Planta 151, 347-352 (1981)

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Freezing Injury in Cold-acclimated and Unhardened Spinach Leaves II. Effects of Freezing on Chlorophyll Fluorescence and Light Scattering Reactions Rupert J. Klosson and Gotthard H. Krause Botanisches Institut der Universitfit D/isseldorf, Universitfitsstrage 1, D-4000 Dfisseldorf 1, Federal Republic of Germany

Abstract. Leaves from cold-acclimated and from unhardened spinach plants (Spinacia oleracea L.) were subjected to a freezing/thawing procedure in which varying minimum temperatures were reached. Subsequently, the chlorophyll fluorescence induction signal (Kautsky phenomenon) and the light-induced apparent absorbance changes at 535 nm (light-scattering changes indicative of the proton gradient, and absorbance changes induced by the membrane potential) of the leaves were studied to obtain information on the course and mechanism of frost damage to the photosynthetic apparatus. Membrane energization as indicated by these signals was related in a complex way to the inactivation of CO2 assimilation due to the progressing impact of freezing: In the absence of CO2, the maximum energization of the thylakoids was progressively decreased. According to altered fluorescence signals, the electron transport system was affected in parallel. In the presence of CO2, energization frequently appeared increased when the leaves had been partially damaged, i.e., when the CO2 assimilation rates were lowered. The results suggest that the primary frost injury in chloroplasts of intact leaves consists of an inhibition of the energy conserving photosynthetic processes and, in addition, of a partial inactivation of the carbon reduction cycle. The pattern of freezing injury was no different in frost-hardened and unhardened leaves. Key words: Chlorophyll, fluorescence - CO z assimilation - Freezing injury - Light scattering (leaves) Membrane potential - Spinacia.

Introduction In the first part of this study (Klosson and Krause 1981) thylakoid membranes were isolated from frostAbbreviation: Chl= chlorophyll

injured leaves in order to localize the damage arising in the course of freezing and thawing in the photosynthetic apparatus. Additional evidence regarding the mechanism of freezing injury of the chloroplasts in situ was obtained by the present investigation of the physical phenomena, such as chlorophyll fluorescence, light-scattering, and membrane potential-dependent absorbance changes, exhibited by the whole leaves. This approach allows for the examination of various reactions in the injured leaf without disruption of the tissue. The physical parameters studied are known to be closely related to photosynthetic electron transport and to the concurrent energization of the thylakoid membrane required for photophosphorylation. In leaves, the Kautsky effect of chlorophyll a fluorescence induction, i.e., the rise of fluorescence emission in several phases to a maximum and the subsequent decline to a low steady state value, is a complex phenomenon, and its normal course depends on undisturbed electron transport through photosystem II (see Papageorgiou 1975). The slow decline of fluorescence from the peak to the steady state (fluorescence quenching) can, in part, be explained by the reoxidation of the quencher Q of photosystem II, but it is, additionally, significantly influenced by ion gradients built up across the energized membrane (Krause 1973, 1974). Recent studies with isolated thylakoids (Briantais et al. 1979) have shown that fluorescence quenching is proportional to the H + concentration in the intrathylakoid space, when Q is kept in a largely reduced state. Similarly, light-scattering changes observed at about 535 nm seem to reflect the light-induced proton versus metal cation exchange at the thylakoid membrane (Heber 1969; Krause 1973, 1974). Fast absorbance changes with a maximum at about 520 nm are supposed to be caused by the photoinduced membrane potential (Junge 1977). Taken together, these parameters should give in-

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348

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

formation, in particular, on the energetic state of the illuminated thylakoids situated in frost-injured leaves. As in the foregoing paper, freezing injury is studied in cold-acclimated and in nonhardened spinach leaves.

