Effects of freezing on the structure of chloroplast membranes

CRYOBIOLOGY

21, 465-473 (1984)

Effects of Freezing MANFRED *Fachbtwich

Biologic.

on the Structure JENSEN”

AND

of Chloroplast

WALTER

Membranes

OETTMEIER?’

UnivrrJitiit Essrn, Essen, West Grrmuny and ?‘Lehrstuhl Biochemie Ruhruniwrsitiir Bochum, Bochurrr, West German?;

der Pjlanz,en,

Electron spin resonance (ESR) spectra of stearic acid spin labels incorporated into spinach thylakoids can be used to monitor membrane changes during freezing. Changes in the ESR parameters can be directly correlated to the extent of functional freeze damage. Freeze-induced changes in the ESR parameters strongly depend on the osmotic conditions of the incubation medium. Similar changes as on freezing can be observed by transferring thylakoids from an isotonic to a hypotonic medium, i.e., by swelling osmotically flattened thylakoids. This and computer simulations of spin label ESR spectra, which allow for variation of vesicle shape, lead to the conclusion that freeze-induced ESR spectral changes are due to swelling of the thylakoids. Indeed, van’t Hoff plots of thylakoid packed volume indicate a freeze-induced increase in the apparent number of osmotically active molecules within the intrathylakoid lumen. During freezing, salt and/or sugar leak into the lumen. Simultaneously, proton channels are irreversibly opened. As the structural alterations obtained upon freezing are not accompanied by a change in bulk fluidity, these data are interpreted in terms of a focnf action of cryotoxic agents on critical microstructures, possibly at the rims of the thylakoid membranes.

Biological material exposed to stress during freezing is often damaged or killed. It is generally accepted that the primary sites of freezing injury are at the membrane level, and many investigations into the biochemical alterations of isolated biological membrane systems have been reported (4, 10, 15). Particularly for isolated chloroplast thylakoids the effect of cryotoxic agents on the inactivation of photosynthetic phosphorylation and electron transport has been analyzed (9, 10, 21, 26). Structurally, the inactivation process is associated with a detachment of extrinsic proteins from the membrane (4, 24) and the loss of semipermeability (9). However, protein release does not occur during the first stages of

Received August 1, 1983; accepted November 28. 1983. ’ To whom correspondence should be addressed: Lehrstuhl fiir Biochemie der Pflanzen, Ruhruniversitat Bochum, Postfach 10 21 48, D-4630 Bochum 1, WestGermany.

membrane damage (26), i.e., it cannot be the cause of primary membrane damage. We have recently studied the participation of the lipid phase in freezing injury using stearic acid spin labels in order to probe the lipid interior of chloroplast membranes (I 2). Electron spin resonance (ESR) spectroscopy of fatty acid spin labels incorporated into biological membranes is a sensitive method for estimation of molecular motion and fluidity within the lipid part of the host membranes (7). Particularly, it can be used to detect temperature-dependent phase changes, like the transition of membrane lipids from the liquid-crystalline state to the gel state. For isolated thylakoids, our spin label data have indicated small phase changes at + 15 and - 10°C. For vesicles formed from extracted thylakoid lipids, however, a main phase transition melting zone has been found at about -36°C (12). Since the temperature in a standard freezing experiment (and in na25”C, ture) normally is not lower than

466

JENSEN AND OETTMEIER

phase transition phenomena are very unlikely to play a role in freezing injury to thylakoids. In addition, after damaging freeze-thaw cycles no fluidity changes could be detected. Hence, one would conclude that the organization of the lipid phase is unaltered during and after freezing. However, freezing injury is often accompanied by changes in the line shapes of ESR spectra of the spin label 2-(3-carboxypropyl) - 4,4-dimethyl-2-tridecyl-2-oxazolidinyloxyl (5doxyl) and by an increase in thylakoid packed volume (12). In this paper we want to demonstrate that these ESR spectral changes monitor shape changes of the thylakoids rather than membrane damage itself. Our data suggest that a temporary leakage of salt ions and/or sugar molecules into the thylakoid interior space occurs during a freeze-thaw cycle. This process coincides with the irreversible opening of H+ channels and impairment of photophosphorylation capacity. MATERIALS

AND METHODS

c--

~~

ii-/q:

