Planta
Planta (1984) 161:555-561
9 Springer-Verlag 1984
Effective cryoprotection of thylakoid membranes by ATP Kurt A. Santarius Botanisches Institut der Universit/it, Universiffitsstrasse 1, D-4000 Dfisseldorf, Federal Republic of Germany
Abstract. Freezing of isolated spinach thylakoids in the presence of NaC1 uncoupled photophosphorylation from electron flow and increased the permeability of the membranes to protons. Addition of ATP prior to freezing diminished membrane inactivation. On a molar basis, ATP was at least 100 times more effective in protecting thylakoids from freezing damage than low-molecularweight carbohydrates such as sucrose and glucose. The cryoprotective effectiveness of ATP was increased by M g 2+. In the absence of carbohydrates, preservation of thylakoids during freezing in 100 mM NaC1 was saturated at about 1-2 mM ATP, but under these conditions membranes were not fully protected. However, in the presence of small amounts of sugars which did not significantly prevent thylakoid inactivation during freezing, ATP concentrations considerably lower than 0.5 mM caused nearly complete membrane protection. Neither ADP nor AMP could substitute for ATP. These findings indicate that cryoprotection by ATP cannot be explained by a colligative mechanism. It is suggested that ATP acts on the chloroplast coupling factor, either by modifying its conformation or by preventing its release from the membranes. The results are discussed in regard to freezing injury and resistance in vivo. Key words: ATP - Colligative theory - Cryoprotection - Freezing injury - Spinacia (freezing) Thylakoid membrane.
Introduction
Freezing of isolated thylakoids in the presence of inorganic salts causes a drastic increase in the electrolyte concentration in the surroundings of the Abbreviations. CF 1= chloroplast coupling factor; Hepes = 4-(2hydroxyethyl)-l-piperazineethanesulfonic acid; PMS=phenazinc methosulfate; Tris=2-amino-2-(hydroxymethyl)-l,3-propandiol
membranes which leads to an almost complete uncoupling of photophosphorylation from electron flow (Heber and Santarius 1964, 1973 ; Thebud and Santarius 1981). Concurrent with uncoupling, an increase in the permeability to protons (Heber 1967) and a release of proteins from the membranes (Garber and Steponkus 1976; Volger et al. 1978; Mollenhauer etal. 1983) have been observed. According to Lineberger and Steponkus (1980) inactivation of photophosphorylation during freezing could be a consequence of the loss and inactivation of the chloroplast coupling factor (CF1). If cryoprotectants such as various carbohydrates, certain amino acids and specific proteins are simultaneously present in sufficient amounts during ice formation, membrane inactivation can be avoided (Heber and Santarius 1964, 1973; Heber et al. 1971; Volger and Heber 1975; Santarius 1982a). Protection occurs by unspecific colligative solute action and in part by specific membrane stabilization (Santarius 1982b; Santarius and Bauer 1983; Santarius and Giersch 1983a). This indicates that cryoprotectants affect both the electrolyte concentration in the surroundings of the thylakoids and the interrelationship between the CF 1 and the membranes. Adenylates are known to protect isolated CF 1 from inactivation in a medium of high ionic strength at 0~ (Posorske and 3agendorf 1976). Moreover, ATP and ADP also diminish loss of phosphorylation activity of isolated thylakoids under these conditions (Santarius 1984). In this communication the ability of adenylates to stabilize isolated thylakoid membranes during freezing is reported. Material and methods Plant material. Spinach (Spinacia oleracea L. cv. Monatol) was grown in greenhouses with 10-h light and 14-h dark periods. Leaves were harvested from 5- to 7-week-old plants.
556
Isolation of thylakoids in a Tris-NaCl medium. Washed leaves (100 g) were ground at 4 ~ C for 15 s in a Waring blendor in 375 ml of a medium containing 50 m M 2-amino-2-(hydroxymethyl)-l,3-propandiol (Tris)-HC1 (pH 8.0), 350 mM NaC1, 10 mM KHzPO4, 10 mM sodium ascorbate, 3.3 mM cysteine and 50 mM 2-mercaptoethanol. The homogenate was filtered through eight layers of cheesecloth and centrifuged for I rain at 250 g. The supematant fraction was subjected to 3 min centrifugation at 1500 g. The pellet was resuspended in a medium containing 50 mM Tris-HC1 (pH 8.0) and 350 m M NaC1 and centrifuged for 3 min at 2000 g. The sedimented chloroplasts were ruptured in distilled water and the released thylakoids were centrifuged for 6 min at 20000 g in a solution containing 20 mM NaC1. The washed membranes were resuspended in NaC1 solutions at the concentrations indicated and stored at 0 ~ C.
Isolation of thylakoids in a Tris-sorbitol medium. Leaves were blended in a medium containing 50 m M Tris-HC1 (pH 7.5), 330 mM sorbitol, 10 mM NaC1, 2 m M ethylenediaminetetraacetic acid (EDTA), 1 m M MgCI2, 1 mM MnCI2, 0.5 mM KHzPO 4, 1 0 r a m sodium ascorbate, 3.3 mM cysteine and 5 0 m M 2-mercaptoethanol under conditions as described above. After filtration, the homogenate was centrifuged for 1 min at 250 g. The chloroplasts were sedimented by centrifugation for 3 min at 2000 g. The pellet was resuspended in the above medium, but without ascorbate, cysteine and 2-mercaptoethanol, and again centrifuged for 3 rain at 2000 g. This washing procedure was repeated once more. The sedimented chloroplasts were broken by suspending them in 5 m M MgC12 ; after approx. 30 s, freed thylakoids were transferred to a medium containing 5 0 m M 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (Hepes)-NaOH (pH 7.6), 330 mM sorbitol, 10 mM NaC1 and 5 m M MgC12 and stored at 0 ~ C.
Chlorophyll determination. Chlorophyll was determined according to Arnon (1949).
Freezing of thylakoids. For freezing of isolated membranes in the presence of various concentrations of NaC1, glucose, sucrose and adenylates, samples (0.5 ml each) of thylakoid suspensions in glass tubes were placed into a cryostat kept at - 1 5 ~ C. After freezing for 3-5 h, samples were thawed rapidly in a water bath at room temperature. Controls were kept at 0 ~ C for the same time.
Cyclic photophosphorylation. The activity of phenazine methosulfate (PMS)-mediated photophosphorylation before and after freezing was determined either in high-intensity white light as described recently (Santarius 1982a) or under the same conditions in nearly saturating red light (RG 630 cut-off filter, Schott & Gen., Mainz, FRG). Ferricyanide reduction. Thylakoids equivalent to 30 gg of chlorophyll were added to 2 ml of a reaction medium containing 25 mM Tris-HC1 (pH 7.8), 2.2 mM KHzPO 4, 2.2 mM ADP, 4 mM MgC12 and 0.4 m M K3[Fe(CN)6 ]. Potassium-hexacyanoferrate(III) reduction was recorded at room temperature with a Zeiss spectrophotometer (Zeiss, Oberkochen, FRG) at 400 nm during illumination with nearly saturating red light (RG 630 cut-off filter).
Methyl viologen reduction and noncyclic photophosphorylation. Light-dependent Oz uptake in the presence of methyl viologen was measured polarographically with a Clark-type electrode at 20 ~ C. Thylakoids isolated in a Tris-sorbitol medium and corresponding to 50 gg of chlorophyll were suspended in 1 ml
K.A. Santarius: Cryoprotection of thylakoids by ATP of a medium containing 50 mM Hepes-NaOH (pH 7.6), 330mM sorbitol, 1 0 m M KHzPO4, 5 m M MgClz, 1 mM NaN 3 and 25 gM methyl viologen. The reaction was started by illumination with nearly saturating red light as given above. After addition of small amounts of ADP (50 to 200 nmol each), electron transport was recorded in the presence (state 3) and after consumption of this nucleotide (state 4). The rates of methyl viologen reduction (corresponding to state 3) and ATP formation and the photosynthetic control and ADP/O ratios were calculated according to Robinson and Wiskich (1976).
Light-dependent proton gradient. Proton movement across the thylakoid membrane during dark-light and light-dark transition was determined by measurement of 9-aminoacridine fluorescence. Thylakoids were isolated in a Tris-NaC1 medium. Membranes equivalent to 20 gg of chlorophyll were suspended in 2.1 ml of a medium containing 30 mM N-[2-hydroxy-l,1bis(hydroxymethyl)ethyl]glycine (Tricine)-NaOH (pH 7.6), 100 mM NaC1, 2 mM MgC12, 25 gM methyl viologen and 10 gM 9-aminoacridine and placed into a spectrofluorometer (Eppendorf, Netheler & Hinz GmbH, Hamburg, FRG) at room temperature. Fluorescence of 9-aminoacridine was excited by 313- to 366-nm light of low intensity, and the emitted light was recorded by a photomultiplier which was protected against red actinic light by an appropriate filter combination (Corning 5030 and 9782). After a dark period of at least 1 min, thylakoids were irradiated for 3 min with saturating red actinic light (RG 630 cut-off filter) which was again followed by darkness. From the quenching of the 9-aminoacridine fluorescence in the light (steady state), the ApH was calculated according to Briantais et al. (1979; see also Tillberg et al. 1977).