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The growth of plant material, cold-acclimation of plants, frost treatment of detached leaves, and CO2 gas exchange measurements were carried out as outlined in part I of this study (Klosson and Krause 1981). In all leaf samples, photosynthetic CO2 uptake at a limiting (about half saturating) light intensity (45-50 W m 2, half band width 630-680 nm) was determined after the frost treatment. In the presentation of the results, the rates of COz assimilation are included as references regarding the extent of freezing injury. Chlorophyll a fluorescence and light-scattering (apparent absorbance at 535 nm) were recorded at room temperature as described previously (Krause 1973, 1974). Fast changes of the absorbance signal at 535 nm represented a defined proportion (60-70%) of the membrane potential-dependent absorbance change (maxlmum at 520 nm); fast absorbance changes seen upon darkening were taken as a relative measure of the photoinduced membrane potential in the steady state.

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Fig. 1 a-f. Signals of chlorophyll fluorescence induction of spinach leaves, recorded after frost treatment (minimum temperatures given in the graph). Fluorescence was excited in CO2-free air subsequent to 3 rain dark periods by 36 W m - 2 red light (half band width 630-680 nm) and recorded in the far-red band at 740 nm. a Unhardened control; b-c frost-injured unhardened leaves; d frost-hardened control; e-f frost-injured hardened leaves. S denotes stationary fluorescence emission after 2 min in the light. Rates of photosynthesis were (in ~tmol CO2 uptake/rag Chl h):72 (a), 4 (b), 0 (e) 46 (d), 7 (e), and 0 (f)

fluorescence. As demonstrated in Fig. l, only this second phase appears to be affected by freezing and is finally eliminated. The sigmoidal shape of the induction curve is thereby lost. Since at room temperature chlorophyll fluorescence is mainly emitted by photosystem II, these changes signalize inactivation of this photosystem, or of the electron donation to it. (b)

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Response of Chlorophyll Fluorescence to Frost Treatment. Freezing injury of the leaves, as manifested by reduced rates of photosynthesis, strongly affected the characteristics of fluorescence induction (Figs. 1 and 2). With progressing inactivation of the leaf, the variable part of fluorescence emission in the maximum, Fv(peak), gradually declines, whereas the initial fluorescence (Fo) remains largely unchanged. The decline in Fv(peak) is accompanied by a decrease in the maximal slope of the fluorescence rise, leading to the peak, i.e., the second phase of the rise of variable

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Fig. 2a, b. Effects of frost treatment on the chlorophyll fluorescence induction of unhardened (a) and hardened (b) spinach leaves. The following parameters of the induction signal (in relative units) are plotted versus minimum temperatures of frost treatment: -- ~-., variable fluorescence emission in the peak (Fv(peak)); -.. A .... initial (fast) fluorescence (Fo) ; --X--, maximal slope of the fluorescence rise to the peak. In addition, the course of inactivation of photosynthetic CO 2 uptake by the leaves is depicted ( , - , rates of photosynthesis). For experimental details see Fig. 1

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strongly injured leaves. The course of these changes with progressing injury is depicted in Fig. 4. It has been shown before (Krause 1973) that the effects of carbon dioxide on chlorophyll fluorescence lowering of the fluorescence peak and of the extent of quenching - depend on the CO2 fixing activity of the leaf, Supposedly, the fast onset of CO2 uptake and thus of NADPH consumption prevents the full reduction of Q in the induction period. Therefore, in the presence of CO2, the increased quenching frequently observed in partly damaged leaves (Figs. 3 and 4) may indicate that the electron transport is limited by the lowered capacity of carbon reduction. This interpretation is supported by the finding that the highest fluorescence peak was found after a dark period of 1 h (when the Calvin cycle has become fully dark-inactivated); then neither an effect of CO2 on the peak height nor a differential effect of freezing on fluorescence induction in the presence and absence of CO2 were observed (data not shown). In the absence of CO;, the decline of fluorescence quenching due to freezing represents a general decrease in photosynthetic activity of the thylakoid membrane, but, because of the complex nature of this signal, it cannot be attributed to a specific inhibition. It should be noted that freezing affects chlorophyll fluorescence similarly in hardened and unhardened leaves.

