Freezing of Isolated Thylakoid Membranes in Complex Media. VII. The Effect of Bovine Serum Albumin

Biochem. Physiol. Pflanzen 187,149-162 (1991) Gustav Fischer Verlag lena

Freezing of Isolated Thylakoid Membranes in Complex Media. VII. The Effect of Bovine Serum Albumin KURT A. SANTARIUS Botanisches Institut, Universitat Dusseldorf, Dusseldorf, F.R.G. Key Term Index: Bovine serum albumin, cryopreservation, freezing injury, photosynthetic reactions, thylakoid membrane; Spinacia oleracea

Summary The cryoprotective properties of bovine serum albumin (BSA) were investigated with thylakoid membranes isolated from spinach leaves (Spinacia oleracea L. cv . Monatol). The membranes were frozen in a complex salt medium containing the predominant inorganic electrolytes of the chloroplast stroma. Under mild freezing conditions, only partial stabilization of PSII-dependent photosynthetic activities but efficient cryopreservation of PSI-mediated photophosphorylation took place in the presence of BSA. The lower the freezing temperature, the less protein was necessary for optimum protection of light-induced proton uptake and ATP synthesis . At higher initial BSA concentrations or more severe freezing , i.e. when the protein concentration in the residual unfrozen fraction exceeded a critical limit, rapid inactivation of photophosphorylation occurred in parallel with the ice crystal formation . This damage was due to an increase in the proton permeability of the membranes , while the capacity of linear whole-chain electron transport remained unaffected or even rose relative to unfrozen membranes. The effects of the solutes on thylakoid activities can be explained in part by colligative action, i.e. each solute acts on different membrane sites and reduces the concentration of the others in the residual unfrozen liquid. In addition, damage and protection of the membranes was influenced by the quantity of ice formed, the final volume of the unfrozen solution , the final membrane concentration in this fraction and the duration time of exposure to a given temperature . Extremely high concentrations of BSA and thylakoids in the frozen system caused inactivation of photophosphorylation regardless of the volume of the residual liquid and the freezing temperature. When chloroplast membranes were kept at 0 °C, BSA effectively stabilized cyclic photophosphorylation but only slightly reduced the progressive inactivation of linear photosynthetic electron flow.

Introduction It has been indicated in numerous studies that proteins playa role in acclimation of plants to freezing temperatures. On the one hand, cold hardening induces changes in the soluble protein patterns in various plant tissues (e.g., MEZA-BASSO et al. 1986; JOHNSON-FLANAGAN and SINGH 1987; MOHAPATRA et al. 1987; ROBERTSON et al. 1988; KURKELA et al. 1988; GILMOUR et al. 1988; GUY and HASKELL 1989; PERRAS and SARHAN 1989). However, from such data it cannot be gathered whether these alterations directly influence frost resistance, i.e. whether they are primarily associated with the development of freezing tolerance . Abbreviations : 9-AA, 9-aminoacridine; BSA , bovine serum albumin ; CFl, peripheral part of chloroplast coupling factor; ChI, chlorophyll; DCCD, N,N'-dicyclohexyIcarbodiimide; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; MV, methyl viologen ; PMS, phenazine methosulfate; qAA , light-induced quenching of 9-aminoacridine fluorescence BPP 187 (1991) 2