Results
The effect of freezing on isolated chloroplast membranes in the presence of membrane-toxic compounds and cryoprotectants has been studied intensively in various laboratories. It was found that at a given freezing temperature, membrane preservation was mainly determined by the ratio of cryoprotectant to cryotoxic compounds (Heber and Santarius 1964, 1973; Santarius and Giersch 1983a). For instance, if washed thylakoids were suspended in solutions containing solely glucose and NaC1 and freezing took place at temperatures around - 1 5 ~ a molar ratio of sugar to salt above 1 was necessary for appreciable membrane protection. The experiment depicted in Fig. 1 shows that in the presence of 100 m M NaC1 about 200 m M of glucose was needed to maintain 50% of the phosphorylating capacity during freezing. However, if 0.5 m M of ATP was present, much less glucose was necessary for comparable membrane protection. Moreover, if thylakoids were provided with 2 m M MgC12 plus 0.5 m M ATP, the sugar concentration sufficient for 50% membrane preservation was nearly a tenth of that necessary in the absence of Mg z § and ATP, and cyclic photophosphorylation became partly protected even in the absence of glucose. Addition of 2 m M
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Fig. 2A, B. Protection of spinach thylakoid membranes during freezing in the presence of ATP (A) and sucrose and glucose (B) as a function of the concentration of the cryoprotectants. Thylakoids isotated in a Tris-NaC1 medium and suspended in 5 m M Hepes-NaOH (pH 7.5), 100 m M NaCI, 2 m M MgC12 and variable concentrations of ATP (o, o), sucrose (% A) and glucose 0% n), respectively, as indicated on the abscissae, were frozen for 3-4 h at - 15 ~ C. After thawing, PMS-mediated photophosphorylation ( , open symbols) and ferricyanide reduction (- . . . . , closed symbols) were measured in nearly saturating red light. Photophosphorylation and electron transport were plotted as % of the unfrozen control (unfrozen control = 100%). Activities of the unfrozen control: photophosphorylation 778 pmol ATP m g - 1 chlorophyll h - 1 ; electron transport 402 pmol ferricyanide r a g - 1 chlorophyll h - 1
MgC12 in the absence of ATP did not appreciably alter membrane preservation. The experiment shown in Fig. 1 was carried out at a pH around 6.7 and in the absence of buffer. When the pH was adjusted to 7.5 prior to freezing
by use of 5 m M Hepes-NaOH, similar results were obtained (not shown). Under these conditions, somewhat less glucose was needed for a comparable degree of cryoprotection. Moreover, partial protection of thylakoids in the absence of glucose was already observed when ATP only was added to the membrane suspension and was considerably more pronounced in the presence of ATP plus Mg 2§ (see also Figs. 2, 4, 5). These differences could be due to both altered pH and a protective effect of the buffer molecules. Uncoupling of photophosphorylation during freezing of isolated thylakoids is accompanied by stimulation of the noncyclic electron transport under phosphorylating conditions (Heber and Santarius 1964; Heber et al. 1973; Volger et al. 1978; Thebud and Santarius 1981). The addition of ATP protects cyclic photophosphorylation and decreases the stimulation of ferricyanide reduction to a similar extent (Fig. 2). Considerable membrane preservation was reached in the presence of relatively small amounts of ATP, without addition of any other common cryoprotectant. On a molar basis, ATP was at least 100 times more effective than sucrose and glucose. However, preservation by ATP was saturated at 1-2 raM, at 50-70% protection, whereas membranes were 95-100% protected by low-molecular-weight carbohydrates. This is similar to data obtained with cryoprotective proteins (Volger and Heber 1975). However, in combination with small amounts of sugars which are insufficient for detectable membrane stabilization, almost complete membrane protection was reached when very low concentrations of either ATP or proteins were added prior to freezing (see Figs. 1, 3 and Volger and Heber 1975). The high cryoprotective effectiveness of ATP on thylakoids was even more striking in regard to coupling of noncyclic photophosphorylation to photosynthetic electron transport. To obtain a high degree of coupling, chloroplast membranes were isolated and stored in a Tris-sorbitol medium. In this case, the cryoprotective action of sorbitol was gradually abolished by the addition of increasing concentrations of NaC1. As a result, the decrease in the activity of noncyclic photophosphorylation was accompanied by a drastic stimulation of the electron flow which finally reached rates exceeding four to five times those of the unfrozen controls (Fig. 3). This indicates that ADP/O ratios dropped to zero and photosynthetic control was no longer observed (not shown). If small amounts of ATP were added prior to freezing, a considerably higher salt concentration was necessary for a comparable degree of uncoupling. For example,
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Fig. 3. Activities of photosynthetic electron transport and noncyclic photophosphorylation of spinach thylakoid membranes after freezing in the presence and absence of ATP as a function of the initial NaC1 concentration. Thylakoids were isolated in a Tris-sorbitol medium and suspended in 20 m M Hepes-NaOH (pH 7.4), 132 m M sorbitol, 2 m M MgC12 and variable concentrations of NaCI as indicated on the abscissa. Aliquots were frozen for 3-5 h at - 1 5 ~ directly (without ATP, v, v) and in the presence of 0.1 m M (o, e) and 0.25 m M ATP (% m), respectively. After thawing, light-dependent 0 2 uptake in the presence of methyl viologen was measured at 20 ~ C. Noncyclic photophosphorylation ( , open symbols) and methyl viologen reduction in the presence of A D P ( - - , closed symbols) were plotted as % of the unfrozen controls (unfrozen controls = 100%). Activities of the unfrozen controls: photophosphorylation 192 gmol ATP synthesized mg 1 chlorophyll h - 1 ; electron transport 104 gmol O2 consumed m g - 1 chlorophyll h - 1 ; A D P / O ratio 0.9; photosynthetic control 3.2
Fig. 4. The effect of freezing on cyclic photophosphorylation and light-dependent proton gradient of spinach thylakoid membranes as a function of the ATP concentration. Thylakoids isolated in a Tris-NaC1 medium and suspended in 5 m M HepesN a O H (pH 7.5), 100 m M NaC1, 2 m M MgC12 and variable concentrations of ATP as indicated on the abscissa were frozen for 3-5 h at - 1 5 ~ C. After thawing, PMS-mediated photophosphorylation ( o ) and the quenching of the 9aminoacridine fluorescence were measured. The ApH values (-9 - - - ) calculated from the fluorescence signals were plotted by use of an extended scale as indicated on the righthand-side ordinate (for explanation see text). Activities of the unfrozen control: photophosphorylation in nearly saturating red light 646 gmol ATP m g - 1 chlorophyll h - t ; ApH 4.12. Note that ApH calculated here cannot be taken as an absolute measure, since the internal volume of the thylakoids was not determined
to obtain similar alterations in the photochemical activities of thylakoids in the course of a freezethaw cycle, nearly twice as much NaC1 had to be added in the presence of only 0.1 m M ATP compared with membranes frozen in the absence of the adenylate. It should be mentioned that freezing in the presence of very low initial NaC1 concentrations, e.g. 4 m M in Fig. 3, also resulted in some uncoupling, i.e. stimulation of the photosynthetic electron transport and decrease in photophosphorylation and, thus, a decline in the ADP/O ratio and photosynthetic control value. This is in agreement with earlier findings that cryoprotection of chloroplast membranes is diminished at very low salt concentrations (Santarius and Giersch 1983 b; see also Santarius 1982a; Santarius and Giersch 1984). Uncoupling of photophosphorylation of isolated thylakoids during freezing is related to a breakdown of the ability to accumulate protons in the light (Heber 1967). The question arises whether the addition of ATP prior to freezing prevents changes in the permeability of the mem-
branes to protons. According to Schuldiner et al. (1972) the quenching of the 9-aminoacridine fluorescence in the light can be used to estimate the size of the proton gradient across the thylakoid membrane. As recently shown by Hangarter and Good (1982), under certain conditions a threshold value of the ApH is required for photophosphorylation of isolated thylakoid membranes. In the experiment shown in Fig. 4, thylakoids were incubated at high concentrations of ions; under these conditions a threshold ApH of 2 to 3 is expected (Hangarter and Good 1982). Taking this into account, cryoprotection of photophosphorylation by increasing concentrations of ATP seems to be closely correlated with alterations in ApH (Fig. 4). When ADP and A M P were added to the membranes prior to freezing, little or no protection was observed (Fig. 5). This is valid for adenylate concentrations up to 5 m M (not shown). In some experiments very small amounts of ADP exhibited a distinct but small cryoprotective effect which was not enhanced at ADP concentrations exceeding 1 mM.