&

FIG. 1. ESR spectrum of the spin label 5-doxyl before (top) and after freezing for 3 hr at - 18°C (bottom). The incubation medium contained 100 mM NaCI, 50 mM sorbitol, 4 mM MgCl,, and 5 mM Tritine, pH 8.

measurement of ESR spectra, and packed volume measurements were performed as described recently (12). For determination of the proton gradient across the thylakoid membrane, the quenching of 9-aminoacridine fluorescence, in the presence of 50 pM methylviologen, was measured as described by Tillberg et al. (25). The reaction medium contained, in a volume of 3 ml, 5 FM 9-aminoacridine, 0.1 M sorbitol, 10 mM KH,PO,, pH 7.7, 5 mM MgCl,, 20 mM KCl, 1 mM KCN, 50 p,M methylviologen, and thylakoids corresponding to 100 Fg chlorophyll. Red actinic light was provided by a halogen lamp and filtered through 8 cm water, 1 mm Caltlex C (Balzers, Liechtenstein), 3 mm RG 630 (Schott, Mainz), and K65 from Balzers. For calculation of ApH (23) the intrathylakoid space was estimated according to a formula given by Nobel (19): V2/Vi = osmolarity of the medium/2 x molar chlorophyll concentration, where V, = external volume and Vi = internal volume. The half time of H+ efflux from the thylakoids after switching off the light was determined by half-logarithmic plots of the decay kinetics of the 9-aminoacridine fluorescence signal and a least-squares fit. The correlation coefficients were greater than

Spinach was grown in a greenhouse in 12hr day (20°C) and 12-hr night (14°C) cycle. The killing point of the plant material was -6°C. Chloroplasts were isolated in a medium containing 0.05 M Tris buffer, pH 8, 0.35 M NaCl, 10 mM KH,PO,, 3 mM cysteine, and 40 mM mercaptoethanol. After washing in 50 mM Tris, pH 8, and 0.35 M NaCl, the thylakoids were released from the chloroplasts by osmotic shock and washed twice in the final storage medium. The storage media were fitted according to the special requirements of the particular experiment and are indicated in the legends of the respective figures. After isolation, samples of the thylakoid suspension were mixed with concentrated sugar/salt solutions, placed into a freezer at - 25°C for at least 3 hr, and allowed to thaw at room temperature. Measurement of photosynthetic activities (photophosphorylation and electron transport from water to methylviologen), 0.995.

467

EFFECTS OF FREEZING ON CHLOROPLASTS

FIG. 2. Dependence of the freeze-induced changes on the ESR parameter h+lh, of the spin label 5-doxyl on the type of cryoprotectant added: 100 h +/ho before freezing (left); 100 h+/h,, after thawing (right). Note that the h+lh, values before freezing are on a low level only when an osmotically active cryoprotectant was added before freezing. The last washing medium during isolation of the thylakoids contained 100 mM NaCI, 5 mM Tricine, pH 8, the chlorophyll concentration was between 2 and 3 mg chlorophyll/ml. Samples were incubated for 20 hr at - 18°C.

RESULTS AND DISCUSSION

Freeze-Induced Changes of the 20°C 5-Doxyl ESR Spectra When the spin label 5-doxyl has been incorporated into isolated thylakoids and ESR spectra are recorded before and after freezing, the spectral line shapes are usually modified (12). A spectrum typical for intact thylakoids suspended in a medium at nearly isotonic conditions and a spectrum typical for freeze-damaged thylakoids are shown in Fig. 1. Characteristic changes can be seen, particularly in the line widths and relative line heights. In order to quantify the effect of media of varying cryoprotective capacity on the spectral modifications, we have recently introduced the empirical parameter h+lh, (12), i.e., the ratio of the height of a low field line component over the height of the central ESR line (see Fig. 1). First, the effect of sorbitol and several sugars, previously shown to be cryoprotective agents, was investigated (Fig. 2). When

1 NaC I

NoF 175

mM

NoNO C+llO

NClBr

NOI

mM SUCROSE)