149

On the other hand, distinct proteins which were isolated from various frost-hardy plant material are highly effective in preservation of thylakoid membranes during freezing in vitro (HEBER and KEMPFLE 1970; VOLGER and HEBER 1975; RosAs et al. 1986; HINCHA et al. 1989, 1990). Likewise, animal proteins such as BSA protect isolated chloroplast membranes against the deleterious effect of a freeze-thaw treatment (SANTARIUS 1986b). The significance of proteins for cryopreservation and their protective mechanism are still unclear. In the present study, the effect of BSA on thylakoid membranes isolated from spinach leaves was investigated in detail. Membranes were frozen in a complex medium consisting of the predominant inorganic electrolytes of the chloroplast stroma (SANTARIUS 1986b); under these conditions the pattern of membrane inactivation is comparable to that produced during freezing of intact leaves (SANTARIUS 1986c, 1990a; see also GRAFFLAGE and KRAUSE 1986). The results are discussed with regard to the importance of proteins in membrane stabilization during freezing. Materials and Methods Material Spinach (Spinacia oleracea L. cv Monatol) was grown for 5 -7 weeks in a greenhouse with 9 h light periods. Isolation of thylakoid membranes The isolation procedure was recently described in detail (SANTARIUS 1990a). After rupture of the washed chloroplasts by suspending them for 30 s in a medium consisting of 5 mM MgCl2 and 5 mM Hepes /KOH (PH 7.5), a solution was added leading to a complex salt medium with final concentrations of 70 mM KCl, 30 mM NaN0 3 , 20 mM K2 S04 , 5 mM MgCI 2 , and 5 mM Hepes/KOH (PH 7.5). Thylakoids were again sedimented by centrifugation for 3 min at 2000 g. The pellet was resuspended in the complex salt medium. In some experiments (Figs. 7-9) the concentration of the complex salt medium was increased up to the twofold level and/or reduced up to one tenth prior to freezing. Freeze-thaw treatment Freezing of chloroplast membranes took place in the complex salt medium (above) in the presence of various concentrations of BSA (Boehringer, Mannheim, FRG; high quality preparation, albumine content 98%, catalogue No. 238040). Aliquots of the thylakoid suspensions were transferred into glass tubes and either kept at 0 °C or rapidly frozen in the dark in cryostats adjusted to different temperatures. After storage for various times, frozen samples were quickly thawed in a water bath at room temperature. Light-induced thylakoid membrane reactions The activities of various photosynthetic reactions were measured under conditions recently described in detail. Phosphorylating (presence of ADP and Pi) and uncoupled (addition of 5 mM NH4 Cl) whole-chain photosynthetic electron transport from water to MY was determined as O2 uptake (SANTARIUS 1990a). Light-induced proton uptake into the intrathylakoid space was monitored by means of quenching of 9-AA fluorescence (SANTARIUS 1990b). Light-induced fluorescence quenching (qAA) was quantified by the ratio t.F/(F-t.F), where t.F is quenched fluorescence in the steady-state during illumination and F is the maximum fluorescence in the dark. Fluorescence measurements took place in the absence and presence of 6 ~M DCCD. Noncyclic photophosphorylation was recorded as pH change in a slightly buffered medium at pH 8.0 (SANTARIUS 1990b). PMS-mediated photosynthetic ATP formation was assayed either in high-intensity white light (Figs. 1, 5, 7-9) or red light (Fig. 6) as described earlier (SANTARIUS 1986a, 1990b). Since sulfate is

150

BPP 187 (1991) 2

competitive with phosphate in phosphorylation (for literature see SANTARIUS 1987), rates of photophosphorylation are dependent on the concentration of sulfate present during illumination of the thylakoids. Therefore, when variable amounts of inorganic salts were transferred with the thylakoids into the reaction medium (Figs. 7 -9), in each experiment the differences in the concentrations of these solutes were equalized resulting in a constant electrolyte level during illumination of the membranes.

Results 1. Freezing at various temperatures Isolated thylakoid membranes suspended in the complex salt medium were subjected to a freeze-thaw cycle in the presence of variable amounts of BSA. Photosynthetic activities of the membranes measured after temperature treatment were widely dependent on the initial protein concentration and the freezing temperature. The lower the freezing temperature the less protein was necessary for comparable degrees of protection of PMS-mediated photophosphorylation (Fig. 1 A). E.g., optimum preservation of cyclic photophosphorylation was observed after exposure of membranes for 24 h at temperatures around -15°C in the presence of about 3% BSA (Fig. 1; see also Fig.9A). Deviation from these optimum conditions by lowering of the freezing temperature (Fig. 1 B) or by increase of the initial protein concentration (see Fig. 9 A) diminished membrane protection. At temperatures above -15°C, optimum survival of photophosphorylation was reached with BSA concentrations higher than 3% (data not shown). Under more severe freezing conditions, both the extent of thylakoid preservation and the protein concentration for maximal protection declined with decreasing freezing temperature (Fig. 1 B). At temperatures close to -30°C, cyclic photophosphorylation became completely inactivated regardless of the amount of protein added to the thylakoid suspension prior to freezing. Storage of thylakoids for 24 h at O°C in the absence of BSA resulted in a slight depression of the phosphorylating activity which was nearly prevented when protein was present in the medium (Fig. 1 A). Compared to cyclic photophosphorylation, linear photosynthetic electron transport behaved differently (Fig. 2). When thylakoids were kept for 24 h at O°C, the capacity of whole-chain electron flow decreased by 50-60% relative to that of freshly isolated membranes (see also SANTARIUS 1990a), independent of the amount of BSA present in the medium. Freezing of thylakoids for the same time in the absence of BSA intensified damage to the electron transport system almost irrespective of the freezing temperature. When BSA was added prior to freezing, enhanced protection of the electron transfer capacity was obtained with rise of the initial protein concentration and decrease of the freezing temperature. Under mild freezing conditions, the stabilizing effect of BSA was much smaller than at lower freezing temperatures. After severe freezing in the presence of higher concentrations of BSA, rates of uncoupled electron flow even considerably exceeded the activity of freshly isolated thylakoid membranes. Comparison of the activities of phosphorylating (coupled) and uncoupled electron transport and noncyclic photophosphorylation of thylakoids after freeze-thaw treatment in the absence of BSA revealed that coupling was completely abolished independent of the freezing temperature (Fig. 3). When BSA was added prior to freezing, the lower the freezing temperature the less protein was necessary for partial protection of coupling. Similar to the results obtained for PMS-mediated photophosphorylation (Fig. 1), optimum curves were BPP 187 (1991) 2