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Fig. 5. The effect of various adenylates on spinach thylakoid membranes during freezing. Thylakoids were isolated in a TrisNaC1 medium and frozen for 3-4 h at - 1 5 ~ C in the presence of 5 m M Hepes-NaOH (pH 7.1), 100 m M NaC1, 2 m M MgC12 and variable concentrations of ATP (o), A D P ( , ) and A M P (D), respectively, as indicated on the abscissa. After thawing, PMS-mediated photophosphorylation was measured in saturating white light. The activity of the unfrozen controls was 982 pmol ATP m g - t chlorophyll h 1
Discussion
Very recently it was shown that cryoprotection of isolated thylakoid membranes mainly depends on the ratio of sugar to salt, the total solute concentration prior to freezing and the freezing temperature (Santarius and Giersch 1983a, b, 1984; Hincha et al. 1984). Apart from extreme conditions such as very low or high initial solute concentrations and freezing temperatures below - 2 0 ~ C, predominantly the colligative action of the solutes contributes to membrane preservation, i.e., cryoprotectants prevent the increase in the concentration of potentially membrane-toxic salts during freezing up to a critical limit (Heber and Santarius 1973; Heber et al. 1979). However, cryoprotection by high-molecular-weight compounds such as proteins (Volger and Heber 1975) and dextrans (Santarius 1982 a, b) cannot be explained by the colligative concept, because these compounds are already effective at very low concentrations. At present, the mechanism of cryopreservation of biomembranes by high-molecular-weight solutes is not understood. The data presented in this paper clearly demonstrate that ATP most effectively stabilizes isolated chloroplast membranes in the course of a freezethaw cycle. Obviously, cryoprotection by ATP cannot be explained by colligative action as the effective concentrations are extremely low in relation
to the total concentration of other solutes present in the membrane suspension. Rather, membrane stabilization during freezing may be caused by specific interactions between ATP and the thylakoid membranes. Possibly, the results can be widely interpreted by the action of ATP on the CF t. It is known that removal of the CFt from the membranes results in uncoupling, i.e., in inactivation of photophosphorylation accompanied by an enhancement of photosynthetic electron flow (Avron 1963; Hesse et al. 1976; Telfer et al. 1980). If sufficient concentrations of cations, in particular of highly effective Mg 2 § or Ca 2 -- which achieve binding of CF 1 to the membrane, are present in the medium, uncoupling can be avoided (Hesse et al. 1976; Telfer et al. 1980). Presumably, the release of CF 1 opens channels for protons in the membranes (McCarty and Racker 1966; Schmid and Junge 1974). Any leakage of H + caused by permeability changes in the chloroplast membranes would impair the efficiency of photophosphorylation and simultaneously loosen the control of electron transport by the ApH. This hypothesis is supported by various data in the literature. In earlier investigations carried out at temperatures above freezing, isolated CF 1 was found to be cold-labile mainly in the presence of high concentrations of salts (Lien et al. 1972). Effective protection against cold inactivation of CF~ at high NaC1 levels occurred in the presence of Mg; + and very low concentrations of ATP (Posorske and Jagendorf 1976); it has been suggested that binding of ATP to the CF I decreases the proton permeability of thylakoid membranes and, thereby, causes a more strongly coupled condition (see also McCarty et al. 1971; Telfer and Evans 1972). Taking this into consideration, it can be inferred that ATP may cause similar effects on thylakoid membranes during freezing. Release of subunits of the CF t related to thylakoid inactivation by freezing was repeatedly observed (Garber and Steponkus 1976; Steponkus et al. 1977; Volger et al. 1978; Lineberger and Steponkus 1980; Hincha et al. 1984). In the present contribution, it was shown that at a given NaC1 concentration which caused uncoupling, addition of ATP prior to freezing completely or at least partly prevents inactivation of photophosphorylation and stimulation of electron flow in the presence of very low concentrations of carbohydrates (Figs. 1, 3) and even in the total absence of common cryoprotectants (Figs. 2, 4, 5). The protective action of ATP (Fig. 1) is improved by Mg 2§ Concurrent with the cryoprotective effect on photophosphorylation, ATP diminishes the effect of
560
freezing on the H § permeability of the membranes (Fig. 4). All these results support the view that ATP acts at the CF a. This action could be either a modification of the CF 1 that leads to conformational changes and thus stabilizes the protein complex against cold inactivation, or ATP diminishes the release of CF 1 from thylakoids. As a result, uncontrolled proton leakage across the membranes caused by freezing would be prevented. Possibly, cryoprotection of isolated thylakoid membranes by high-molecular-weight solutes such as specific proteins and dextrans which are likewise effective at very low molar concentrations (see above) can be explained by a similar mechanism. In contrast to data obtained at 0 ~ C (Posorske and Jagendorf 1976; Santarius 1984), ADP does not protect thylakoid membranes during freezing even in the presence of Mg 2 § (Fig. 5). Apparently, ADP is incapable of stabilizing the CF 1 at the membranes under freezing conditions. Uncoupling of photophosphorylation is not observed as a primary effect when freezing of chloroplast membranes takes place in situ (Heber et al. 1973; Thebud and Santarius 1981; Klosson and Krause 1981; Krause et al. 1982). In thylakoid membranes isolated from partly frost-damaged leaves photosynthetic electron transport, noncyclic photophosphorylation and the extent of light-induced H § uptake were lowered to nearly the same degree without an appreciable increase in the permeability of the membranes towards protons (Klosson and Krause 1981). Obviously, damage to thylakoids in situ and in vitro is caused by different mechanisms. In this respect, it is of interest that ATP levels existing in spinach chloroplasts in situ )tre in the magnitude of 0.5 to 2 m M (for references see Krause and Heber 1976) and, in some cases, seem to increase during frost-hardening even in the dark (Sobczyk and Kacperska-Palacz 1978). Moreover, it is well known that in chloroplasts various low- and high-molecular-weight cryoprotectants become accumulated during coldacclimation (Heber 1959, 1970; Heber and Ernst 1967; Kappen and Ullrich 1970; Santarius and Milde 1977; Krause et al. 1982, 1983). Presumably, at freezing temperatures that lead to irreversible cell damage, thylakoids are still sufficiently protected with regard to injury caused by increase in the concentration of cryotoxic, potentially uncoupling solutes such as inorganic salts in the chloroplast stroma. Thus, inactivation of thylakoid membranes during freeze-damage of leaf tissue probably is not caused primarily by modification or release of CF 1 but occurs by others, as yet unknown, alterations.
K.A. Santarius: Cryoprotection of thylakoids by ATP The author is grateful to Professor G.H. Krause for critical reading of the manuscript and to Miss Britta Dietzel for competent technical assistance.