FIG. 3. Dependence of the freeze-induced increase in the ESR parameter h+/h, of the spin label S-doxyl on the type of cryotoxicant. Because the initial values of 100 h ,/h, varied from 2.4 to 7.7, these values were taken as LOO%,and the relative freeze-induced increase in h+/h, is shown. Note that the increase in h +h, and the cryotoxicity of the medium increase from left (NaF) to the right (NaI). After freezing (21 hr at -25”C), only the NaF samples had retained some phosphorylation activity (26 pmol ATP/mg chlorophyllihr). The last washing medium during isolation of the thylakoids contained 110 mM sucrose, 4 mM MgCl?. and 5 mM Tricine, pH 8.

no osmotically active sugar was present or when glycerol was added to the medium, the level of h+lh, was high, i.e., near the upper limit both before and after freezing. If osmotically active sugars were present, the value of h+lh, was reduced. Upon freezing the value of h+lh,, increased to varying extents depending on which osmotic agent was present. The extent of increase appeared to be inversely correlated to the effectiveness of the specific sugars as cryoprotectants. In the presence of raffinose, which is the most protective agent (22), no increase in h+lh, was observed. At the same concentration of sorbitol and glucose h +lh, showed a large increase, nearly to the highest value, while sucrose had an intermediate influence. If the concentration of these less effective cryoprotectants was

468

JENSEN AND OETTMEIER

increased high enough, they prevented the increase in h+lh, induced by freezing (data not shown). Figure 3 demonstrates the dependence of the ESR parameter h+lh, on cryotoxicants. It is known that sodium salts with different anions are cryotoxic to different degrees (10). The cryotoxic activity of F-, Cl-, NO:, Br-, and I- corresponds to Hofmeister’s lyotropic power series. In this series, F- causes the least, and I- the greatest freezing injury. A similar picture emerges for the structural changes as indicated by the ESR spectra of 5-doxyl. The smallest increase in the ESR parameter h +/ h,, was found in the presence of NaF followed by NaCl, NaBr, NaNO,, and NaI. Similar results were also obtained for the membrane perturbing agent phenylpyruvic acid (10). Here, an increase in h+lh, was obtained at concentrations that uncouple (inactivate) photophosphorylation (data not shown) but not at concentrations that leave the membranes intact. Relationship between ESR Spectra and Thylakoid Volume As already pointed out there is in general a correlation between freeze-induced functional membrane inactivation and structural changes, as monitored by changes in the ESR spectra of 5-doxyl. In addition, our earlier results (12) have revealed a relationship between h+lh, ratios and thylakoid packed volume. This might be the reason for high h+lh, levels in the presence of membrane penetrating glycerol (see Fig. 2). In order to investigate the influence of osmolarity on the ESR spectra of 5-doxyl, thylakoids were suspended in media of increasing concentrations of salts and sucrose. The value of h+lh, was extremely dependent upon the osmotic conditions of the medium, both in salt and sugar (Fig. 4). In hypotonic media, where thylakoids swell (28), the value of h+lh, was high, while it was low in isotonic (i.e., 330 osmol/liter) or hypertonic media. Although KC1 and sucrose exert equal effects on a molar rather

1

\Lo r+ SUCROSE KI

o---c

+-.-+ x-*

02

04 concentrotian

KCL

06

08 CM1

FIG. 4. Influence of the osmotic conditions on 5doxyl ESR spectra. The last washing medium during isolation of the thylakoids contained 10 mM MgCI, and 10 mM Tricine, pH 8. The samples were mixed with concentrated salt or sucrose solutions 5 min before recording of ESR spectra. The same thylakoid preparation was used for the KI and sucrose experiments.

than on an osmolar basis, the level of h+/ h, and of the thylakoid volume appear to be closely related. Interestingly, it is not only the value of h+lh, that changes upon variation of the osmolarity, but the whole spectrum changes with the same characteristics as in Fig. 1. According to these data, it seems likely that freeze-thaw injury induces a swelling of the thylakoids (cf. 12)). The variation in the initial values of h+lh, (see legend, Fig. 3) could be explained by slow diffusion of salts into the intrathylakoid space during storage at 4”C, since the ESR experiments were started at different times after the isolation of the thylakoids. Although there is a clear correlation between alterations in 5-doxyl ESR spectra and volume changes, the precise nature of structural changes as monitored by the ESR spectra so far remains unclear. The osmolarity of the medium or freezing does not induce changes in the spectra of 2-(14carboxytetradecyl) -2-ethyl-4,4-dimethyl-3oxazolidinyloxyl (16-doxyl), a stearic acid spin label bearing the nitroxyl group at the terminal end of the fatty acid, and thus

469

EFFECTS OF FREEZING ON CHLOROPLASTS

TABLE 1 ESR Parameters h-/h, (for definition see Fig. 11, Nuclear Hypetfine Tensor 2 I”,, and Central Line Width W, of Calculated Spectra as in Fig. 5.