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Fig. 1. The effect of freezing temperature on cyclic photophosphorylation of isolated chloroplast membranes suspended in the complex salt medium containing various concentrations of BSA. Thylakoids were stored for 22-24 h at temperatures between 0 and -15 °C (A) and -15 and -31 °C (B), respectively. The activity of PMS-mediated photophosphorylation as expressed in % of unfrozen controls is plotted as a function of the initial concentration of BSA. Rates of cyclic photophosphorylation determined immediately after membraJIe isolation (= 100%) : 836 to 1121 !lmol ATP mg- I ChI h- I .

Fig . 2. The effect of freezing temperature on the capacity of the electron transport system of isolated chloroplast membranes suspended in the complex salt medium containing various amounts of BSA . Thylakoids were stored for 22-24 h at various temperatures as indicated . The activity of uncoupled whole-chain electron transport (H 20 --+ MV) determined in the presence of 5 mM NH4 CI and given in % of unfrozen controls is plotted versus the initial concentration of BSA. Rates of the membranes measured immediately after isolation (= 100 %): 303 !lmol O2 mg- I Chi h- I .

obtained when the activity of linear photosynthetic ATP formation was plotted versus the initial BSA concentration (e.g., Fig_3C). The optimum of photophosphorylation correlated with a minimum of uncoupling. Like for cyclic photophosphorylation, severe freezing produced complete inactivation of noncyclic ATP formation, regardless of the amount of protein surrounding the membranes . However, under these conditions electron flow in the presence of ADP and Pi, although already markedly enhanced compared to the rates of unfrozen controls, were still accelerated after addition of an uncoupler (Fig. 3 D). Measurement of light-induced quenching of 9-AA fluorescence (qAA) signified a decrease or loss of light-dependent H+ uptake upon freezing, which led to partial or complete inactivation of the phosphorylating activity of the membranes (compare Fig. 4 with Figs. 1

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Fig. 3. The effect of freezing temperature on phosphorylating and uncoupled electron transport and noncyclic photophosphorylation of isolated chloroplast membranes suspended in the complex salt medium containing various concentrations of BSA. Thylakoids were stored for 22-24 h at -7.2 (A), -15.1 (B), -20.5 (C), and - 30.5 °C (D), respectively. The activities of uncoupled electron transport from water to MV (5 mM NH4 CI; - . - ) and of noncyclic photophosphorylation (- - - V - - -) as given in % of unfrozen thylakoids are plotted as a function of the initial concentration of BSA. Phosphorylating electron transport (presence of ADP and Pi; - 0 - ) is expressed in % of the uncoupled electron flow of the unfrozen control. Activities of unfrozen membranes measured immediately after isolation (= 100 %): uncoupled electron transport, 265 - 304 f.tmol O 2 mg -I ChI h -I; noncyclic photophosphorylation, 188-248 f.tmol ATP mg- I ChI h- I .

and 3). Obviously, reduction of ATP synthesis was due to diminution oflight-induced proton uptake. After mild freezing in the presence of smaller amounts of BSA, the addition of DCCD prior to fluorescence measurements partly restored light-induced proton gradient formation (Fig.4A). As it is known that DCCD blocks channels for protons in the membranes which were opened by release of the CFI (for literature see SANTARIUS 1990b), the result points to the dissociation of this protein complex. In contrast, DCCD was not effective after treatment at lower temperatures (Fig. 4C). The data presented in Figs. 1-4 indicate that inactivation of photophosphorylation under mild and severe freezing conditions and in the presence of low and high initial BSA concentrations was based on different mechanisms.