References Arnon, D.I. (1949) Copper enzymes in isolated chloroplasts. Polyphenol-oxidase in Beta vulgaris. Plant Physiol. 24, 1-15 Avron, M. (1963) A coupling factor in photophosphorylation. Biochim. Biophys. Acta 77, 699-702 Briantais, J.-M., Vernotte, C., Picaud, M., Krause, G.H. (1979) A quantitative study of the slow decline of chlorophyll a fluorescence in isolated chloroplasts. Biochim. Biophys. Acta 548, 128-138 Garber, M.P., Steponkus, P.L. (1976) Alterations in chloroplast thylakoids during an in vitro freeze-thaw cycle. Plant Physiol. 57, 673-680 Hangarter, R.P., Good, N.E. (1982) Energy thresholds for ATP synthesis in chloroplasts. Biochim. Biophys. Acta 681, 397-404 Heber, U. (1959) Beziehungen zwischen der Gr613e von Chloroplasten und ihrem Gehalt an 16slichen Eiweigen und Zukkern im Zusammenhang mit dem Frostresistenzproblem. Protoplasma 51, 284~298 Heber, U. (1967) Freezing injury and uncoupling of phosphorylation from electron transport in chloroplasts. Plant Physiol. 42, 1343-1350 Heber, U. (1970) Proteins capable of protecting chloroplast membranes against freezing. In: Ciba found, symp. on the frozen cell, pp. 175-188, Wolstenholme, G.E.W., O'Connor, M., eds. Churchill, London Heber, U., Ernst, R. (1967) A biochemical approach to the problem of frost injury and frost hardiness. In: Cellular injury and resistance in freezing organisms (Proc. Int. Conf. Low Temp. Sci.), vol. II, pp. 63-77, Asahina, E., ed. Inst. Low Temp. Sci., Hokkaido University, Sapporo Heber, U., Santarius, K.A. (1964) Loss of adenosine triphosphate synthesis caused by freezing and its relationship to frost hardiness problems. Plant Physiol. 39, 712-719 Heber, U., Santarius, K.A. (1973) Cell death by cold and heat and resistance to extreme temperatures. Mechanisms of hardening and dehardening. In: Temperature and life, pp. 232-263, Precht, H., Christophersen, J., Hensel, H., Larcher, W., eds. Springer, Berlin Heidelberg New York Heber, U., Tyankova, L., Santarius, K.A. (1971) Stabilization and inactivation of biological membranes during freezing in the presence of amino acids. Biochim. Biophys. Acta 241, 578 592 Heber, U., Tyankova, L., Santarius, K.A. (1973) Effects of freezing on biological membranes in vivo and in vitro. Biochim. Biophys. Acta 291, 23 37 Heber, U., Volger, H., Overbeck, V., Santarius, K.A. (1979) Membrane damage and protection during freezing. Adv. Chem. Ser. 180, 159 189 Hesse, H., Jank-Ladwig, R., Strotmann, H. (1976) On the reconstitution of photophosphorylation in CFl-extracted chloroplasts. Z. Naturforsch. Teil C 31,445-451 Hincha, D.K., Schmidt, J.E., Heber, U., Schmitt, J.M. (1984) Colligative and non-colligative freezing damage to thylakoid membranes. Biochim. Biophys. Acta 769, 8 14 Kappen, L., Ullrich, W.R. (1970) Verteilung von Chlorid und Zuckern in Blattzellen halophiler Pflanzen bei verschieden hoher Frostresistenz. Ber. Dtsch. Bot. Ges. 83, 265-275 Klosson, R.J., Krause, G.H. (1981) Freezing injury in coldacclimated and unhardened spinach leaves. I. Photosynthetic reactions of thylakoids isolated from frost-damaged leaves. Planta 151,339-346
K.A. Santarius : Cryoprotection of thylakoids by ATP Krause, G.H., Heber, U. (1976) Energetics of intact chloroplasts. In: The intact chloroplast, pp. 171-214, Barber, J., ed. Elsevier/North-Holland Biomedical Press, Amsterdam Krause, G.H., Klosson, R.J., Justenhoven, A., Ahrer-Steller, V. (1983) Effects of freezing stress on the photosynthetic system in vivo. In: Proc. 6th Int. Congr. on Photosynthesis, Brussels (in press) Krause, G.H., Klosson, R.J., Tr6ster, U. (1982) On the mechanism of freezing injury and cold acclimation of spinach leaves. In: Plant cold hardiness and freezing stress, mechanisms and crop implications, vol. 2, pp. 55-75, Li, P.H., Sakai, A., eds. Academic Press, New York Lien, S., Berzborn, R.J., Racker, E. (1972) Partial resolution of the enzymes catalyzing photophosphorylation. IX. Studies on the subunit structure of coupling factor 1 from chloroplasts. J. Biol. Chem. 247, 3520-3524 Lineberger, R.D., Steponkus, P.L. (1980) Effects of freezing on the release and inactivation of chloroplast coupling factor 1. Cryobiology 17, 486-494 McCarty, R.E., Fuhrman, J.S., Tsuchiya, Y. (1971) Effects of adenine nucleotides on hydrogen-ion transport in chloroplasts. Proc. Natl. Acad. Sci. USA 68, 252~2526 McCarty, R.E., Racker, E. (1966) Effect of a coupling factor and its antiserum on photophosphorylation and hydrogen ion transport. Brookhaven Symp. Biol. 19, 202-214 Mollenhauer, A., Schmitt, J.M., Coughlan, S., Heber, U. (1983) Loss of membrane proteins from thylakoids during freezing. Biochim. Biophys. Acta 728, 331-338 Posorske, L., Jagendorf, A.T. (1976) Nucleotide and metal interactions affecting inactivation of spinach chloroplast coupling factor by NaCI in the cold. Arch. Biochem. Biophys. 177, 276-283 Robinson, S.P., Wiskich, J.T. (1976) Factors affecting the ADP/O ratio in isolated chloroplasts. Biochim. Biophys. Acta 440, 131-146 Santarius, K.A. (t 982 a) Cryoprotection of spinach chloroplast membranes by dextrans. Cryobiology 19, 200 210 Santarius, K.A. (1982b) The mechanism of cryoprotection of biomembrane systems by carbohydrates. In : Plant cold hardiness and freezing stress, mechanisms and crop implications, vol. 2, pp. 475-486, Li, P.H., Sakai, A., eds. Academic Press, New York Santarius, K.A. (1984) The role of the chloroplast coupling factor in the inactivation of thylakoid membranes at low temperatures. Physiol. Plant. (in press) Santarius, K.A., Bauer, J. (1983) Cryopreservation of spinach chloroplast membranes by low-molecular-weight carbohydrates. I. Evidence for cryoprotection by a noncolligativetype mechanism. Cryobiology 20, 83-89 Santarius, K.A., Giersch, Ch. (1983a) Cryopreservation of spinach chloroplast membranes by tow-molecular-weight
561 carbohydrates. II. Discrimination between colligative and noncolligative protection. Cryobiology 20, 90-99 Santarius, K.A., Giersch, Ch. (1983 b) Inactivation and protection of isolated thylakoid membranes during freezing. In: Effects of stress on photosynthesis, pp. 201-210, Marcelle, R., Clijsters, H., van Poucke, M., eds. Nijhoff/Jnnk, The Hague Santarius, K.A., Giersch, Ch. (1984) Factors contributing to inactivation of isolated thylakoid membranes during freezing in the presence of variable amounts of glucose and NaC1. Biophys. J. (in press) Santarius, K.A., Milde, H. (1977) Sugar compartmentation in frost-hardy and partially dehardened cabbage leaf cells. Planta 136, 163-166 Schmid, R., Junge, W. (1974) On the influence of extraction and recondensation of CF 1 on the electric properties of the thylakoid membrane. In: Proc. 3rd Int. Congr. on Photosynthesis, vol. II, pp. 821-830, Avron, M., ed. Elsevier, Amsterdam Schuldiner, S., Rottenberg, H., Avron, M. (1972) Determination of ApH in chloroplasts. 2. Fluorescent amines as a probe for the determination of ApH in chloroplasts. Eur. J. Biochem. 25, 64-70 Sobczyk, E.A., Kacperska-Palacz, A. (1978) Adenine nucleotide changes during cold acclimation of winter rape plants. Plant Physiol. 62, 875-878 Steponkus, P.L., Garber, M.P., Myers, S.P., Lineberger, R.D. (1977) Effects of cold acclimation and freezing on structure and function of chloroplast thylakoids. Cryobiology 14, 303-321 Telfer, A., Barber, J., Jagendorf, A.T. (1980) Electrostatic control of chloroplast coupling factor binding to thylakoid membranes as indicated by cation effects on electron transport and reconstitution of photophosphorylation. Biochim. Biophys. Acta 591,331-345 Telfer, A., Evans, M.C.W. 0972) Evidence for chemiosmotic coupling of electron transport to ATP synthesis in spinach chloroplasts. Biochim. Biophys. Acta 256, 625-637 Thebud, R., Santarius, K.A. (1981) Effects of freezing on spinach leaf mitochondria and thylakoids in situ and in vitro. Plant Physiol. 68, 1156-1160 Tillberg, J.-E., Giersch, Ch., Heber, U. (1977) CO2 reduction by intact chloroplasts under a diminished proton gradient. Biochim. Biophys. Acta 461, 31-47 Volger, H.G., Heber, U. (1975) Cryoprotective leaf proteins. Biochim. Biophys. Acta 412, 335-349 Volger, H., Heber, U., Berzborn, R.J. (1978) Loss of function of biomembranes and solubilization of membrane proteins during freezing. Biochim. Biophys. Acta 511,455-469 Received 5 January; accepted 17 February 1984
Planta (1984) 161:555-561
9 Springer-Verlag 1984
Effective cryoprotection of thylakoid membranes by ATP Kurt A. Santarius Botanisches Institut der Universit/it, Universiffitsstrasse 1, D-4000 Dfisseldorf, Federal Republic of Germany
Abstract. Freezing of isolated spinach thylakoids in the presence of NaC1 uncoupled photophosphorylation from electron flow and increased the permeability of the membranes to protons. Addition of ATP prior to freezing diminished membrane inactivation. On a molar basis, ATP was at least 100 times more effective in protecting thylakoids from freezing damage than low-molecularweight carbohydrates such as sucrose and glucose. The cryoprotective effectiveness of ATP was increased by M g 2+. In the absence of carbohydrates, preservation of thylakoids during freezing in 100 mM NaC1 was saturated at about 1-2 mM ATP, but under these conditions membranes were not fully protected. However, in the presence of small amounts of sugars which did not significantly prevent thylakoid inactivation during freezing, ATP concentrations considerably lower than 0.5 mM caused nearly complete membrane protection. Neither ADP nor AMP could substitute for ATP. These findings indicate that cryoprotection by ATP cannot be explained by a colligative mechanism. It is suggested that ATP acts on the chloroplast coupling factor, either by modifying its conformation or by preventing its release from the membranes. The results are discussed in regard to freezing injury and resistance in vivo. Key words: ATP - Colligative theory - Cryoprotection - Freezing injury - Spinacia (freezing) Thylakoid membrane.
