The fluorescence trace, on the other hand, shows only the slow phase. Thus, it may be that only the slow phase of the structural changes is involved in control of spillover. When 0.67 mM CaCl, was added first, a slight decrease in chlorophyll a fluorescence was observed. Similar results have been observed by Wydrinski et al. (16). This slight decrease in fluorescence is accompanied by an increase in turbidity. When 3.3 mM NaCl was added after the CaCl,, no further change in either chlorophyll a fluorescence or turbidity was ob-
FIG. 1. Timecourse of cation-induced changes in chlorophyll a fluorescence and turbidity. The effect of addition of either 3.3 mt.r NaCl or 0.67 mM CaCl, on the chlorophyll a fluorescence level and turbidity (AA,,,) were examined. Other conditions were as described in Materials and Methods.
462
GROSS AND PRASHER
served, indicating that divalent cations do something to the chloroplast membrane which prevents the action of monovalent cations. Furthermore, the Ca’+-induced structural changes alone are not sufficient to induce the fluorescence increases. The monovalent cations are also required. Concentration dependence of the structural changes. If the turbidity decreases
shown in Fig. 1 are related to the decreases in chlorophyll a fluorescence, both phenomena should have the same salt concentration dependence. This prediction is confirmed by the results presented in Fig. 2. Other cations (Table I) such as K+ and tetraethylammonium also cause turbidity decreases in the same concentration range. Thus both the turbidity decreases and the chlorophyll a fluorescence decreases (9) show no selectivity for one monovalent cation over another. pH dependence of Na+-induced decreases in turbidity and chlorophyll a fluorescence. If the turbidity decreases are related to the chlorophyll a fluorescence
decreases, they should show the same dependence on pH. When the pH is lowered (Fig. 3), the chlorophyll a fluorescence level decreases both in the presence and absence of 3.3 mM NaCl. These results agree with those obtained by Wraight et al. (17) obtained for uncoupled chloroplasts or chloroplasts in which the internal pH was lowered by illumination (18-20). However, our results are the opposite of those obtained by Mohanty et al. (5) in which lowering the pH to 3.8 caused an increase in fluorescence from Photosystem II compared to Photosystem I at 77°K. The reason for this difference is not clear. An increase in turbidity accompanied the decrease in fluorescence observed as the pH was lowered. Similar increases in turbidity have previously been observed by Packer et al. (21) and Gross and Libbey (14). Between pH 8 and 6, the fluorescence level obtained in the absence of NaCl decreased with decreasing pH whereas that obtained in the presence of 3.3 mM NaCl remained essentially constant. Therefore, the effect of NaCl on the fluorescence level (which is represented by the difference between the value obtained in the absence of NaCl and
[NaCl](mM)
FIG. 2. The NaCl
concentration dependences changes in turibidity and chlorophyll a fluorescence chloroplast suspensions. Various concentrations
of of
of NaCl were added to chloroplast suspensions and the effects on turbidity (AA,,,) and chlorophyll a fluorescence were recorded. The final fluorescence or turbidity levels obtained after salt addition were calculated as percent of the initial control level obtained prior to salt addition. Other conditions were as described in Materials and Methods. (0) Fluorescence, (0) AA,,,. TABLE I CATION
SPECIFICITY OF MONOVALENT CATION-INDUCED STRUCTURAL CHANCES
Cation NaCl KC1 Na-tricine (pH 8) TEA+ Cl
Lx (o/o) decrease 0.6 i 0.2 0.8 zt 0.3 0.7 f 0.3 0.5 f 0.2
12 11 7 11
aThe fluorescence yield was determined as a function of salt concentration after which the concentrations required for half-maximal response (C, ,..) and the maximal per cent decrease (Ii..) were calculated. Each value represents the average of five separate determinations. Other conditions were as described for Fig. 2 except that 3-(3,4-dichlorophenyl)-I,ldimethylurea was omitted from the reaction mixture.
that obtained in its presence) decreased with decreasing pH over this pH range. A similar result was obtained for the turbidity changes, confirming that the two effects are related. Wavelength dependence of Na+ ioninduced structural changes in chloroplasts.