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Fig. 4. The effect of freezing temperature on the light-induced proton gradient of isolated chloroplast membranes suspended in the complex salt medium containing various amounts of BSA. Thylakoids were stored for 22-24 h at -7.1 (A), -15.2 (B) and -22.2 °C (C), respectively. Light-induced 9AA fluorescence quenching, expressed as qAA = A F/(F - A F), was measured in the absence (0) and presence (.) of DCCD and plotted versus the initial concentration of BSA. qAA of unfrozen thylakoids determined directly after isolation (= 100 %): 1.38-1.61 in the absence and 1.26-1.41 in the presence of DCCD.

2. Time course of freeze inactivation When thylakoids were subjected to moderate freezing temperatures in the absence of BSA, membrane injury took a biphasic course (Fig. SA). An initial rapid decrease of the phosphorylating activity during the crystallization process was followed by a progressive decline of the rate of ATP formation gradually leading to complete inactivation. A shift to milder freezing conditions resulted in diminution of the fast component but acceleration of the slow course of damage. E.g., at -6°C, inactivation of photophosphorylation gradually proceeded with freezing time. The initial rapid component of membrane injury was enlarged by lowering the freezing temperature. In the presence of BSA, the rate of inactivation of the phosphorylating system was drastically diminished at mild and moderate freezing temperatures (Fig. SB). The protective effect of BSA was related to both a diminution of the slow component particularly at mild freezing and a reduction or disappearance of the rapid phase. The fast breakdown of the proton gradient and ATP synthesis and the stimulation of linear photosynthetic electron transport reactions, which was observed at severe freezing conditions in the presence of BSA, occurred almost simultaneously with the ice crystal formation (Fig. 6).

3. Variations in the concentrations of the complex salt medium and thylakoid membranes Damage and protection of chloroplast membranes during a given period of freeze-thaw treatment are largely determined by the amount of ice formed, the final volume of the residual unfrozen solution and final concentrations of solutes and membranes in this fraction (SANTARIUS and GIERSCH 1983, 1984; HINCH A et al. 1984). These factors are widely dependent on the freezing temperature, but also on the initial concentrations of solutes and membranes and the ratios between the various compounds. In the following, lS4

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Fig. 7. The effect of dilution of distinct components of a suspension of isolated chloroplast membranes during freezing. Samples were kept for 22-24 h at O°C (- - -0- - -) and frozen at -15°C (---.-), respectively. Starting from the complex salt medium containing 3 % BSA, different dilutions were carried out prior to temperature treatment. A: The total thylakoid suspension was diluted with distilled water, i. e. all samples exhibited a constant ratio of BSAlsalts/membranes. B: The suspension medium only (salts plus BSA) was diluted, whereas the thylakoid concentration was kept constant, i. e. all samples exhibited a constant ratio of BSAlsalts but decreasing ratios of solutes/membranes. C: The concentrations of salts and thylakoids were reduced simultaneously, but the initial BSA concentration was kept at 3 %, i. e. all samples exhibited a constant ratio of salts/membranes but increasing ratios of BSAlsalts and BSAlmembranes. The activity of PMS-mediated photophosphorylation is plotted versus the extent of dilution of the respective components of the membrane suspension prior to temperature treatment. Factor 'I' corresponds to the undiluted membrane suspension containing 70 mM KCI, 30 mM NaN0 3 , 20 mM K 2S0 4 , 5 mM MgCI 2 , 5 mM Hepes/KOH (pH 7.5), 3 % BSA and thylakoids corresponding to 1 mg Chi ml- 1 , factor '0.1' denotes a dilution of the respective component(s) to one tenth of the initial concentration.

