Planta

Planta 151, 339-346 (1981)

© Springer-Verlag 1981

Freezing Injury in Cold-acclimated and Unhardened Spinach Leaves I. Photosynthetic Reactions of Thylakoids Isolated from Frost-damaged Leaves Rupert J. Klosson and Gotthard H. Krause Botanisches Institut der Universit/it Dfisseldorf, Universitfitsstrage 1, D-4000 D/isseldorf 1, Federal Republic of Germany

Abstract. Spinach plants (Spinacia oleracea L.) were frost-hardened by cold-acclimation to I ° C or kept in an unhardy state at 20°/14 ° C in phytotrons. Detached leaves were exposed to temperatures below 0 ° C. Rates of photosynthetic CO2 uptake by the leaves, recorded after frost treatment, served as a measure of freezing injury. Thylakoid membranes were isolated from frost-injured leaves and their photosynthetic activities tested. Ice formation occurred at about - 4 ° to - 5 ° C, both in unhardened and coldacclimated leaves. After thawing, unhardened leaves appeared severely damaged when they had been exposed to - 5° to - 8 ° C. Acclimated leaves were damaged by freezing at temperatures between - 1 0 ° to - 14 ° C. The pattern of freezing damage was complex and appeared to be identical in hardened and unhardened leaves: 1. Inactivation of photosynthesis and respiration of the leaves occurred almost simultaneously. 2. When the leaves were partly damaged, the rates of photosynthetic electron transport and noncyclic photophosphorylation and the extent of lightinduced H + uptake by the isolated thylakoids were lowered at about the same degree. The dark decay of the proton gradient was, however, not stimulated, indicating that the permeability of the membrane toward protons and metal cations had not increased. 3. As shown by partial reactions of the electron transport system, freezing of leaves predominantly inhibited the oxygen evolution, but photosystem II and photosystem I-dependent electron transport were also impaired. 4. Damage of the chloroplast envelope was indicated by a decline in the percentage of intact chloroplasts found in preparations from injured leaves. The results are discussed in relation to earlier studies on freezing damage of thylakoid membranes occurring in vitro. Abbreviations: ChI=chlorophyll; DCPIP=2,6-dichlorophenol indophenol; HEPES=N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid; MES=2(N-morpholino) ethane sulfonic acid

Key words: Chloroplast and freezing - Freezing injury - Frost tolerance - Photosynthesis and freezing

Spinacia. Introduction Chloroplast membranes isolated from spinach leaves have been frequently used as model systems to investigate the mechanism of freezing injury and to elucidate the basis of frost hardiness of plant leaves (for a recent review see Heber et al. 1979a). In these studies, suspensions of thylakoids were subjected under various conditions to a freezing-thawing procedure in vitro, and their photosynthetic activities were subsequently tested. It has been convincingly shown (Heber and Santarius 1964; Heber 1967; Santarius 1973) that a primary membrane damage caused by freezing consists of an increased proton permeability, leading to uncoupling of photophosphorylation. In more severely damaged membranes, the photosynthetic electron transport is also inhibited. The watersplitting apparatus appeared as the most freezingsensitive part of the electron transport system (Heber et al. 1973). Comparatively little is known to date about the mechanism of damage inflicted upon intact leaves by the freezing-thawing process (cf. Heber et al. 1973). In the present study we have tried to clarify whether inactivation of photosynthesis in intact leaves by freezing follows the same pattern of membrane damage as observed upon freezing of isolated thylakoids. In frost-treated leaves, reduced rates of photosynthetic CO2 uptake and of respiration were taken as manifestations of freezing injury. Photosynthetic reactions of thylakoids isolated from those leaves were studied to elucidate the primary causes of inactivation. In order to answer the question as to possible differences in the mechanism of freezing injury of leaves which differ in frost resistance, freezing-sensitive plants were compared with hardened plant material.

0032-0935/81/0151/0339/$01.60

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Materials and Methods Plant Material. Plants of Spinacia oleracea L. were cultivated in a greenhouse for about 4 weeks and for another week in a phytotron at a temperature of 20° C (day)/14 ° C (night) and 75% relative humidity. The light period was 9 h, the light intensity 25.000 lx. A batch of the plants was then hardened by acclimation to low temperatures using a variation of the procedure described by Critchley (1976): The light periods were reduced to 8 h and the temperature of the light and dark periods was kept for consecutive 2-day intervals at 15° C/I0 ° C, 10° C/3 ° C, 10° C/1 ° C, and 1° C/ I°C.