To obtain more information concerning the nature of the Na+ ion-induced structural changes, effects on the total visible absorption spectra were examined. The results (Fig. 4) are presented as difference spectra in which the chloroplasts in the reference cuvette were suspended in 100 mM sucrose
MONOVALENT
CATION-INDUCED
STRUCTURAL
463
CHANGES
absorption changes of the same sign. Addition of CaCl, first (Fig. 4B) caused an increase in turbidity accompanied by a decrease in pigment absorption, which agrees with previous results (14). Addition of NaCl after the CaCl, caused no change in the turbidity @A,,,), which agrees with the results presented in Fig. 1, but did cause a further decrease in pigment absorption at 435, 470, and 680 nm. Our absorption spectra (Fig. 4) show that addition of NaCl caused a decrease both in turbidity and pigment absorption. However, the question arises as to whether the two effects are related. If they are, they should show the same kinetics upon NaCl 6.0 70 so 9.0 addition. In order to test this hypothesis, PH kinetics of the salt-induced absorption FIG. 3. pH dependence of monovalent cationchanges were determined at three waveinduced changes in turbidity and chlorophyll a fluo- lengths (435,470, and 540 nm). When NaCl rescence. Chloroplasts were incubated in 100 mM was added before the CaCl, (Fig. 5), it sucrose + 10 FM 3-(3,4-dichlorophenyl)-l,l-dimethylcaused decreases in absorbance at all three urea in the presence or absence of 3.3 mM NaCl. The wavelengths. The kinetics are similar original pH was about 6.2. The chloroplasts were then showing an initial rapid decrease followed titrated to the pH values indicated with either HCl or
-I050
Tris base. Then, the levels of turbidity @A,,,) and chlorophyll a fluorescence were determined as described in Materials and Methods.
+ 0.2 mM Tris base and those in the sample compartment were suspended in same medium with the addition of the salts in question. Absorption changes in region between 500 and 650 nm are attributable primarily to light-scattering changes whereas those in the absorption bands are due to “absorption flattening” or “seiving” effects (22, 23), resulting from a change in the spatial location of pigments. Addition of 3.3 mM NaCl caused a decrease both in turbidity (at 540 nm) and in pigment absorption (at 430, 470, and 680 nm). This result is surprising since a decrease in turbidity is usually accompanied by an increase in pigment absorption and vice-versa (see Fig. 4B and Refs. 14, 23, and 24). Addition of 0.67 mM CaCl, after the NaCl caused an increase in turbidity (confirming the results of Fig. 1) and a further decrease in pigment absorption. Thus the effects on Na+ and Ca2+ ions on turbidity and pigment absorption changes are quite different. They cause light-scattering changes of opposite sign but pigment
+10r
0
,/--
-10 '\
CO.-Me
r___-----
,-'.I
8
-20 P
9’ 2 +Kl
r
I 400
500
600
700
x h-d
FIG. 4. Difference spectra for cation-induced structural changes in chloroplasts. Chloroplasts (at 10 &ml chlorophyll)were suspended in 100 mM sucrose + 0.2 mM Tris base. Salts as indicated were added to the sample cuvette and the resulting difference spectra were determined. (A) 3.3 mM NaCl (final concentration) was added first (dashed line) followed by 0.67 mM CaCl, (solid line). (B) 0.67 mM CaCl, was added first (dashed line) followed by 3.3 mM NaCl (solid line). Other conditions were as described in Materials and Methods.
464
GROSS AND PRASHER
I
AA=002
~,,,“, :?c FIG. 5. Comparison of the kinetics of the cationinduced absorption changes at 435, 470, and 540 nm.