the effect of vanatlOns of the suspension medium on the phosphorylating activity of thylakoids is investigated after freeze-thaw treatment at constant temperature and exposure time. As shown above, when thylakoids were suspended in the complex salt medium in the presence of 3 % BSA, optimum cryoprotection was observed during exposure for 24 h at -15°C (Fig. 1). Dilution of this membrane suspension with distilled water prior to freezing led to a decrease in membrane 'survival' (Fig. 7 A). It is generally accepted that dilution results in an increase of the total amount of ice formed and, in parallel, reduction in the volume of the unfrozen fraction, but does not affect the final concentration of solutes and membranes reached in the residual liquid. Inactivation of photophosphorylation was even more pronounced when only the medium, i.e. salts plus 3% BSA, was diluted prior to freezing, but the initial membrane concentration was kept constant (Fig. 7B). In this case, thylakoid packing drastically increased in the residual unfrozen fraction. Strongest depression of the phosphorylating activity with progressive dilution was observed when the latter was extended to salts and membranes, whereas an initial BSA concentration of 3% was maintained throughout (Fig. 7C). Under these conditions, all frozen samples exhibited an equal ratio of salts/membranes, but the BSA concentration in the residual liquid drastically 156

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rose with progressive dilution. This clearly shows that increasing inactivation of photophosphorylation with dilution of the medium as outlined in Fig. 7B cannot be explained by a decrease in the availability of BSA per thylakoid membrane. The results shown in Figs. 7B and 7C indicate that changes of the thylakoid concentration relative to that of saIts and BSA influence membrane 'survival' during freeze-thaw treatment. This is confirmed by Fig. 8: an increase of the thylakoid concentration prior to freezing in the complex salt medium containing 3 % BSA was correlated with a decline of the phosphorylating capacity. In contrast, high thylakoid concentrations rather improve membrane stabilization during storage at O°C. The relationship of the amount of ice crystals formed, the volume of the unfrozen solution and the final concentrations of inorganic salts and protein on membrane damage and 'survival' was represented more clearly in Fig. 9. In these experiments constant amounts of membranes were subjected to a freeze-thaw cycle in the presence of various initial osmolarities of the complex salt medium and different concentrations of BSA. As expected from variation of the freezing temperature (Fig . 1), optimum curves were obtained when the activity of cyclic photophosphorylation was plotted as a function of the initial concentrations of BSA (Fig. 9A) and the complex salt medium (Fig. 9C) and versus the BSAIsait ratios (Figs.9B, 9D), respectively. Fairly independent of the initial electrolyte concentration, optimum membrane protection occurred at similar BSAIsalt ratios (Fig. 9B). At lower initial BSA concentrations, most favourable membrane 'survival' was reached at lower protein/salt ratios (Fig. 9D). Proceeding from these optimum proportions to lower BSAIsalt ratios, on the one hand and higher ratios on the other, inactivation of photophosphorylation was obviously due to different mechanisms . Discussion

1. The effect of BSA on various membrane sites Storage of isolated thylakoid membranes for 24 h at O°C in the complex salt medium in the absence of BSA resulted in a marked depression of whole-chain linear photosynthetic BPP 187 (1991) 2

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and concentrated up to the twofold initial concentration (factor '2'), i. e. the factors denote the respective salt concentration. Ratios of BSAlsalts result from the BSA concentration (in %) divided by the concentration of the complex salt medium (as expressed by the factors from 0..25 to 2). A and B: Definite initial concentrations of the complex salt medium and variable amounts of BSA. Salt concentration factors: 0, 0..25; "',0..5; 0, 0..75;., 1; \l, 1.5;.,2. C and D: Given initial amounts ofBSA and variable concentrations of the salts. BSA concentrations (in %):.,0.2; 0,0.5; "',1; 0, 2; . , 3; \l, 4. The initial thylakoid concentration corresponding to ca. 1 mg ChI ml- 1 was kept constant throughout. Freezing took place for 22-24 h at -15°C. The activity of PMS-mediated photophosphorylation is plotted as a function of the initial concentrations of BSA (A), the complex salt medium (C) and the ratios of BSAlsalts (B, D). Note that the BSAlsalt ratios are plotted on a logarithmic scale. Two different experiments are presented for AlB·land C/D, respectively.

electron transport (Fig. 2). This inactivation was due to damage of the PSII-mediated electron flow, while coupling was partially maintained and PSI activity remained fairly unimpaired (SANTARIUS 1990a). Thus, PMS-mediated ATP formation was only slightly affected during long-term storage at O°C (Fig. 1 A). In the presence 'of BSA, efficient stabilization of cyclic photophosphorylation took place (Figs. 1 A, 7, 8), but only insignificant preservation of the electron transport system was reached under nonfreezing conditions (Fig. 2). When thylakoids were subjected for 24 h to freeze-thaw treatment without BSA in the complex salt medium, a strong depression of the capacity of linear photosynthetic electron transport, abolition of coupling and almost complete inactivation of light-induced H+ uptake 158