Frost Treatment. Samples of detached spinach leaves, placed into a cryostate, were cooled from + 5° C at a rate of 3° C/h (or 6° C/h) to a certain minimum temperature arid, after staying at this temperature for 2 h (or 4 h), were warmed up at the same rate back to +5 ° C. The extent of damage was not visibly influenced by the faster rate of cooling and heating or by the longer period at the minimum temperature. Control leaves were kept at ÷ 4 ° C for the duration of the frost treatment. C O 2 Gas Exchange and Transpiration. One leaf of each sample was placed into a translucent cuvette, where a round section (16 cm 2) of the leaf blade was illuminated with red light (half band width about 630-680 nm) and exposed to an air stream (300 ~tl 1-1 COz), passing the lower and upper surface of the leaf at a flow rate of 20 1/h (cf. Krause 1973). Respiratory COz evolution in the dark and photosynthetic CO2 uptake in the light were monitored at about 22° C with a Maihak (UNOR 5N1) infrared analyzer. Rates of photosynthesis given in the figures were calculated from the difference of COz content in the air stream in the dark and light state (10 min preillumination), i.e., they are approximations of true steady-state photosynthesis. The intensity of actinic light, 45-50 W m - 2 was about half-saturating for photosynthesis. Transpiration was determined by weight loss during storage of the leaves at room temperature.

Ice Formation in the leaves was controlled by visible changes in the appearance of the leaves and by the temperature jump of a thermocouple connected to the sample.

Isolation of Thylakoid Membranes. Frost-treated and control leaves (6-10 g) were homogenized in a Starmix blender at 4° C in a solution (40 ml) containing 0.33 M sorbitol, 10 mM NaC1, 5 mM MgC12, 10 mM sodium ascorbate, 50 mM L-cystein, 0.4% bovine serum albumin, and 40 mM MES buffer, adjusted to pH 6.1 with NaOH. The homogenate was filtered through surgical gauze and centrifuged for 1 rain at 2,000 g. To rupture the chloroplast envelope, the pellet was suspended in 20 ml of 5 mM MgClz, and the chloroplasts were brought back to an isotonic medium by adding an equal volume of a "double strength" solution containing 0.66 M sorbitol, 20 mM NaC1, 5 mM MgC12, 0.8% serum albumin, and 80 mM HEPES, pH 6.7 (NaOH). The suspension was centrifuged for 1 min at 200 g and the resulting supernatant again for 1 rain at 2,000 g. The chloroplast pellet was resuspended in 0.3 to 0.5 ml of the same isotonic medium (pH 6.7) and stored at 0°C.

Isolation of Intact Chloroplasts. The procedure of Jensen and Bassham (1966) was slightly modified (see Heber 1973). Bovine serum albumin (0.4%) was added to the isolation medium. The integrity of chloroplasts in the final suspension was determined as described by Heber and Santarius (1970). In addition, the integrity was controlled by phase contrast microscopy. In order to calculate the percentage of intactness, the total chlorophyll content of the leaf homogenate was considered, because the supernatants of centrifugation almost exclusively contain broken chloroplasts.

R.J. Klosson and G.H. Krause: Freezing Injury of Spinach Leaves. I

Chlorophyll Determination in leaves and chloroplast suspensions was carried out according to Arnon (1949).

Photosynthetic Electron Transport and Noncyclic Photophosphorylation. Electron transport in the presence of K3Fe(CN)6 (1 raM) and ADP (3 raM) was measured at 20° C, essentially as described by Volger et al. (1978), by recording the change in absorbance at 400 nm with a ZEISS spectrophotometer equipped with a cross beam actinic light source. After two minutes of illumination, the reaction was terminated with HC104 (0.5 M) and ATP formation was determined enzymatically by reduction of NADP + in the presence of glucose, hexokinase (EC 2.7.1.1), and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) (Lamprecht and Trautschold 1974). Alternatively, electron transport was measured polarographically as light-dependent O, uptake in the presence of 25 ~tM methylviologen and 1 mM NAN3; ATP formation was calculated from the time required for phosphorylation of 200 nmol of ADP per 100 ~tg Chl, as indicated by the photosynthetic control of electron transport (Robinson and Wiskich 1976). The two methods gave comparable results. To test activities of photosystem I and II and of water splitting, partial reactions of the electron transport chain were measured as given by Heber et al. (1973). The basic medium of all reactions contained 0.33 M sorbitol, 10 mM KH2PO4, 5 mM MgClz, and 50 mM HEPES, pH 7.6 (NaOH), to which chloroplasts equivalent to 100 ~g Chl per 3 ml were added. Saturating actinic red light was used (6 cm water filter; 1 mm infrared absorbing filter Calflex C, Balzers/Liechtenstein; 3 mm cutoff filter RG 630, Schott/Mainz, FRG). The intensity was 300 W m 2.