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Fig. 3a-d. Long-term chlorophyll fluorescence signals from frosttreated unhardened spinach leaves, recorded in the presence and absence of 300 gll-1 CO2 in air. The intensity of exciting red light was 45 Wm 2 (c.f. legend to Fig. 1). All signals were recorded after I0 rain preillumination in the presence of CO2 and a dark period of 3 min. a Control, kept at +4 C; b-d Freezing-injured leaves; minimum temperatures of frost treatment given in the graph. Rates of photosynthesis (pmol COz uptake/rag Chl h) were 63 (a), 48 (b), 3 (c), and 0 (d)

Effects of Freezing on Light-Scattering and Absorbance Changes. In the signals recorded at 535 nm (Fig. 5), fast changes in the dark/light and light/dark transition denote membrane-dependent absorbance changes (maximum at 520 nm) ; slow changes of apparent absorbance represent light-scattering changes. In Fig. 6, the extent of the slow changes in the light/dark transition (regarded as a respresentation of the steady state in the light) is shown as a function of the minimum temperature of frost treatment. In intact leaves, the strong effect of COz on the

The long-term fluorescence signals of unhardened leaves, depicted in Fig. 3, show two conspicuous effects of frost treatment: first, the extent of quenching decreases as the fluorescence peak is lowered (see also Fig. 1); second, the differences between signals in normal and in CO2-free air, recorded after 3-rain dark periods, are diminished and disappear in more I

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Fig. 5. Signals of light-scattering and absorbance changes at 535 n m in the presence and absence of CO2, observed after frost treatment of unhardened spinach leaves. Fast changes seen upon illumination and darkening represent absorbance changes induced by the membrane potential ( m a x i m u m at about 520 nm); slow changes indicate altered light-scattering. For experimental details and rates of photosynthesis see legend to Fig. 3

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R.J. Klossonand G.H. Krause: FreezingInjury of Spinach Leaves. II that in those leaves sufficient photosynthetic energy is still provided by electron transport, leading to the build-up of ion gradients across the thylakoid membrane; but this energy apparently cannot be fully utilized in carbon reduction. More drastic frost treatment leads to a parallel decline of the light-scattering changes both in the presence and absence of CO2, indicating the gradual inhibition of photoinduced membrane energization. As shown in Fig. 7, the potential-dependent absorbance change is affected by freezing in much the same way as the light-scattering signal. In undamaged leaves, active CO2 assimilation keeps the absorbance change at a low level; partial damage leads to a stimulation of the signal in the presence of CO2. The final decline, which is independent of CO2, suggests that in more strongly injured leaves not only the proton gradient but also the membrane potential is impaired. A residual light-dependent membrane potential, however, is apparently still present in fully damaged leaves (see also Fig. 5). Again, cold-acclimation of the leaves only shifted the temperature range of inactivation to lower values, but did not alter the effects of freezing on these energy-dependent signals. Discussion

The results presented above, together with those obtained by the analysis of isolated thylakoids (Klosson and Krause 1981), support the concept that freezing injury of leaves is traceable primarily to membrane damage. According to earlier investigations (Heber and Santarius 1964; Santarius 1969) and to our data, the thylakoid membranes belong to the freezing-sensitive sites in the cell. In spinach leaves, the tolerance to freezing is limited by a well-defined temperature range that is widened in a reproducible manner by cold-acclimation. Beyond this limit, thylakoid membranes are affected by freezing in various ways. Changes in the induction signal of chlorophyll fluorescence indicate damage in or near photosystem II. Because of the complex nature of fluorescence induction it is difficult at present to interprete these changes in detail. The most conspicuous effect of frost treatment is the loss of the second phase of the fluorescence rise from F o to the peak, accompanied by a lowering of the maximal emission of variable fluorescence. As shown by the preceding study of isolated thylakoids, this does not seem to be caused by increased activity of photosystem I relative to photosystern II, or by changed distribution of excitation energy between the photosystems. Possible causes of lowered variable fluorescence are inactivation of photosystem II trapping centers, inhibited electron donation to