FIG. 5. ESR spectra and macrosconic orientation performed by computer simulations with the program EULORZ. A low value of (T means a high degree of alignment of the spin label molecules, a high value of o a low degree of alignment. The following input parameters were used: A,, = 6.3. A,,. = 5.8, A;; = 33.6. G, = 2.0088, G,, = 2.0061, G;; = 2.0027, NORNT = 10000, FREQ = 9.5, THETA0 = 0, ATILT = 0, PERG = 0, RLH(1,2,3) = (0.7, 1.0, 0.55), WC = 3, WV = 1, GAMMA = 37 (wobble model).

0

100 /2+/h,

32 36 40 44 48 52 56 60 64 68 12

0.05 3.2 7.2 11.3 14.9 11.7 20.0 21.7 23.1 24.2 25.0

2 T’n (G)

W” (G)

57.5 57.0 57.0 56.5 56.5 56.0 56.0 56.0 56.0 55.5 55.5

6.5 6.0 5.5 5.5 5.0 4.5 4.0 4.0 3.5 3.5 3.0

probing a different environment than 5doxyl within the thylakoid membrane. Furthermore, neither osmolarity nor freezing affects the ESR parameter 2 T;, for 5-doxyl. This seems to exclude an interpretation in terms of fluidity changes. It is known that strong magnetic fields, like those in ESR spectrometers, induce a macroscopic ordering, i.e., photosynthetic membranes tend to orient with their planes perpendicular to the direction of the magnetic field (2, 5). It appeared reasonable, therefore, to simulate ESR spectra taking into account the macroscopic factor. This was done using the computer program EULORZ as described by Libertini et al. (14). This program can vary an orientation parameter (u) to account for the degree of alignment of the molecules without affecting the overall mobility parameters of the spin label molecules. In Fig. 5, (T is varied from a low value corresponding to a high degree of alignment as in osmotically flattened thylakoids to a high value as in spherical vesicles. The ESR parameters h+l h,, nuclear hyperfine tensor 2 T;, and line width of the central line W,, of the calculated spectra are presented in Table 1. It can be seen that 100 h+/h, varies from 0.05 to 25, W, from 6.5 to 3 G, while the parameter 2 T;,, associated with the fluidity of the

470

JENSEN

AND

OETTMEIER

200 r membrane, changes as little as in the experimental spectra (from 57.5 to 55.5 G). Comparison of the experimental spectra in Fig. 1 and the simulated spectra in Fig. 5 yields a remarkable coincidence in line shapes. Therefore, an interpretation of the freeze-thaw-induced changes in 5-doxyl ESR spectra in terms of macroscopic, i.e., shape changes of the host membranes, the thylakoids, is a very attractive possibility. Electron microscopy pictures of Murakami and Packer (17) indicate that osmotically produced volume changes of thylakoids are associated with shape changes and not with changes of the surface area. Thus, the connection between the observed ESR spectral --LI -I-. I 15 20 5 IO changes and the observed volume changes CI/OSMOLALITYI 1 /P can easily be explained, since both are diFIG. 6. Van? Hoff plots of thylakoid packed volume rectly linked to shape changes of the thy- versus Uosmolality. The thylakoids were incubated in lakoids. 100 mM NaCI, 50 mM sucrose, 4 mM MgCl,, and 5

Solute Influx and Irreversible Opening of Proton Channels Both the so-called packed volume and the shape and orientation of thylakoids which are responsible for the ESR line shapes, may not only depend on the true internal volume of thylakoids but may also be influenced by intervesicular interactions (27). It is justified to ask the question, whether freezing changes only the partitioning or the true volume. Osmotic forces cause true volume changes only when the number of osmotically active solutes in the inner thylakoid lumen change. Whether freezing really does support solute influx is an essential question. This was tested by diluting freeze-damaged samples in sucrose solutions of different osmolalities and sedimenting the thylakoids in order to obtain packed volume values. According to the Boyle-van’t Hoff law (V-b = n X R x T/p, where p = osmotic pressure, V = volume of the organelle, b = so-called “nonosmotic” volume, n = apparent number of osmotically active moles in Vb, R = gas constant, T = absolute temperature), a plot of volume versus l/osmo-