Light-induced ApH and H + Uptake. To determine the proton gradient, the quenching of 9-amino-acridine fluorescence in the presence of 25 ~M methylviologen was measured as described by Tillberg et al. (1977). The same basic reaction medium and actinic light source, as given above, were used. For calculation of the ApH according to Schuldiner et al. (I972), the intrathylakoid space was assumed to be 10 ~tl/mg Chl. Light-induced H + uptake was measured with a glass electrode at about pH 7 in the absence of HEPES buffer and KH2PO4. Samples contained 100 gg Chl in a 3 ml volume. Spectra of Chlorophyll Fluorescence at 77 K were recorded as described by Ben-Hayyim and Krause (1980).

Results Sensitivity and Tolerance T o w a r d Freezing. T h e r a t e s o f p h o t o s y n t h e t i c CO2 u p t a k e by the leaves, determ i n e d at limiting light intensities within 1 2 h after f r o s t t r e a t m e n t , s e r v e d as a c o n v e n i e n t m e a s u r e o f t h e e x t e n t o f f r e e z i n g i n j u r y . A s will b e s h o w n , inacti-v a t i o n o f p h o t o s y n t h e s i s b y f r e e z i n g o c c u r r e d in c l o s e parallel to m o s t o t h e r p a r a m e t e r s m e a s u r e d a n d was also closely related to externally visible d a m a g e , such as i n f i l t r a t i o n o f t h e leaves. I n s o m e i n s t a n c e s , i n h i b i t i o n o f p h o t o s y n t h e s i s e v e n a p p e a r e d as t h e m o s t sensitive response to freezing. Leaves that seemed undamaged immediately after frost treatment did not show a temporary depression of CO2 assimilation, n o r did they d e v e l o p any visible i n d i c a t i o n o f injury w h e n s t o r e d for 7 days at 4 ° C or for 2 days at a b o u t 20 ° C. Figure 1 depicts the inactivation of photosynthesis and respiration of unhardened and hardened leaves

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When partly damaged leaves were stored after treatment for 24 h at about 23 ° C, rates of CO2 uptake and release, respectively, were further decreased, as compared to the unfrozen controls. Storage for 48 h at 4 ° C was without measurable effect (data not shown). From measurements of transpiration, stomatal closure, possibly caused by the freezing procedure, can be excluded as a cause of reduced photosynthetic activity. This is shown in Fig. 2. Transpiration of partially damaged leaves was not decreased, but rather stimulated, which may be due to the infiltration of part of the tissue by water. In all the following figures, the inactivation curve of photosynthesis of the respecitve experiments is included as a reference to the temperature region in which frost damage does occur.

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Fig. 2a-c. Effect of frost treatment on transpiration of unhardened (b) and frost-hardened (d) spinach leaves. Columns show loss of water in % of fresh weight, as observed during storage at room temperature for 15 rain, 1 h and 3 h after frost treatment. Numbers denote the minimum temperature of treatment in centigrades. Rates of photosynthesis exhibited by the unhardened (a) and hardened leaves (e) after treatment are given to demonstrate the extent of frost damage

as a function of the minimum temperature of frost treatment. It can be seen that both processes are inactivated within a narrow temperature range of 2 4 degrees. The inset shows the relation between freezing injury and ice formation in the leaves, conceivably occurring in the intercellular space (see Heber ct al. 1979a). The unhardened spinach leaves possess little frost tolerance: Freezing coincides with partial damage. In the hardened leaves, ice formation occurred at the same temperature, but the leaves did tolerate freezing within a temperature range of about 5-6 degrees. The frost injury manifested by reduced rates of photosynthesis and respiration was irreversible.

Electron Transport and Photophosphorylation. Figure 3 shows rates of photosynthetic electron transport from water to ferricyanide and of concurrent phosphorylation of ADP (ATP determined enzymatically) of thylakoid membranes isolated from frost-treated unhardened and hardened leaves. In a similar experiment (Fig. 4), electron transport was mediated by methylviologen and rates of ATP formation were determined indirectly from the kinetics of O2 uptake upon addition of limiting amounts of ADP (Robinson and Wiskich 1976). It should be noted that the chloroplast isolation procedure was designed to collect a high percentage and thus a representative portion of the chloroplasts present in the leaf, rather than to obtain optimal photosynthetic rates. In both experiments, electron transport and noncyclic photophosphorylation by the thylakoids were inactivated approximately in conjunction with photosynthesis of the whole leaves. The course of inactivation although occurring at different temperature ranges - was very similar in hardened and unhardened plants: In partly damaged leaves, both electron transport and photophosphorylation were affected to about the same ex-

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lope membranes was decreased when the leaves had been injured by freezing. This is shown for partially hardened leaves in the experiment of Fig. 9, indicating that the chloroplast envelope becomes damaged when photosynthesis is inactivated by freezing. It should be noted that the percentage of intact chloroplasts in the unfrozen control was low, because all chloroplasts present in the homogenate were considered, whereas in the usual isolation procedure intact chloroplasts are selected.