Introduction
Freezing of isolated thylakoids in the presence of inorganic salts causes a drastic increase in the electrolyte concentration in the surroundings of the Abbreviations. CF 1= chloroplast coupling factor; Hepes = 4-(2hydroxyethyl)-l-piperazineethanesulfonic acid; PMS=phenazinc methosulfate; Tris=2-amino-2-(hydroxymethyl)-l,3-propandiol
membranes which leads to an almost complete uncoupling of photophosphorylation from electron flow (Heber and Santarius 1964, 1973 ; Thebud and Santarius 1981). Concurrent with uncoupling, an increase in the permeability to protons (Heber 1967) and a release of proteins from the membranes (Garber and Steponkus 1976; Volger et al. 1978; Mollenhauer etal. 1983) have been observed. According to Lineberger and Steponkus (1980) inactivation of photophosphorylation during freezing could be a consequence of the loss and inactivation of the chloroplast coupling factor (CF1). If cryoprotectants such as various carbohydrates, certain amino acids and specific proteins are simultaneously present in sufficient amounts during ice formation, membrane inactivation can be avoided (Heber and Santarius 1964, 1973; Heber et al. 1971; Volger and Heber 1975; Santarius 1982a). Protection occurs by unspecific colligative solute action and in part by specific membrane stabilization (Santarius 1982b; Santarius and Bauer 1983; Santarius and Giersch 1983a). This indicates that cryoprotectants affect both the electrolyte concentration in the surroundings of the thylakoids and the interrelationship between the CF 1 and the membranes. Adenylates are known to protect isolated CF 1 from inactivation in a medium of high ionic strength at 0~ (Posorske and 3agendorf 1976). Moreover, ATP and ADP also diminish loss of phosphorylation activity of isolated thylakoids under these conditions (Santarius 1984). In this communication the ability of adenylates to stabilize isolated thylakoid membranes during freezing is reported. Material and methods Plant material. Spinach (Spinacia oleracea L. cv. Monatol) was grown in greenhouses with 10-h light and 14-h dark periods. Leaves were harvested from 5- to 7-week-old plants.
556
Isolation of thylakoids in a Tris-NaCl medium. Washed leaves (100 g) were ground at 4 ~ C for 15 s in a Waring blendor in 375 ml of a medium containing 50 m M 2-amino-2-(hydroxymethyl)-l,3-propandiol (Tris)-HC1 (pH 8.0), 350 mM NaC1, 10 mM KHzPO4, 10 mM sodium ascorbate, 3.3 mM cysteine and 50 mM 2-mercaptoethanol. The homogenate was filtered through eight layers of cheesecloth and centrifuged for I rain at 250 g. The supematant fraction was subjected to 3 min centrifugation at 1500 g. The pellet was resuspended in a medium containing 50 mM Tris-HC1 (pH 8.0) and 350 m M NaC1 and centrifuged for 3 min at 2000 g. The sedimented chloroplasts were ruptured in distilled water and the released thylakoids were centrifuged for 6 min at 20000 g in a solution containing 20 mM NaC1. The washed membranes were resuspended in NaC1 solutions at the concentrations indicated and stored at 0 ~ C.
Isolation of thylakoids in a Tris-sorbitol medium. Leaves were blended in a medium containing 50 m M Tris-HC1 (pH 7.5), 330 mM sorbitol, 10 mM NaC1, 2 m M ethylenediaminetetraacetic acid (EDTA), 1 m M MgCI2, 1 mM MnCI2, 0.5 mM KHzPO 4, 1 0 r a m sodium ascorbate, 3.3 mM cysteine and 5 0 m M 2-mercaptoethanol under conditions as described above. After filtration, the homogenate was centrifuged for 1 min at 250 g. The chloroplasts were sedimented by centrifugation for 3 min at 2000 g. The pellet was resuspended in the above medium, but without ascorbate, cysteine and 2-mercaptoethanol, and again centrifuged for 3 rain at 2000 g. This washing procedure was repeated once more. The sedimented chloroplasts were broken by suspending them in 5 m M MgC12 ; after approx. 30 s, freed thylakoids were transferred to a medium containing 5 0 m M 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (Hepes)-NaOH (pH 7.6), 330 mM sorbitol, 10 mM NaC1 and 5 m M MgC12 and stored at 0 ~ C.
Chlorophyll determination. Chlorophyll was determined according to Arnon (1949).
Freezing of thylakoids. For freezing of isolated membranes in the presence of various concentrations of NaC1, glucose, sucrose and adenylates, samples (0.5 ml each) of thylakoid suspensions in glass tubes were placed into a cryostat kept at - 1 5 ~ C. After freezing for 3-5 h, samples were thawed rapidly in a water bath at room temperature. Controls were kept at 0 ~ C for the same time.
Cyclic photophosphorylation. The activity of phenazine methosulfate (PMS)-mediated photophosphorylation before and after freezing was determined either in high-intensity white light as described recently (Santarius 1982a) or under the same conditions in nearly saturating red light (RG 630 cut-off filter, Schott & Gen., Mainz, FRG). Ferricyanide reduction. Thylakoids equivalent to 30 gg of chlorophyll were added to 2 ml of a reaction medium containing 25 mM Tris-HC1 (pH 7.8), 2.2 mM KHzPO 4, 2.2 mM ADP, 4 mM MgC12 and 0.4 m M K3[Fe(CN)6 ]. Potassium-hexacyanoferrate(III) reduction was recorded at room temperature with a Zeiss spectrophotometer (Zeiss, Oberkochen, FRG) at 400 nm during illumination with nearly saturating red light (RG 630 cut-off filter).
Methyl viologen reduction and noncyclic photophosphorylation. Light-dependent Oz uptake in the presence of methyl viologen was measured polarographically with a Clark-type electrode at 20 ~ C. Thylakoids isolated in a Tris-sorbitol medium and corresponding to 50 gg of chlorophyll were suspended in 1 ml
K.A. Santarius: Cryoprotection of thylakoids by ATP of a medium containing 50 mM Hepes-NaOH (pH 7.6), 330mM sorbitol, 1 0 m M KHzPO4, 5 m M MgClz, 1 mM NaN 3 and 25 gM methyl viologen. The reaction was started by illumination with nearly saturating red light as given above. After addition of small amounts of ADP (50 to 200 nmol each), electron transport was recorded in the presence (state 3) and after consumption of this nucleotide (state 4). The rates of methyl viologen reduction (corresponding to state 3) and ATP formation and the photosynthetic control and ADP/O ratios were calculated according to Robinson and Wiskich (1976).
Light-dependent proton gradient. Proton movement across the thylakoid membrane during dark-light and light-dark transition was determined by measurement of 9-aminoacridine fluorescence. Thylakoids were isolated in a Tris-NaC1 medium. Membranes equivalent to 20 gg of chlorophyll were suspended in 2.1 ml of a medium containing 30 mM N-[2-hydroxy-l,1bis(hydroxymethyl)ethyl]glycine (Tricine)-NaOH (pH 7.6), 100 mM NaC1, 2 mM MgC12, 25 gM methyl viologen and 10 gM 9-aminoacridine and placed into a spectrofluorometer (Eppendorf, Netheler & Hinz GmbH, Hamburg, FRG) at room temperature. Fluorescence of 9-aminoacridine was excited by 313- to 366-nm light of low intensity, and the emitted light was recorded by a photomultiplier which was protected against red actinic light by an appropriate filter combination (Corning 5030 and 9782). After a dark period of at least 1 min, thylakoids were irradiated for 3 min with saturating red actinic light (RG 630 cut-off filter) which was again followed by darkness. From the quenching of the 9-aminoacridine fluorescence in the light (steady state), the ApH was calculated according to Briantais et al. (1979; see also Tillberg et al. 1977).