mM Tricine, pH 8, for 4 hr at -25”C, 1 hr at O”C, or 6 hr at 0°C. After thawing the samples were diluted IO-fold in sucrose solutions of graded concentrations in order to obtain a final osmolality of 52, 64, 86, 128, 263 mosmoliliter (data from “Handbook of Chemistry and Physics”). Besides sucrose, the solutions contained 5 mM Tricine, pH 8, and 4 mM MgCI,. Photosynthetic activity was measured as described elsewhere (12). The ATPI2e ratio is the number of ATP molecules, which are formed in the light while two electrons are transported from water to methylviologen.

lality of the resulting medium should yield a straight line (18-20, 28). From the slope of this line the number of moles of osmotically active solutes in the intrathylakoid space can be estimated (20). In Fig. 6, the van? Hoff plots for freeze-damaged thylakoids are compared to those of unfrozen controls. As can be seen freeze-damaged thylakoids occupy a greater volume than 0°C controls. For determination of packed volumes, conditions were chosen in a way to avoid further damage beyond that due to freezing injury, i.e., the ion milieu (4 mM MgCl,, 10 mM NaCl, and 5 mA4Tricine, pH 8) was balanced so as to obtain stable stacked thylakoid grana. The nonlinearity of the van’t Hoff curves shown in Fig. 6

EFFECTS OF FREEZING ON CHLOROPLASTS

may be explained by the packing properties of stacked grana under varying osmotic conditions. This may lead to a concomitant variation in the proportion of interstitial fluid trapped in the pellet after centrifugation (cf. (18)). The curves were not corrected for this variable. It is easy to see, however, that the curve of the freeze-damaged thylakoids is much steeper than that of the controls. It seems likely that accelerated solute influx occurs during freeze-thaw treatment. It cannot be completely excluded, however, that the increase in slope of the van’t Hoff curve of the frozen sample (Fig. 6) might be due to some variation in the trapped fluid. The notion that indeed solute influx occurs is substantiated by experiments with model membranes. Morris and McGrath (16) have observed reversible opening of hydrophilic channels during freezing and thawing in liposomes. As Fig. 6 shows, solute influx also occurs during incubation at 0°C for 6 hr, but this process is much slower, andwhat is important-does not produce any damage. Such influx can also be observed

30 mM Nor,1

70 (-:lC

471

after freeze-thaw treatment of completely cryoprotected samples, but it never reaches the magnitude of the influx in unprotected thylakoids. It has to be emphasized that the thylakoids are not leaky toward sucrose after an intermediately severe frost treatment, i.e., when ATP synthesis, but not electron transport, is inactivated. On the other hand, it is known that the ability of thylakoids to generate a pH gradient in the light may be completely lost after such treatment (3, 24). The correlation between inactivation of ATP synthesis, solute influx, and irreversible opening of H+ channels is of special interest. In Fig. 7 the cryotoxicity of the incubation medium was increased by increasing the concentration of NaCl at a constant concentration of the cryoprotecting agent sucrose (110 mM). At a concentration less than 90 mM NaCl no frost inactivation could be detected; the sucrose concentration was sufficient for a complete cryoprotection of the thylakoids. In Fig. 7 this range is marked phase 1. Phase 3, beyond 160 mM NaCl, repre-

4 IO TM

150

I30

SUCSCSt)

FIG. 7. pH gradient, H+ permeability, and photosynthetic activity of thylakoids after a freeze-thaw cycle. Thylakoids were incubated at -25°C for 6 hr in a medium containing a fixed concentration of cryoprotectant (110 mM sucrose) and a variable content of cryotoxicant (NaCI). The pH gradient was calculated from the 9-aminoacridine (9-AA) fluorescence quenching (23). H+ permeability is assumed to be inversely related to the half time of decay of the 9-aminoacridine signal in the dark. In phase 1 the ratio of (cryoprotective) sucrosei(cryotoxic) NaCl is high enough to prevent freeze damage. Phase 2 represents the zone of primary freeze damage (30% inactivation of ATP synthesis). In phase 3 the sucrose/NaCl ratio is so low that photosynthetic electron transport is uncoupled and ATP production becomes completely inhibited.