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Fig. 9. Integrity of the chloroplast envelope in chloroplast preparations from frost-treated spinach leaves (--[]--). The right-hand ordinate denotes the percentage of intact chloroplasts present in the leaf homogenate prepared after frost treatment at the temperatures given on the abscissa. For reference, the rates of photosynthesis of the frost-treated leaves are given ( - • - )

Discussion

Our data indicate that freezing injury of spinach leaves is complex. Initial signs of injury consisted of decreased rates of photosynthesis and respiration of the leaves and of inhibited electron transport, photophosphorylation, and light-induced proton uptake

R.J. Klosson and G.H. Krause: Freezing Injury of Spinach Leaves. I

by the isolated thylakoids. In addition, the stability of the choroplast envelope was affected by freezing. Occasionally, CO2 assimilation by the whole leaves appeared to be slightly more sensitive to freezing than the other parameters (cf. Figs. 4 and 5). This raises the possibility that the Calvin cycle is also affected; for instance, damage of the chloroplast envelope may disturb the light activation of certain enzymes of the cycle (see Heber et al. 1979b). In thylakoids frozen in vitro, the water-splitting system was the most affected site of the electron transport chain. However, in intact leaves, photosystem I and photosystem lI-dependent reactions were also impaired, even when the leaves were only partially damaged (Fig. 5). Chlorophyll fluorescence spectra at 77 K showed that the damage was not predominantly located at one of the two photosystems (Fig. 6). In contrast, freezing in vitro has been observed to inactivate photosystem II more than photosystem I; reactions of the latter may even be stimulated by freezing, while reactions involving photosystern II are inhibited (Heber et al. 1973; Volger et al. 1978). In earlier investigations it was shown that freezing of isolated thylakoids primarily lowered the lightinduced H ÷ gradient and consequently the rates of photophosphorylation, but did not inhibit electron transport proportionally (Heber and Santarius 1964; Heber 1967). Depending on experimental conditions, noncyclic electron transport was even stimulated. However, the parallel breakdown of electron transport and phosphorylation in partly damaged leaves (Figs. 3 and 4) suggests that uncoupling was not a primary effect of frost treatment in vivo. This is supported by the relative insensitivity of the ApH and by the constancy of the decay kinetics of the 9amino-acridine fluorescence signal (Figs. 7 and 8). It may be argued that thylakoids isolated from damaged leaves do not represent the state of injury inflicted by the freeze-thaw process, but are altered by the preparation. However, as will be shown in the following contribution, signals of chlorophyll fluorescence and light scattering of the frost-treated leaves indicate that the injury of thylakoids does indeed occur in situ. The discrepancy between the freezing injury occurring in vivo and in vitro is not fully understood at present. It was noticed by Heber et al. (1973) that effects of freezing on the thylakoids in vivo were similar to those observed in vitro in the presence of phenylpyruvate, an amphiphilic substance that seems to interact with the membranes when its concentration is raised by freezing. In thylakoids frozen with phenylpyruvate, all electron transport reactions tested were strongly inhibited. However, photophosphorylation was still considerably stronger affected. Also, it ap-