Results
The effect of freezing on isolated chloroplast membranes in the presence of membrane-toxic compounds and cryoprotectants has been studied intensively in various laboratories. It was found that at a given freezing temperature, membrane preservation was mainly determined by the ratio of cryoprotectant to cryotoxic compounds (Heber and Santarius 1964, 1973; Santarius and Giersch 1983a). For instance, if washed thylakoids were suspended in solutions containing solely glucose and NaC1 and freezing took place at temperatures around - 1 5 ~ a molar ratio of sugar to salt above 1 was necessary for appreciable membrane protection. The experiment depicted in Fig. 1 shows that in the presence of 100 m M NaC1 about 200 m M of glucose was needed to maintain 50% of the phosphorylating capacity during freezing. However, if 0.5 m M of ATP was present, much less glucose was necessary for comparable membrane protection. Moreover, if thylakoids were provided with 2 m M MgC12 plus 0.5 m M ATP, the sugar concentration sufficient for 50% membrane preservation was nearly a tenth of that necessary in the absence of Mg z § and ATP, and cyclic photophosphorylation became partly protected even in the absence of glucose. Addition of 2 m M
K.A. Santarius: Cryoprotection of thylakoids by ATP
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Fig. 1. The effect of ATP and Mg 2+ on spinach thylakoid membranes during freezing in the presence of variable ratios of glucose/NaCl. Thylakoids were isolated in a Tris-NaCi medium and suspended in 100 m M NaC1 and variable concentrations of glucose as indicated on the abscissa; the pH was 6.7. Aliquots were frozen for 3-4 h at - 16 ~ C without additional compounds (1) and in the presence of 2 m M MgC12 (n), 0.5 m M ATP (,v) and 2 m M MgC12 plus 0.5 m M ATP (o), respectively. After thawing, PMS-mediated photophosphorylation was measured in saturating white light. The activity of the unfrozen controls was 716 pmol ATP mg 1 chlorophyll h-1. chl = chlorophyll
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Fig. 2A, B. Protection of spinach thylakoid membranes during freezing in the presence of ATP (A) and sucrose and glucose (B) as a function of the concentration of the cryoprotectants. Thylakoids isotated in a Tris-NaC1 medium and suspended in 5 m M Hepes-NaOH (pH 7.5), 100 m M NaCI, 2 m M MgC12 and variable concentrations of ATP (o, o), sucrose (% A) and glucose 0% n), respectively, as indicated on the abscissae, were frozen for 3-4 h at - 15 ~ C. After thawing, PMS-mediated photophosphorylation ( , open symbols) and ferricyanide reduction (- . . . . , closed symbols) were measured in nearly saturating red light. Photophosphorylation and electron transport were plotted as % of the unfrozen control (unfrozen control = 100%). Activities of the unfrozen control: photophosphorylation 778 pmol ATP m g - 1 chlorophyll h - 1 ; electron transport 402 pmol ferricyanide r a g - 1 chlorophyll h - 1
MgC12 in the absence of ATP did not appreciably alter membrane preservation. The experiment shown in Fig. 1 was carried out at a pH around 6.7 and in the absence of buffer. When the pH was adjusted to 7.5 prior to freezing
by use of 5 m M Hepes-NaOH, similar results were obtained (not shown). Under these conditions, somewhat less glucose was needed for a comparable degree of cryoprotection. Moreover, partial protection of thylakoids in the absence of glucose was already observed when ATP only was added to the membrane suspension and was considerably more pronounced in the presence of ATP plus Mg 2§ (see also Figs. 2, 4, 5). These differences could be due to both altered pH and a protective effect of the buffer molecules. Uncoupling of photophosphorylation during freezing of isolated thylakoids is accompanied by stimulation of the noncyclic electron transport under phosphorylating conditions (Heber and Santarius 1964; Heber et al. 1973; Volger et al. 1978; Thebud and Santarius 1981). The addition of ATP protects cyclic photophosphorylation and decreases the stimulation of ferricyanide reduction to a similar extent (Fig. 2). Considerable membrane preservation was reached in the presence of relatively small amounts of ATP, without addition of any other common cryoprotectant. On a molar basis, ATP was at least 100 times more effective than sucrose and glucose. However, preservation by ATP was saturated at 1-2 raM, at 50-70% protection, whereas membranes were 95-100% protected by low-molecular-weight carbohydrates. This is similar to data obtained with cryoprotective proteins (Volger and Heber 1975). However, in combination with small amounts of sugars which are insufficient for detectable membrane stabilization, almost complete membrane protection was reached when very low concentrations of either ATP or proteins were added prior to freezing (see Figs. 1, 3 and Volger and Heber 1975). The high cryoprotective effectiveness of ATP on thylakoids was even more striking in regard to coupling of noncyclic photophosphorylation to photosynthetic electron transport. To obtain a high degree of coupling, chloroplast membranes were isolated and stored in a Tris-sorbitol medium. In this case, the cryoprotective action of sorbitol was gradually abolished by the addition of increasing concentrations of NaC1. As a result, the decrease in the activity of noncyclic photophosphorylation was accompanied by a drastic stimulation of the electron flow which finally reached rates exceeding four to five times those of the unfrozen controls (Fig. 3). This indicates that ADP/O ratios dropped to zero and photosynthetic control was no longer observed (not shown). If small amounts of ATP were added prior to freezing, a considerably higher salt concentration was necessary for a comparable degree of uncoupling. For example,
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Fig. 3. Activities of photosynthetic electron transport and noncyclic photophosphorylation of spinach thylakoid membranes after freezing in the presence and absence of ATP as a function of the initial NaC1 concentration. Thylakoids were isolated in a Tris-sorbitol medium and suspended in 20 m M Hepes-NaOH (pH 7.4), 132 m M sorbitol, 2 m M MgC12 and variable concentrations of NaCI as indicated on the abscissa. Aliquots were frozen for 3-5 h at - 1 5 ~ directly (without ATP, v, v) and in the presence of 0.1 m M (o, e) and 0.25 m M ATP (% m), respectively. After thawing, light-dependent 0 2 uptake in the presence of methyl viologen was measured at 20 ~ C. Noncyclic photophosphorylation ( , open symbols) and methyl viologen reduction in the presence of A D P ( - - , closed symbols) were plotted as % of the unfrozen controls (unfrozen controls = 100%). Activities of the unfrozen controls: photophosphorylation 192 gmol ATP synthesized mg 1 chlorophyll h - 1 ; electron transport 104 gmol O2 consumed m g - 1 chlorophyll h - 1 ; A D P / O ratio 0.9; photosynthetic control 3.2
Fig. 4. The effect of freezing on cyclic photophosphorylation and light-dependent proton gradient of spinach thylakoid membranes as a function of the ATP concentration. Thylakoids isolated in a Tris-NaC1 medium and suspended in 5 m M HepesN a O H (pH 7.5), 100 m M NaC1, 2 m M MgC12 and variable concentrations of ATP as indicated on the abscissa were frozen for 3-5 h at - 1 5 ~ C. After thawing, PMS-mediated photophosphorylation ( o ) and the quenching of the 9aminoacridine fluorescence were measured. The ApH values (-9 - - - ) calculated from the fluorescence signals were plotted by use of an extended scale as indicated on the righthand-side ordinate (for explanation see text). Activities of the unfrozen control: photophosphorylation in nearly saturating red light 646 gmol ATP m g - 1 chlorophyll h - t ; ApH 4.12. Note that ApH calculated here cannot be taken as an absolute measure, since the internal volume of the thylakoids was not determined
to obtain similar alterations in the photochemical activities of thylakoids in the course of a freezethaw cycle, nearly twice as much NaC1 had to be added in the presence of only 0.1 m M ATP compared with membranes frozen in the absence of the adenylate. It should be mentioned that freezing in the presence of very low initial NaC1 concentrations, e.g. 4 m M in Fig. 3, also resulted in some uncoupling, i.e. stimulation of the photosynthetic electron transport and decrease in photophosphorylation and, thus, a decline in the ADP/O ratio and photosynthetic control value. This is in agreement with earlier findings that cryoprotection of chloroplast membranes is diminished at very low salt concentrations (Santarius and Giersch 1983 b; see also Santarius 1982a; Santarius and Giersch 1984). Uncoupling of photophosphorylation of isolated thylakoids during freezing is related to a breakdown of the ability to accumulate protons in the light (Heber 1967). The question arises whether the addition of ATP prior to freezing prevents changes in the permeability of the mem-
branes to protons. According to Schuldiner et al. (1972) the quenching of the 9-aminoacridine fluorescence in the light can be used to estimate the size of the proton gradient across the thylakoid membrane. As recently shown by Hangarter and Good (1982), under certain conditions a threshold value of the ApH is required for photophosphorylation of isolated thylakoid membranes. In the experiment shown in Fig. 4, thylakoids were incubated at high concentrations of ions; under these conditions a threshold ApH of 2 to 3 is expected (Hangarter and Good 1982). Taking this into account, cryoprotection of photophosphorylation by increasing concentrations of ATP seems to be closely correlated with alterations in ApH (Fig. 4). When ADP and A M P were added to the membranes prior to freezing, little or no protection was observed (Fig. 5). This is valid for adenylate concentrations up to 5 m M (not shown). In some experiments very small amounts of ADP exhibited a distinct but small cryoprotective effect which was not enhanced at ADP concentrations exceeding 1 mM.