The absorbance changes at 435,470, and 540 nm were monitored upon addition of either 3.3 mM NaCl or 0.67 mM CaCl,. Other conditions were as for the Materials and Methods section except that 3-(3,4dichlorophenyl)-1 , I-dimethylurea was omitted from the reaction mixture.
concentrations of NaCl caused a decrease in turbidity (AA,,,) but higher concentrations reversed this effect causing an increase in turbidity once more. These results parallel those obtained for chlorophyll a fluorescence (9). The increases in turbidity observed at higher NaCl concentrations also confirm previous results (5, 14). On the other hand, the absorbance at 435 nm showed a steady decrease with increasing NaCl concentration. Thus, pigment absorption at 430 nm decreases with increasing NaCl concentration regardless of the direction of the Na+-induced changes in turbidity and chlorophyll a fluorescence. These results suggest that both chlorophyll a fluorescence and turbidity changes reflect one aspect of the structural changes whereas the pigment absorption changes (monitored at 430 nm) reflect another aspect of the structural changes. It is of interest that Govindjee and Vander Muellen (25) observed that Na+ and Ca2+ both cause an increase in a-anilinonapthalene-1-sulfonic acid fluorescence. Thus, these fluorescence increases may be more closely correlated with the pigment absorption decreases than with changes in turbidity or chlorophyll a fluorescence.
by a slower phase which required 30 set for completion. Addition of CaCl, after the NaCl caused an increase in turbidity (AA,,,) together with a decrease in absorbance at 435 nm. The absorbance changes observed at both wavelengths were biphasic. No change was observed at 470 nm. Ultrastructure of chloroplasts These results are consistent with those presented in Fig. 4 for the corresponding with mono- and divalent cations. steady-state absorption spectra. When the nature of the Na-induced CaCl, was added before the NaCl, it caused an increase in absorbance at 540 nm accompanied by decreases at 470 and 435 nm. These results are consistent with those previously obtained (see Fig. 4 and Ref. 14). Addition of NaCl after CaCl, caused a further decrease in absorbance at 435 and 470 nm similar to that shown in Fig. 4. The similarity of the kinetics observed at 435, 470, and 540 nm for the NaCl-induced structural changes suggests that they re-0.1 flect the same underlying process. The effect of NaCl concentration on 2o ~aCl]~M,*O absorbance
changes at 435 and 540 nm.
Previously (14), we have observed that NaCl caused an increase in turbidity associated with a decrease in pigment absorption albeit at higher NaCl concentrations. A complete concentration curve for the NaCl-induced changes in absorbancy at 435 and 540 nm (Fig. 6) showed that low
treated
To clarify structural
+5
-20
FIG. 6. NaCl concentration dependence of absorbance changes at 435 and 540 nm. Chloroplasts were incubated in the presence of the NaCl concentrations indicated and the absorbances at 435 and 540 nm were determined. Conditions were as described in Materials and Methods except that 3-(3,4-dichlorophenyl)-1, 1-dimethylurea was omitted from the reaction medium. (0) AA,,,, (0) AA,,,.
MONOVALENT
CATION-INDUCED
change, we used electron microscopy. Figures 7 and 8 show chloroplasts which were incubated in 100 mM sucrose + 0.2 mM Tris base and either no additions (Fig. 7A), 3 mM NaCl (Fig. 7B), 1 mM CaCl, (Fig. 8A), and/or both added together (Fig. 8B). Approximately 95% of the chloroplasts observed had lost their outer membranes. However, the inner membranes are still connected together. Chloroplasts fixed in the absence of either NaCl or CaCl, show grana stacking’ but are somewhat swollen. The observation of the existence of grana stacks under extremely low ionic strength at first glance disagrees with the results of Izawa and Good (26), Okhi et al. (27), and Murakami and Packer (4), who observed no grana stacking under what they called “low ionic strength” conditions. However, the difference can probably be accounted for by the difference in conditions employed. Murakami and Packer (4) suspended their chloroplasts in distilled water, thereby adding osmotic stress to low ionic strength. Since we had 100 mM sucrose present, we were only looking at ionic effects. Both Good and Izawa (26) and Okhi et al. (27) had 50 mM tricine present. Therefore, their conditions did not correspond to our very low ionic strength conditions but may correspond to our chloroplasts incubated in 3 mM NaCl (see below). Also, we can rule out the hypothesis (28) that unstacking of the thylakoids is the result of repulsion of negative charges unmasked by the low ionic strength conditions since our chloroplasts remain stacked at low ionic strength. Addition of 3 mM NaCl to the low ionic strength chloroplasts causes unstacking of the thylakoids (Fig. 7B). This corresponds to the case in which spillover is promoted. It is these chloroplasts that resemble those incubated in the presence of tricine buffer (26, 27, 29) which has also been shown to promote spillover (9). Thus, it is not low ionic strength per se but the presence of low concentrations of NaCl or tricine buffer which causes unstacking. 1In the absence of salts, there sometimes appear to be fewer grana stacks than observed in the presence of CaCl,. However, there is no evidence that this is statistically significant.