351 photosystem II, or, more likely, structural changes of the pigment system leading to stimulated thermal deactivation of excited states. (Such structural alterations are known to occur upon acidification of the thylakoid interior; see Briantais et al. 1979). Notably, heat inactivation of chloroplasts (Krause and Santarius 1975; Schreiber and Armond 1978) is accompanied by similar effects on variable fluorescence; however, upon heating, the F o level is increased, whereas Fo was not significantly changed even by lethal frost treatment. This confirms evidence (Santarius 1975) that the mechanisms of heat and frost inactivation of the photosynthetic apparatus are not identical. Although the slow fluorescence decline from the peak to the steady state is caused in part by the energization of the membrane (Krause 1973), the effect of freezing on this quenching process (measured in the absence of COz) results mostly from the lowering of the fluorescence peak. The effect of frost treatment on the energy state in terms of light-induced ion gradients and membrane potential is clearly reflected by the light-scattering reactions and potentialdependent absorbance changes in the leaf. The gradual decline of these signals (under CO2-free conditions) occurred almost in parallel to the inactivation of photophosphorylation and light-induced proton uptake by the isolated thylakoids (c.f. Klosson and Krause 1981). Noticeably, frost treatment diminished or abolished the effect of CO2 on all signals indicative of membrane energization (including the fluorescence quenching). This means that the difference between maximal energization in the light (CO2-free condition) and membrane energization in the steady state during CO2 fixation is diminished; thus, in partly damaged leaves, the capacity of the Calvin cycle to utilize the energy provided by photosynthetic light reactions seems impaired. This apparently leads to an increased energization in the presence of CO2. Two alternative interpretations of this effect are possible: 1) Freezing causes an energy transfer inhibition; this would, inspite of a high proton motive force, diminish the amount of available ATP. 2) Frost treatment affects the capacity of the carbon reduction cycle independently; in intermediate states of injury, ATP would be provided in excess of consumption. The data available at present favor the second hypothesis. The Calvin cycle is known to be regulated by complex control mechanisms. It is feasible that frost treatment disturbs the normal light activation of the cycle, e.g., damage of the chloroplast envelope may prevent the alkalization of the chloroplast stroma in the light, which is required for activation (see Heber et al. 1979). Indeed, in several experiments (c.f. Figs. 6 and 7), CO2 fixation ap-