K.A. Santarius: Cryoprotection of thylakoids by ATP
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Fig. 5. The effect of various adenylates on spinach thylakoid membranes during freezing. Thylakoids were isolated in a TrisNaC1 medium and frozen for 3-4 h at - 1 5 ~ C in the presence of 5 m M Hepes-NaOH (pH 7.1), 100 m M NaC1, 2 m M MgC12 and variable concentrations of ATP (o), A D P ( , ) and A M P (D), respectively, as indicated on the abscissa. After thawing, PMS-mediated photophosphorylation was measured in saturating white light. The activity of the unfrozen controls was 982 pmol ATP m g - t chlorophyll h 1
Discussion
Very recently it was shown that cryoprotection of isolated thylakoid membranes mainly depends on the ratio of sugar to salt, the total solute concentration prior to freezing and the freezing temperature (Santarius and Giersch 1983a, b, 1984; Hincha et al. 1984). Apart from extreme conditions such as very low or high initial solute concentrations and freezing temperatures below - 2 0 ~ C, predominantly the colligative action of the solutes contributes to membrane preservation, i.e., cryoprotectants prevent the increase in the concentration of potentially membrane-toxic salts during freezing up to a critical limit (Heber and Santarius 1973; Heber et al. 1979). However, cryoprotection by high-molecular-weight compounds such as proteins (Volger and Heber 1975) and dextrans (Santarius 1982 a, b) cannot be explained by the colligative concept, because these compounds are already effective at very low concentrations. At present, the mechanism of cryopreservation of biomembranes by high-molecular-weight solutes is not understood. The data presented in this paper clearly demonstrate that ATP most effectively stabilizes isolated chloroplast membranes in the course of a freezethaw cycle. Obviously, cryoprotection by ATP cannot be explained by colligative action as the effective concentrations are extremely low in relation
to the total concentration of other solutes present in the membrane suspension. Rather, membrane stabilization during freezing may be caused by specific interactions between ATP and the thylakoid membranes. Possibly, the results can be widely interpreted by the action of ATP on the CF t. It is known that removal of the CFt from the membranes results in uncoupling, i.e., in inactivation of photophosphorylation accompanied by an enhancement of photosynthetic electron flow (Avron 1963; Hesse et al. 1976; Telfer et al. 1980). If sufficient concentrations of cations, in particular of highly effective Mg 2 § or Ca 2 -- which achieve binding of CF 1 to the membrane, are present in the medium, uncoupling can be avoided (Hesse et al. 1976; Telfer et al. 1980). Presumably, the release of CF 1 opens channels for protons in the membranes (McCarty and Racker 1966; Schmid and Junge 1974). Any leakage of H + caused by permeability changes in the chloroplast membranes would impair the efficiency of photophosphorylation and simultaneously loosen the control of electron transport by the ApH. This hypothesis is supported by various data in the literature. In earlier investigations carried out at temperatures above freezing, isolated CF 1 was found to be cold-labile mainly in the presence of high concentrations of salts (Lien et al. 1972). Effective protection against cold inactivation of CF~ at high NaC1 levels occurred in the presence of Mg; + and very low concentrations of ATP (Posorske and Jagendorf 1976); it has been suggested that binding of ATP to the CF I decreases the proton permeability of thylakoid membranes and, thereby, causes a more strongly coupled condition (see also McCarty et al. 1971; Telfer and Evans 1972). Taking this into consideration, it can be inferred that ATP may cause similar effects on thylakoid membranes during freezing. Release of subunits of the CF t related to thylakoid inactivation by freezing was repeatedly observed (Garber and Steponkus 1976; Steponkus et al. 1977; Volger et al. 1978; Lineberger and Steponkus 1980; Hincha et al. 1984). In the present contribution, it was shown that at a given NaC1 concentration which caused uncoupling, addition of ATP prior to freezing completely or at least partly prevents inactivation of photophosphorylation and stimulation of electron flow in the presence of very low concentrations of carbohydrates (Figs. 1, 3) and even in the total absence of common cryoprotectants (Figs. 2, 4, 5). The protective action of ATP (Fig. 1) is improved by Mg 2§ Concurrent with the cryoprotective effect on photophosphorylation, ATP diminishes the effect of
560
freezing on the H § permeability of the membranes (Fig. 4). All these results support the view that ATP acts at the CF a. This action could be either a modification of the CF 1 that leads to conformational changes and thus stabilizes the protein complex against cold inactivation, or ATP diminishes the release of CF 1 from thylakoids. As a result, uncontrolled proton leakage across the membranes caused by freezing would be prevented. Possibly, cryoprotection of isolated thylakoid membranes by high-molecular-weight solutes such as specific proteins and dextrans which are likewise effective at very low molar concentrations (see above) can be explained by a similar mechanism. In contrast to data obtained at 0 ~ C (Posorske and Jagendorf 1976; Santarius 1984), ADP does not protect thylakoid membranes during freezing even in the presence of Mg 2 § (Fig. 5). Apparently, ADP is incapable of stabilizing the CF 1 at the membranes under freezing conditions. Uncoupling of photophosphorylation is not observed as a primary effect when freezing of chloroplast membranes takes place in situ (Heber et al. 1973; Thebud and Santarius 1981; Klosson and Krause 1981; Krause et al. 1982). In thylakoid membranes isolated from partly frost-damaged leaves photosynthetic electron transport, noncyclic photophosphorylation and the extent of light-induced H § uptake were lowered to nearly the same degree without an appreciable increase in the permeability of the membranes towards protons (Klosson and Krause 1981). Obviously, damage to thylakoids in situ and in vitro is caused by different mechanisms. In this respect, it is of interest that ATP levels existing in spinach chloroplasts in situ )tre in the magnitude of 0.5 to 2 m M (for references see Krause and Heber 1976) and, in some cases, seem to increase during frost-hardening even in the dark (Sobczyk and Kacperska-Palacz 1978). Moreover, it is well known that in chloroplasts various low- and high-molecular-weight cryoprotectants become accumulated during coldacclimation (Heber 1959, 1970; Heber and Ernst 1967; Kappen and Ullrich 1970; Santarius and Milde 1977; Krause et al. 1982, 1983). Presumably, at freezing temperatures that lead to irreversible cell damage, thylakoids are still sufficiently protected with regard to injury caused by increase in the concentration of cryotoxic, potentially uncoupling solutes such as inorganic salts in the chloroplast stroma. Thus, inactivation of thylakoid membranes during freeze-damage of leaf tissue probably is not caused primarily by modification or release of CF 1 but occurs by others, as yet unknown, alterations.
K.A. Santarius: Cryoprotection of thylakoids by ATP The author is grateful to Professor G.H. Krause for critical reading of the manuscript and to Miss Britta Dietzel for competent technical assistance.