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and photophosphorylation took place nearly independent of the freezing temperature (Figs. 1-4). As shown earlier, uncoupling is a main target during freezing of thylakoids in the presence of a single potentially cryotoxic electrolyte (see, e.g., SANTARIUS 1984), but plays a minor role when membranes were suspended in a complex salt medium (SANTARIUS 1986c; GRAFFLAGE and KRAUSE 1986), because sulfate is highly effective in stabilizing CF t binding during freezing (SANTARIUS 1987). In agreement, light-induced H+ uptake was only insignificantly restored when DCCD was added prior to fluorescence measurements (Fig. 4), although about one third of the electron transport capacity of the membranes was left after a 24 h freezing in the absence of BSA (Figs. 2, 3). The effect of BSA on thylakoid membranes during freezing was largely dependent on the initial protein concentration and the freezing temperature. According to the freezing conditions, BSA either caused partial or complete protection of photosynthetic membrane reactions or, at higher protein concentration, in part decreased or even abolished this effect. At mild freezing, only little protection of the electron transfer capacity took place (Fig. 2), but BSA in part maintained coupling (Fig. 3A). As a result, protection of photophosphorylation was observed (Figs. 1 A, 3 A). Interestingly, in the presence of BSA, sulfate did not prevent dissociation of the CF t at mild freezing conditions (Fig. 4A). The more efficient protection of PMS-mediated ATP formation (Fig. I A) relative to noncyclic photophosphorylation (Figs. 3 A, 3 B) at mild and moderate freezing temperatures can probably be explained by an almost complete preservation of PSI activity but incomplete stabilization of psn (SANTARIUS 1990a). Under more severe freezing conditions, protection of the capacity of whole-chain photosynthetic electron transport by BSA increased with lowering the temperature (Figs. 2, 3, 6). Presumably, this stimulation may be related to structural changes of the thylakoid membrane which may be a consequence of the severe dehydration. In contrast, when the phophorylating activity of the membranes after freeze-thaw treatment was plotted versus the initial BSA concentration, optimum curves were obtained (Figs. 1 B, 3C, 9A; see also Fig. 4C). The lower the freezing temperature, the lower was the BSA concentration for optimum membrane preservation (Fig. 1). At more severe freezing, the maximum protection achieved with BSA decreased with lowering the temperature (Fig. 1 B). It is likely that 'mechanical' damage, which led to an increase in the proton permeability of the membranes (Fig. 4C) became manifest when the protein concentration in the residual unfrozen fraction exceeded a critical limit (see next section). These membrane alterations were initiated immediately upon the ice formation (Figs.5B, 6) and led to uncoupling (Figs. 3C, 3D, 6). However, these changes were probably not due to dissociation of the CF t (Fig. 4C). 2. Factors contributing to inactivation and protection of photophosphorylation

When thylakoids were subjected to a freeze-thaw cycle, the concentrations of inorganic salts and BSA reached in the residual unfrozen solution of the system seems to be crucial for damage or stabilization of the phosphorylating activity. Starting from optimum cryoprotection in the presence of a given ratio of BSAIsalts which corresponds to definite protein and salt concentrations in the residual unfrozen portion, the degree of damage at lower protein salt levels is predominantly determined by the final concentration of the inorganic salts, while at higher ratios the rise in the protein concentration seems to enhance inactivation of BPP 187 (1991) 2