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JENSEN AND OETTMEIER

sents the zone of intermediate frost damage, where photophosphorylation is inactivated (either partly or completely), the pH gradient is diminished, and H+ permeability is greatly increased (the half time of decay of 9-aminoacridine fluorescence is decreased (8)). In contrast, electron transport (which is controlled by internal pH (29)) is not inactivated, but even is strongly stimulated (uncoupled), Phase 2, representing the zone of primary damage, extends from 90 to 160 mM NaCl. It is characterized by a constant rate of coupled electron transport, while other photosynthetic activities are already significantly diminished. From the half time of decay of the 9-aminoacridine fluorescence quenching, it is clear that the increase in proton permeability is a very early event in freezing injury. It increases by 20% when ATP synthesis is inactivated by 30%. Simultaneously the pH gradient is diminished (Fig. 7), perhaps explaining the partial inactivation of photophosphorylation (6). Since shape changes of the thylakoids and solute influx also occur at an early stage of freeze damage, there is a remarkable coincidence between structural and activity alterations. However, slight structural changes as monitored by ESR spectra of 5-doxyl and packed volume measurements are not always deleterious (cf. Fig. 6). Thus, the molecular mechanisms of primary freeze inactivation of thylakoid membranes are still unclear. CONCLUDING

REMARKS

For isolated thylakoids the freezing stress consists of a combination of osmotic, low-temperature stress and the stress exerted by high concentrations of cryotoxic solutes. Osmotic and low-temperature stress as such are physiologically and structurally tolerated, as long as the high concentrations of cryoprotectants in the medium stabilize both structure and function. Overall

lipid-lipid interactions are not affected by freezing damage and the concomitant volume increase. No fluidity changes are detectable by means of ESR spectroscopy. This may be understood if one takes into account that thylakoids can change their shape by shrinking and swelling. According to Murakami and Packer (17), membrane thickness and surface area are not altered, and hence the packing of the lipid matrix molecules is very flexible. Obviously, freezing damage does not affect the lipid matrix as a whole. Therefore, the irreversible opening of H+ channels and solute influx during freezing must result from the local action of cryotoxicants. Critical structures or microenvironments presumably are situated in the highly curved rims of the thylakoids because chloroplast ATP synthetase CF, and the underlying proton permeable CFOare situated in this area (13). The high curvature in the rims of flattened thylakoids reaches molecular dimensions, i.e., it represents a structure which is difficult to stabilize by lipid molecules and therefore may be potentially labile (1, 11). ACKNOWLEDGMENTS This work was supported by Deutsche Forschungsgemeinschaft and Minister fur Wissenschaft und Forschung des Landes Nordrhein-Westfalen. REFERENCES 1. Crowe, J. H., and Crowe, L. M. Induction of anhydrobiosis: Membrane changes during drying. Cryobiology

19, 317-328 (1982).

2. Dismukes, G. C., and Sauer, K. The orientation of membrane bound radicals: An EPR investigation of magnetically ordered spinach chloroplasts. Biochim. Biophys. Acta 504, 431-445 (1978). 3. Farkas, D. L., and Malkin, S. Cold storage of isolated class C chloroplasts. Plant Physiol. 64, 942-947 (1979). 4. Garber, M. P, and Steponkus, P. L. Alterations

in chloroplast thylakoids during an in vitro freeze-thaw cycle. Plant Physio!. 57, 673-680 (1976). 5. Geacintov, N. E., van Nostrand, E, Becker, J. E,