References Arnon, D.I. (1949) Copper enzymes in isolated chloroplasts. Polyphenol-oxidase in Beta vulgaris. Plant Physiol. 24, 1-15 Avron, M. (1963) A coupling factor in photophosphorylation. Biochim. Biophys. Acta 77, 699-702 Briantais, J.-M., Vernotte, C., Picaud, M., Krause, G.H. (1979) A quantitative study of the slow decline of chlorophyll a fluorescence in isolated chloroplasts. Biochim. Biophys. Acta 548, 128-138 Garber, M.P., Steponkus, P.L. (1976) Alterations in chloroplast thylakoids during an in vitro freeze-thaw cycle. Plant Physiol. 57, 673-680 Hangarter, R.P., Good, N.E. (1982) Energy thresholds for ATP synthesis in chloroplasts. Biochim. Biophys. Acta 681, 397-404 Heber, U. (1959) Beziehungen zwischen der Gr613e von Chloroplasten und ihrem Gehalt an 16slichen Eiweigen und Zukkern im Zusammenhang mit dem Frostresistenzproblem. Protoplasma 51, 284~298 Heber, U. (1967) Freezing injury and uncoupling of phosphorylation from electron transport in chloroplasts. Plant Physiol. 42, 1343-1350 Heber, U. (1970) Proteins capable of protecting chloroplast membranes against freezing. In: Ciba found, symp. on the frozen cell, pp. 175-188, Wolstenholme, G.E.W., O'Connor, M., eds. Churchill, London Heber, U., Ernst, R. (1967) A biochemical approach to the problem of frost injury and frost hardiness. In: Cellular injury and resistance in freezing organisms (Proc. Int. Conf. Low Temp. Sci.), vol. II, pp. 63-77, Asahina, E., ed. Inst. Low Temp. Sci., Hokkaido University, Sapporo Heber, U., Santarius, K.A. (1964) Loss of adenosine triphosphate synthesis caused by freezing and its relationship to frost hardiness problems. Plant Physiol. 39, 712-719 Heber, U., Santarius, K.A. (1973) Cell death by cold and heat and resistance to extreme temperatures. Mechanisms of hardening and dehardening. In: Temperature and life, pp. 232-263, Precht, H., Christophersen, J., Hensel, H., Larcher, W., eds. Springer, Berlin Heidelberg New York Heber, U., Tyankova, L., Santarius, K.A. (1971) Stabilization and inactivation of biological membranes during freezing in the presence of amino acids. Biochim. Biophys. Acta 241, 578 592 Heber, U., Tyankova, L., Santarius, K.A. (1973) Effects of freezing on biological membranes in vivo and in vitro. Biochim. Biophys. Acta 291, 23 37 Heber, U., Volger, H., Overbeck, V., Santarius, K.A. (1979) Membrane damage and protection during freezing. Adv. Chem. Ser. 180, 159 189 Hesse, H., Jank-Ladwig, R., Strotmann, H. (1976) On the reconstitution of photophosphorylation in CFl-extracted chloroplasts. Z. Naturforsch. Teil C 31,445-451 Hincha, D.K., Schmidt, J.E., Heber, U., Schmitt, J.M. (1984) Colligative and non-colligative freezing damage to thylakoid membranes. Biochim. Biophys. Acta 769, 8 14 Kappen, L., Ullrich, W.R. (1970) Verteilung von Chlorid und Zuckern in Blattzellen halophiler Pflanzen bei verschieden hoher Frostresistenz. Ber. Dtsch. Bot. Ges. 83, 265-275 Klosson, R.J., Krause, G.H. (1981) Freezing injury in coldacclimated and unhardened spinach leaves. I. Photosynthetic reactions of thylakoids isolated from frost-damaged leaves. Planta 151,339-346
K.A. Santarius : Cryoprotection of thylakoids by ATP Krause, G.H., Heber, U. (1976) Energetics of intact chloroplasts. In: The intact chloroplast, pp. 171-214, Barber, J., ed. Elsevier/North-Holland Biomedical Press, Amsterdam Krause, G.H., Klosson, R.J., Justenhoven, A., Ahrer-Steller, V. (1983) Effects of freezing stress on the photosynthetic system in vivo. In: Proc. 6th Int. Congr. on Photosynthesis, Brussels (in press) Krause, G.H., Klosson, R.J., Tr6ster, U. (1982) On the mechanism of freezing injury and cold acclimation of spinach leaves. In: Plant cold hardiness and freezing stress, mechanisms and crop implications, vol. 2, pp. 55-75, Li, P.H., Sakai, A., eds. Academic Press, New York Lien, S., Berzborn, R.J., Racker, E. (1972) Partial resolution of the enzymes catalyzing photophosphorylation. IX. Studies on the subunit structure of coupling factor 1 from chloroplasts. J. Biol. Chem. 247, 3520-3524 Lineberger, R.D., Steponkus, P.L. (1980) Effects of freezing on the release and inactivation of chloroplast coupling factor 1. Cryobiology 17, 486-494 McCarty, R.E., Fuhrman, J.S., Tsuchiya, Y. (1971) Effects of adenine nucleotides on hydrogen-ion transport in chloroplasts. Proc. Natl. Acad. Sci. USA 68, 252~2526 McCarty, R.E., Racker, E. (1966) Effect of a coupling factor and its antiserum on photophosphorylation and hydrogen ion transport. Brookhaven Symp. Biol. 19, 202-214 Mollenhauer, A., Schmitt, J.M., Coughlan, S., Heber, U. (1983) Loss of membrane proteins from thylakoids during freezing. Biochim. Biophys. Acta 728, 331-338 Posorske, L., Jagendorf, A.T. (1976) Nucleotide and metal interactions affecting inactivation of spinach chloroplast coupling factor by NaCI in the cold. Arch. Biochem. Biophys. 177, 276-283 Robinson, S.P., Wiskich, J.T. (1976) Factors affecting the ADP/O ratio in isolated chloroplasts. Biochim. Biophys. Acta 440, 131-146 Santarius, K.A. (t 982 a) Cryoprotection of spinach chloroplast membranes by dextrans. Cryobiology 19, 200 210 Santarius, K.A. (1982b) The mechanism of cryoprotection of biomembrane systems by carbohydrates. In : Plant cold hardiness and freezing stress, mechanisms and crop implications, vol. 2, pp. 475-486, Li, P.H., Sakai, A., eds. Academic Press, New York Santarius, K.A. (1984) The role of the chloroplast coupling factor in the inactivation of thylakoid membranes at low temperatures. Physiol. Plant. (in press) Santarius, K.A., Bauer, J. (1983) Cryopreservation of spinach chloroplast membranes by low-molecular-weight carbohydrates. I. Evidence for cryoprotection by a noncolligativetype mechanism. Cryobiology 20, 83-89 Santarius, K.A., Giersch, Ch. (1983a) Cryopreservation of spinach chloroplast membranes by tow-molecular-weight
561 carbohydrates. II. Discrimination between colligative and noncolligative protection. Cryobiology 20, 90-99 Santarius, K.A., Giersch, Ch. (1983 b) Inactivation and protection of isolated thylakoid membranes during freezing. In: Effects of stress on photosynthesis, pp. 201-210, Marcelle, R., Clijsters, H., van Poucke, M., eds. Nijhoff/Jnnk, The Hague Santarius, K.A., Giersch, Ch. (1984) Factors contributing to inactivation of isolated thylakoid membranes during freezing in the presence of variable amounts of glucose and NaC1. Biophys. J. (in press) Santarius, K.A., Milde, H. (1977) Sugar compartmentation in frost-hardy and partially dehardened cabbage leaf cells. Planta 136, 163-166 Schmid, R., Junge, W. (1974) On the influence of extraction and recondensation of CF 1 on the electric properties of the thylakoid membrane. In: Proc. 3rd Int. Congr. on Photosynthesis, vol. II, pp. 821-830, Avron, M., ed. Elsevier, Amsterdam Schuldiner, S., Rottenberg, H., Avron, M. (1972) Determination of ApH in chloroplasts. 2. Fluorescent amines as a probe for the determination of ApH in chloroplasts. Eur. J. Biochem. 25, 64-70 Sobczyk, E.A., Kacperska-Palacz, A. (1978) Adenine nucleotide changes during cold acclimation of winter rape plants. Plant Physiol. 62, 875-878 Steponkus, P.L., Garber, M.P., Myers, S.P., Lineberger, R.D. (1977) Effects of cold acclimation and freezing on structure and function of chloroplast thylakoids. Cryobiology 14, 303-321 Telfer, A., Barber, J., Jagendorf, A.T. (1980) Electrostatic control of chloroplast coupling factor binding to thylakoid membranes as indicated by cation effects on electron transport and reconstitution of photophosphorylation. Biochim. Biophys. Acta 591,331-345 Telfer, A., Evans, M.C.W. 0972) Evidence for chemiosmotic coupling of electron transport to ATP synthesis in spinach chloroplasts. Biochim. Biophys. Acta 256, 625-637 Thebud, R., Santarius, K.A. (1981) Effects of freezing on spinach leaf mitochondria and thylakoids in situ and in vitro. Plant Physiol. 68, 1156-1160 Tillberg, J.-E., Giersch, Ch., Heber, U. (1977) CO2 reduction by intact chloroplasts under a diminished proton gradient. Biochim. Biophys. Acta 461, 31-47 Volger, H.G., Heber, U. (1975) Cryoprotective leaf proteins. Biochim. Biophys. Acta 412, 335-349 Volger, H., Heber, U., Berzborn, R.J. (1978) Loss of function of biomembranes and solubilization of membrane proteins during freezing. Biochim. Biophys. Acta 511,455-469 Received 5 January; accepted 17 February 1984