352 p e a r e d m o r e strongly i m p a i r e d t h a n the energy-dep e n d e n t signals r e c o r d e d with the same leaf in the absence o f CO2. O n the o t h e r h a n d , t h y l a k o i d s isolated f r o m i n j u r e d leaves ( K l o s s o n a n d K r a u s e 1981) d i d n o t suggest an i n h i b i t i o n o f energy transfer. Thus, f r o m this a n d the p r e c e d i n g study, the following picture o f freezing injury o f s p i n a c h leaves emerges: A s the earliest i n d i c a t i o n o f injury, rates o f CO2 a s s i m i l a t i o n m a y be r e d u c e d b y a yet unk n o w n m e c h a n i s m ( p e r h a p s due to changes in the c h l o r o p l a s t envelope), w h e n the system s u p p l y i n g p h o t o s y n t h e t i c energy is still largely intact. This effect is f o l l o w e d closely (or even in parallel) by an inactivation o f the t h y l a k o i d m e m b r a n e t h a t is expressed by v a r i o u s p a r a m e t e r s . P r i m a r i l y , p h o t o s y n t h e t i c elect r o n t r a n s p o r t , b o t h at p h o t o s y s t e m II a n d I, declines; this leads to r e d u c e d rates o f p r o t o n p u m p i n g a n d therefore o f p h o s p h o r y l a t i o n . I n the CO2-free system, this b e c o m e s a p p a r e n t as a decline o f m e m b r a n e energization. Preferential i n a c t i v a t i o n o f the water-splitting system is i n d i c a t e d in m o r e severely i n j u r e d leaves. Progressing injury is finally m a n i f e s t e d by unc o u p l i n g effects t h a t a b o l i s h p h o t o p h o s p h o r y l a t i o n , whereas low residual rates o f electron t r a n s p o r t a n d i n d i c a t i o n s o f a low l i g h t - i n d u c e d m e m b r a n e p o t e n t i a l r e m a i n even in fatally i n j u r e d leaves. The p h o t o s y n thetic p i g m e n t systems o b v i o u s l y are n o t d e g r a d e d to a large extent, as seen by the residual fast rise o f v a r i a b l e fluorescence a n d p a r t i c u l a r l y by the unc h a n g e d l o w - t e m p e r a t u r e fluorescence spectra o f the t h y l a k o i d s . The described changes are irreversible. I d e n t i c a l effects o f freezing have been o b s e r v e d in f r o s t - h a r d e n e d a n d u n h a r d e n e d leaves: c o l d acclimation does n o t seem to alter the p a t t e r n o f i n a c t i v a t i o n processes. F u r t h e r e x p e r i m e n t s are u n d e r w a y to verify - or to m o d i f y - this hypothesis. The authors thank Professor K.A. Santarius and Professor U. Heber for discussion and Mr. M. Jensen for providing a plotting program. The paper contains part of the graduate and thesis work of R.J. Klosson. The study was supported by the Deutsche Forschungsgemeinschaft.

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

References 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 Heber, U.W., 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. (1969) Conformational changes of chloroplasts induced by illumination of leaves in vivo. Biochim. Biophys. Acta 180, 302-319 Heber, U., Enser, U., Weis, E., Ziem, U., Giersch, C. (1979) 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 Junge, W. (1977) Membrane potentials in photosynthesis. Annu. Rev. Plant Physiol. 28, 503-536 Klosson, R.J., Krause, G.H. (1981) Freezing injury in cold-acclimated and unhardened spinach leaves. I. Photosynthetic reactions of thylakoids isolated from frost-damaged leaves. Planta 151, 339 346 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 Krause, G.H. (1974) Changes in chlorophyll fluorescence in relation to light-dependent cation transfer across thylakoid membranes. Biochim. Biophys. Acta 333, 301-313 Krause, G.H., Santarius, K.A. (1975) Relative thermostability of the chloroplast envelope. Planta 127, 285-299 Krause, G.H., Lorimer, G.H., Heber, U., Kirk, M.R. (1978) Photorespiratory energy dissipation in leaves and chloroplasts. In: Photosynthesis '77. Proc. of the 4th Internat. Congress on Photosynthesis, pp. 299-310. Hall, D.O., Coombs, J., Goodwin, T.W., eds. The Biochemical Society London Papageorgiou, G. (1975) Chlorophyll fluorescence: an intrinsic probe of photosynthesis. In: Bioenergetics of photosynthesis, pp. 319-371. Govindjee ed. Academic Press New York Santarius, K.A. (1969) Der Einflul3 von Elektrolyten auf Chloroplasten beim Gefrieren und Trocknen. Planta 89, 23-46 Santarius, K.A. (1975) Sites of heat sensitvity in chloroplasts and differential inactivation of cyclic and noncyclic photophosphorylation by heating. J. Thermal Biol. 1, 101-107 Schreiber, U., Armond, P.A. (1978) Heat-induced changes of chlorophyll fluorescence in isolated chloroplasts and related heat damage at the pigment level. Biochim. Biophys. Acta 502, 138 151 Received 20 August; accepted 14 November 1980