345

pears unlikely that potentially toxic compounds of this type are present in the chloroplasts of intact leaves in concentrations that are sufficient to exert such damaging effects. Obviously, freezing injury taking place in vivo is a much more complex phenomenon than damage occurring in vitro. The basic mechanism of freezing injury to thylakoids in vitro - loss of membrane semipermeability - may apply to various biomembranes in the intact cell. As our data indicate, besides thylakoids other membrane systems are indeed damaged by frost treatment, namely the chloroplast envelope and possibly, as suggested by inactivated respiration, the mitochondria. Due to osmotic stress exerted by the freezing-thawing process, the plasmalemma and tonoplast may also become permeable to solutes or may even rupture (Wiest and Steponkus 1978). A variety of secondary damaging effects could be caused by breakdown of compartmentation in the cell. Conceivably, inhibition of photosynthetic processes, caused for instance by release of phenolic substances from the vacuole, may occur earlier than freezing damage due to increased solute concentration in the environment of the thylakoid membranes, which is observed in vitro. A similar mechanism of freezing damage to spruce chloroplasts has been suggested by Senser and Beck (1977). The picture is further complicated by the fact that different populations of cells and chloroplasts - unimpaired, partly and fully damaged - may be present in one single frost-treated leaf. Notably, in all experiments the course of inactivation was very similar in frost-hardened and unhardened leaves, suggesting that cold-acclimation does not involve an alteration of the mechanism of freezing injury. It is remarkable that in both hardened and unhardened spinach leaves, the temperature range between the limit of frost tolerance and full damage was very narrow ( 2 4 degrees). As indicated by preliminary experiments, this range may be wider in leaves of other species. The authors thank Professor K.A. Santarius for critical reading of the manuscript and Mr. M. Jensen for providing a plotting program. The study contains part of the graduate and thesis work ofR.J. Klosson and was supported by the Deutsche Forschungsgemeinschaft.

References Arnon, D.I. (1949) Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24, 1-15 Ben-Hayyim, G., Krause, G.H. (1980) Transport of mono- and divalent cations across chloroplast membranes mediated by the ionophore A23187. Arch. Biochem. Biophys. 202, 546 557 Butler, W.L., Kitajima, M. (1975) Energy transfer between photosystem II and photosystem I in chloroplasts. Biochim. Biophys. Acta 396, 7~85

346 Critchley, C. (1976) Untersuchungen an Chloroplastenmembranen fiber m6gliche Verfinderungen in den Lipiden im Zusammenhang mit Problemen der Gefrierinaktivierung bzw. der Frostresistenz von Pflanzen. Thesis, University of Dfisseldorf, FRG De Benedetti, E., Garlaschi, F.M. (1977) On the estimation of proton gradient and osmotic volume in chloroplast membranes. J. Bioenerg. Biomembr. 9, 195-201 Heber, U.W., Santarius, K.A. (1964) Loss of a adenosine triphosphate synthesis caused by freezing and its relationship to frost hardiness problems. Plant Physiol. 39, 712-719 Heber, U. (1967) Freezing injury and uncoupling of photophosphorylation from electron transport in chloroplasts. Plant Physiol. 42, 1343-1350 Heber, U., Santarius, K.A. (1970) Direct and indirect transfer of ATP and ADP across the chloroplast envelope. Z. Naturforsch. 25b, 718-728 Heber, U. (1973) Stoichiometry of reduction and phosphorylation during illumination of intact chloroplasts. Biochim. Biophys. Acta 305, 140-152 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. (1979a) Membrane damage and protection during freezing. In: Advances in Chemistry Series, No. 180, Proteins at low temperatures, pp. 159-189, Fennema, O., ed., Am. Chem. Soc. Heber, U., Enser, U., Weis, E., Ziem, U., Giersch, C. (1979b) Regulation of the photosynthetic carbon cycle, phosphorylation and electron transport in illuminated intact chloroplasts. In: Modulation of protein function, pp. 113-137. Atkinson, D.E. ed. Academic Press, New York

R.J. Klosson and G.H. Krause: Freezing Injury of Spinach Leaves. I Jensen, R.G., Bassham, J.A. (1966) Photosynthesis by isolated chloroplasts. Proc. Natl. Acad. Sci. USA 56, 1095-1101 Krause, G.H. (1973) The high-energy state of the thylakoid system as indicated by chlorophyll fluorescence and chloroplast shrinkage. Biochim. Biophys. Acta 292, 715-728 Lamprecht, W., Trautschold, I. (1974) In: Methoden der enzymatischen Analyse, 3rd edition, pp. 2151-2160, Bergmeyer, U. ed., Verlag Chemie, Weinheim 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. (1973) The effect of eutectic crystallization on biological membranes. Biochim. Biophys. Acta 291, 38-50 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 Senser, M., Beck, E. (1977) On the mechanism of frost injury and frost hardening of spruce chloroplasts. Planta 137, 195-201 Tillberg, J.-E., Giersch, C., Heber, U. (1977) CO2 reduction by intact chloroplasts under a diminished proton gradient. Biochim. Biophys. Acta 461, 3147 Volger, H., Heber, U., Berzborn, RJ. (1978) Loss of function of biomembranes and solubilization of membrane proteins during freezing. Biochim. Biophys. Acta 511,455 469 Wiest, S.C., Steponkus, P.L. (1978) Freeze-thaw injury to isolated spinach protoplasts and its simulation at above freezing temperatures. Plant Physiol. 62, 699-705

Received 20 August; accepted 14 November 1980