STRUCTURAL
CHANGES
465
Addition of 1 mM CaCl, to chloroplasts suspended in low ionic strength (Fig. 8A) causes shrinkage in a direction perpendicular to the plane of the lamellae. These results are consistent with the turbidity and absorption flattening changes described above. Addition of 1 mM CaCl, after incubation of the chloroplasts in 3 mM NaCl (not shown) caused the majority of the grana to restack. Thus Caz+ ions can reverse as well as prevent the Na+ ioninduced unstacking of the lamellae. Addition of 1 mM CaCl, prevents the NaClinduced unstacking of the thylakoids (Fig. 8B) as well as the Na-induced fluorescence changes (Fig. 1). Thus, when the grana appear stacked either under very low ionic strength conditions and/or in the presence of divalent cations, spillover is inhibited. When the thylakoids are unstacked, spillover is promoted. Unstacking the grana probably changes the relative positions of the two photosystems, allowing greater spillover. One might question whether such a mechanism for control of spillover could occur in vivo in the presence of a high concentration of cations (30, 31). However, one could imagine local fluctuations in cation concentration which could produce these effects. Also, there may be endogenous agents which, like tricine buffer, allow unstacked thylakoids to exist at higher salt concentrations. Furthermore, a small amount of unstacking which might not require extreme conditions may serve to control the photosynthetic process. The antagonistic effects of mono- and divalent cations on chloroplast structure and chlorophyll a fluorescence are reminescent of similar antagonistic effects on other processes in plants. These include membrane permeability (32, 33) and stomata1 closure (33). CONCLUSIONS
The close correlation observed between monovalent cation-induced decreases in turbidity and chlorophyll a fluorescence indicates that the structural changes are responsible for the increases in spillover. Ultrastructural studies showed that low concentrations of NaCl caused unstacking
FIG. 7. The effect of NaCZ on the ultrastructure ofchlorophts. Chloroplasts were incubated in 100 rn~ sucrose + 0.2 mM Tris base (A) and in the same medium + 3.0 mM NaCl (B) prior to fixation with glutaraldehyde. Other conditions were as described in Materials and Methods. The magnification was 25,000x. 466
FIG. 8. The effect of C&Y, on the ultrastructure of chloroplasts. Chloroplasts were incubated in the presence of 1 mM CaCI, (A) and 1 mM CaCl, + 3 mM NaCl (B) prior to fixation. Other conditions were as described for Fig. 7. The magnification was 25,000x. 467
468
GROSS AND PRASHER