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the phosphorylating system. This was particularly clear when the phosphorylating activity was considered after exposure to a given freezing temperature: the higher the initial salt concentration, the more BSA was necessary for comparable cryoprotection (Figs. 9 A, 9C), i.e. optimum membrane stabilization occurred at a definite BSAIsait ratio (Figs. 9B, 9D). Obviously, high concentrations of both inorganic salts and BSA caused inactivation of photophosphorylation, but suitable combinations of these potentially membrane-toxic compounds can affect cryostability of thylakoids. This indicates a colligative action, i.e. the different solutes act on different membrane sites and each reduces the concentration of the others in the residual unfrozen liquid (HEBER et ;11 . 1971; SANTARIUS 1971, 1986a). The colligative concept was already proved for low-Illllkcular-weight solutes (LINEBERGER and STEPONKUS 1980; SANTARIUS and GIERSCH 1983, 1984) and has been also suggested for polymers (SANTARIUS 1982), which exhibit extreme deviations from ideal thermodynamic behaviour at high concentrations (see, e.g., MONEY 1989). However, various findings cannot be explained by the colligative concept. For instance, to get comparable membrane protection, less protein had to be added prior to freeze-thaw treatment at the lower freezing temperatures (Figs. lA, 3A, 3B, 4A, 4B). This result is in contradiction to earlier data obtained during a 3-5 h freezing ofthylakoids in the presence of variable amounts of glucose and NaCl (SANTARIUS and GIERSCH 1984). But one has to consider that the time course of freeze-thaw injury of isolated chloroplast membranes is approximately biphasic: after an initial rapid damage, thylakoid disintegration was almost linearly dependent on incubation time (HINCHA and SCHMITT 1988). The slow phase reflects a progressive membrane inactivation, which is strongly dependent on the temperature and the composition of the suspending medium. In the absence of cryoprotectants, progressive thylakoid destruction in frozen samples was much more pronounced when only one single inorganic electrolyte was available as compared to a complex medium (SANT ARIUS 1986 c). At mild freezing of thylakoids in the complex salt medium, this gradual membrane disintegration became reduced with both lowering the temperature (Fig. 5A) and addition of limited amounts of BSA prior to freezing (Fig. 5 B). Presumably, a certain low BSA concentration in the residual liquid was sufficient for a drastic decrease of progressive temperature-dependent inactivation of the phosphorylating system. As solutes became concentrated in the residual unfrozen fraction, the lower the freezing temperature, the less BSA had to be added to the complex salt medium in order to reach the effective protein concentration in the surrounding of the membranes. Obviously, the deleterious effect of the simultaneous increase in the total salt concentration in the residual liquid became partly compensated by a temperature-dependent membrane-stabilizing effect of BSA. Other factors which affect the cryostability of biomembranes are the amount of ice formed and the final volume of the residual liquid fraction. A decrease in the total starting concentration of solutes at a given BSAIsalt ratio was accompanied with diminution of the efficiency of thylakoid protection (Fig. 9B), even when the amount of membranes relative to the volume of the residual liquid remained constant (Fig. 7 A). This indicates mechanical damage by the larger amount of ice crystals formed and, in parallel, reduction of the volume of the unfrozen fraction, respectively (SANTARIUS and GIERSCH 1984; HINCHA et al. 1984). Under these conditions, inactivation of photophosphorylation was even enhanced when the diminution of the residual liquid phase was combined with a strong rise in the concentration of BSA (Fig.7C) and membranes (Fig.7B) in this fraction. The deleterious effect of 160

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extremely high thylakod concentrations in the frozen system was shown with either decreasing initial osmolarities (Fig. 7B) or, likewise, increasing initial membrane concentration (Fig. 8) in the presence of constant BSAIsait ratios (see also SANTARIUS and GIERSCH 1984). Obviously, an extremely small portion of the residual liquid as well as very high protein concentrations and a very dense thylakoid packing in this fraction enhanced membrane damage. The rapid phase of membrane damage became visible at moderate freeze-thaw treatment and rose with decrease of the freezing temperature (Figs. 5, 6). In this case, inactivation of photophosphorylation occurred almost in parallel with the crystallization process. It is likely that the rapid component of damage is attributed to membrane rupture caused by large amounts of ice crystals formed and very high concentrations of inorganic electrolytes, BSA and membranes in the residual unfrozen fraction, simultaneously leading to an extremely strong dehydration. When thylakoids were frozen at temperatures around -15 to -23°C in the presence of the complex salt medium and initial amounts of BSA below the concentrations which produced optimum protection of the phosphorylating activity, membrane preservation was solely dependent on the BSAIsait ratio but independent of the temperature (Fig. 1 B). This also does not fit into the colligative concept, because the concentrations of both BSA and inorganic electrolytes in the liquid fraction were higher at the lower freezing temperatures. The mechanism of this effect remains obscure.

Acknowledgements The author is very grateful to Mrs. BRITTA FRINKER for competent technical assistance and to Professor Dr. G. H. KRAUSE for critical reading of the manuscript.

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