EFFECTS OF FREEZING ON CHLOROPLASTS and Tinkel, J. B. Magnetic field induced orientation of photosynthetic systems. B&him. Biophys. Acta 261, 65-79 (1972). 6. Ciraber, P., and Schlodder, E. Regulation of the rate of ATP synthesis/hydrolysis by ApH and A$. In “Photosynthetic Electron Transport and Photophosphorylation” Vol. 2, “Photosynthesis” (G. Akoyonoglou, Ed.). pp. 867-880. Balaban Int. Sci. Services Phila., Glenside. 1981. 7. Griffith, 0. H., and Jost, P. C. Lipid spin labels in biological membranes. In “Spin Labeling Theory and Applications” (L. J. Berliner, Ed.), pp. 453-523, Academic Press, New York, 1976. 8. Harrault, F., and de Kouchkovsky, Y. Measurement of chloroplast internal protons with Y-aminoacridine: Probe binding, dark proton gradient, and salt effects. Biochirn. Biophys. Aciu 592, 153-168 (1980). 9. Heber, U. Freezing injury and uncoupling of phosphorylation from electron transport in chloroplasts. Plunt PhysioL. 42, 1343-1350 (1967). IO. Heber, U., Volger, H., Overbeck, V., and Santarius, K. A. Membrane damage and protection during freezing. In “Advances in Chemistry” (0. Fennema, Ed.), Series 180, pp. 159-189. Amer. Chem. Sot., Washington, D.C., 1979. 11. Israelachvili, J. N., Marcelja, S., and Horn, R. G. Physical principles of membrane organization. Quart. Rev. Biophys. 13, 121-200 (1980). 12. Jensen, M., Heber. U., and Oettmeier, W. Chloroplast membrane damage during freezing: The lipid phase. Cryobiology 18, 322-335 (1981). 13. Kopp, F., Miihletaler, K., and Berzborn. R. J. Mobility of chloroplast coupling factor 1 (CF,) at the thylakoid surface as revealed by freezeetching after antibody labeling. Z. Natwforsch. C 29, 694-699 (1974). 14. Libertini, L. J., Burke, C. A., Jost, P. C., and Griffith, 0. H. An orientation distribution model for interpreting ESR line shapes of ordered spin labels. J. Magn. Reson. 15,460-467 (1974). 15. Lineberger, R. D., and Steponkus, P. L. Cryoprotection by glucose, sucrose, and raffinose to chloroplast thylakoids. Plant Physiol. 65, 298304 (1980). 16. Morris, G. J., and McGrath, J. J. The response of multilamellar liposomes to freezing and thawing. Cqobiology 18, 390-398 (1981).

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17. Murakami, S., and Packer, L. Protonation and chloroplast membrane structure. J. Cell Biol. 47, 332-351 (1970). 18. Nobel. P. S. Light-induced chloroplast shrinkage in vivo detectable after rapid isolation of chloroplasts from Pisum sativum. Plant Physiol. 43, 781-787 (1968). 19. Nobel. P. S. Light-induced changes in the ionic content of chloroplasts in Pisum sativum. Biochirn. Biophys. Acra 172, 134- 143 (1969). 20. Nobel, P. S. The Boyle-Van? Hoff relation. J. Theor. Biol. 23, 375-379 (1969). 21. Santarius. K. A. Der EinfluB von Elektrolyten auf Chloroplasten beim Gefrieren und Trocknen. Pkrritrr 89, 23-46 (1969). 22. Santarius, K. A. The protective effect of sugars on chloroplast membranes during temperature and water stress and its relationship to frost, desiccation and heat resistance. PIrrnru 113, 105-l 14 (1973). 23. Schuldiner, S.. Rottenberg, H., and Avron, M. Determination of ApH in chloroplasts: 2. Fluorescent amines as a probe for the determination of ApH in chloroplasts. Eur. J. Biochem. 25, 64-70 (1972). 24. Steponkus, P. L.. Garber, M. P., Myers, S. P., and Lineberger, R. D. Effects of cold acclimation and freezing on structure and function of chloroplast thylakoids. Cryobiology 14, 303-321 (1977). 25. Tillberg, J., Giersch, Ch., and Heber, U. CO, reduction by intact chloroplasts under a diminished proton gradient. Biochim. Biophys. Acta 461, 31-47 (1977). 26. Volger, H. G., Heber, U., and Berzborn, R. J. Loss of function of biomembranes and solubilization of membrane proteins during freezing. Biochim. Biophys. Actu 511, 455-469 (1978). 27. Weis, E. The influence of metal cations and pH on the heat sensitivity of photosynthetic oxygen evolution and chlorophyll fluorescence in spinach chloroplasts. Planta 813, l-7 (1982). 28. Williams, R. J., and Meryman, H. T. Freezing injury and resistance in spinach chloroplast grana. Plant Physiol. 45, 752-755 (1970). 29. Witt, H. T. Coupling of quanta, electrons, field ions and phosphorylation in the functional membrane of photosynthesis. Q44art. Rev. Biophys. 4, 365-477 (1971).