of the grana lamellae which could be prevented or reversed by addition of salts of divalent cations. Thus, spillover was enhanced when the lamellae were unstacked and inhibited when stacking occurred either in the absence of salts and/or the presence of divalent cations. The unstacking of the lamellae caused by the monovalent cations must either change the mutual orientation of the two photosystems or bring them into closer proximity. Experiments are now in progress to determine the molecular mechanism of this process. ACKNOWLEDGMENTS This work was supported in part by Grant No. GB19156 from the National Science Foundation. We thank Dr. Robert M. Pfister, Dr. Clinton Stoner, and Miss Adele Arar for help with the electron microscopy. We also thank Dr. Stoner for use of the electron microscope. REFERENCES 1. MURATA, N. (1969) Biochim. Biophys. Acta 189, 171-181. 2. MURATA, N., TASHIRO, H., AND TAKAMIYA, A. (1970) Btichim. Biophys. Acta 197, 250-256. 3. MURATA, N. (1971) Biochim. Biophys. Acta 245, 365-372. 4. HOMANN, P. (1969) Plant Physiol. 44,932-936. 5. MURAKAMI, S., AND PACKER, L. (1971) Arch. Biothem. Biophys. 146,4837-347. 6. MOHANTY, P., BRAUN, B. Z., AND GOVINDJIE (1973) Biochim. Biophys. Acta 292,459-476. 7. GROSS, E., AND PACKER, L. (1967) Arch. Biochem. Biophys. 121, 779-789. 8. DILLEY, R. A., AND ROTHSTEIN, A. (1967) Biochim. Biophys. Acta 135, 427-443. 9. GROSS, E. L., AND HESS, S. C. (1973) Arch Biochem. Biophys. 159,832-836. 10. SEELY, G. R. (1973) J. Theor. Biol. 40, 173-187. 11. SEELY, G. R. (1973) J. Z’heor. Biol. 40, 188-198. 12. GROSS, E. L. (1971) Arch. Biochem. Biophys. 147, 77-84.
13. ARNON, D. I. (1949) Plant Physiol. 24, 1-15. 14. GROSS, E. L., AND LIBBEY, J. W. (1972) Arch. Biochem. Biophys. 153,457-467. 15. STONER, C. D., AND SIRAK, H. D. (1973) J. Cell Biol. 56.51-64. 16. WYDRINSKI, T., GROSS, E. L., AND GOVINDJEE, in
preparation. 17. WRAIGHT, C. A., KRAAN, G. P. B., ANDGEFXITS, N. M. (1972). Biochim. Biophys. Acto 283, 259-267.
18. MURATA, N., AND SUGAHARA, K. (1969) Biochim. Biophys. Acta 189, 182-192. 19. COHEN, W. S., AND SHERMAN, L. A. (1971), FEBS Lett. 16, 319-323. 20. WRAIGHT, C. A., AND CROFTS, A. R. (1970) Eur. J. Biochem.
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21. PACKER, L., DEAMER, D. W., AND CROFTS, A. R. (1966) In Energy Conversion by the Photosynthetic Apparatus, pp. 281-302, Brookhaven Symposium No. 19. 22. DUYSENS, L. M. N. (1956) Biochim. Biophys. Acta 19, 1-12. 23. ITOH, M., IZAWA, S., AND SHIBATA, K. (1963) Biochem. Biophys. Acta’69, 130-140. 24. SHAVIT, N., AND AVRON, M. (1967) Biochim. Biophys. Acta 131, 516-525. 25. GOVINDJEE AND VANDER MUELLEN, D., in prepara-
tion. 26. IZAWA, S., AND GOOD, N. E. (1966) Plant Physiol. 41,544-552. 27. OHKI, R., KUNIEDA, R., AND TAKAMIYA, A. (1971) Biochim. Biophys. Acta 226, 144-153. 28. KIRK, J. T. 0. (1971). in Annual Review of
Biochemistry, Vol. 40, pp. 161-196, Annual Reviews Inc., Palo Alto. 29. ANDERSON, J. M., AND VERNON, L. R. (1967) Biochim. Biophys. Acta 143, 363-376. 30. NOBEL, P. S. (1969) Biochim. Biophys. Acta 172, 134-143. 31. GROSS. E. L., AND HESS, S. C. (1974) Biochim. Biophys. Acta (in press). 32. BONNEX, J., AND GLASTON, A. W. (1952) in Princi-
ples of Plant Physiology, p. 95, W. H. Freeman and Co., San Francisco. 33. KLJIPER, R., J., C., (1972) Annu. Reu. Plant Physiol. 23,157-72.