Photosynthesis Research 25: 161-171, 1990. © 1990 Kluwer Academic Publishers. Printed in the Netherlands. Regular paper
Chlorophyll luminescence as an indicator of stress-induced damage to the photosynthetic apparatus. Effects of heat-stress in isolated chloroplasts Wolfgang Bilger I & Ulrich Schreiber Institut fiir Botanik und Pharmazeutische Biologie der Universitiit Wiirzburg, Mittlerer Dallenbergweg 64, 87 Wiirzburg, FRG; iPresent address: Carnegie Institution of Washington, Department of Plant Biology, 290 Panama Street, Stanford, CA 94305, USA Received 16 August 1989; accepted in revised form 4 May 1990
Key words: Delayed light emission, delayed fluorescence, membrane potential, oxygen evolving system, proton gradient, Spinacia oleracea Abstract
A brief review is given of investigations on stress-induced alterations of ms- to s-luminescence yield of chlorophyll in plants. Three different approaches are considered: phytoluminography, luminescencetemperature curves, and luminescence induction curves. The remainder of this article presents new results of the effect of heat stress on luminescence induction curves of isolated chloroplasts. Three parameters with widely different heat resistances were resolved from induction curves. A fast valinomycin sensitive transient, L'i, with a 50% inhibition temperature of 33 to 34°C was correlated with the magnitude of the light-induced membrane potential after heat pretreatment. A slower nigericin sensitive transient, L ' , with a 50% inhibition temperature of 39 to 40°C was mainly correlated with the light-induced proton gradient. An uncoupler resistant part of the induction curve, L0, was enhanced by heat stress (half maximum after pretreatment at 46°C) and was correlated with the degree of inhibition of oxygen evolution. Since L 0 was also raised by other treatments impairing the oxygen evolving enzyme system, and since this rise was inhibited by DCMU and hydroxylamine, this type of luminescence was ascribed to the intrinsic backreaction. We conclude that luminescence induction curves can serve as an useful indicator of the intactness of the membrane potential, the proton gradient, and the oxygen evolving enzyme system in isolated chloroplasts after heat stress.
Abbreviations: 9 - A A - 9-aminoacridine; CCCP-carbonylcyanide m-chlorophenylhydrazone; AA518 light-induced absorbance change at 518 nm; AAo., A A o , - rapid AA518 upon switching actinic light on or off, respectively; Li, t m , L 0 - i n this order, initial spike, main maximum, uncoupler insensitive transient of luminescence induction curve; L'i = L i - L 0 ; L m = L m - L 0 ; P, P680- primary donor of PS II; P F D - photon flux density (400-700 nm); Q A - primary acceptor of PS II
Introduction
In recent years increased attention has been given to effects on plants of adverse environmental conditions whether natural or caused by man. Non-intrusive techniques play an important role
in the detection of stress effects on photosynthetic activity. A well established technique is the measurement of chlorophyll fluorescence (Schreiber and Bilger 1987). Another non-intrusive probe whose yield is highly dependent on membrane energization is chlorophyll luminesc-
162 ence, or delayed light emission (for reviews see Lavorel 1975, Malkin 1977, Jursinic 1986). This probe provides more direct information on membrane energization, which is considered one of the main targets of heat stress (cf. Weis 1981). Luminescence is observed in the dark following an illumination period. There is general agreement that luminescence is emitted by photosystem II (PS II) and caused by the backreaction of the primary stabilized charged pair in PS II, P680 ÷ and QA. The backreaction can create an excited state whose decay can be observed as light emission. The high activation energy barrier of the backreaction can be lowered by the presence of artificial or light-induced electrical or proton gradients across the thylakoid membrane, which considerably enhance the luminescence yield (Mayne and Clayton 1966, Barber and Kraan 1970, Wraight and Crofts 1971). After turning the light off the luminescence decay is multi-exponential (e.g., Malkin 1977). In stressrelated research, it is primarily the luminescence components decaying in the order of milliseconds and seconds that have been used. In the past, attempts to measure stressinduced changes in luminescence emission include many different approaches depending on the particular stress factor under study (Havaux and Lannoye 1985). In the following three approaches will be considered in more detail. Each approach involves the measurement of luminescence after repetitive illumination of a sample using a phosphoroscope. Phytoluminography is a technique that gives a photographic image of the luminescence emission of whole leaves during actinic illumination (Sundbom and Bj6rn 1977). In such images brighter areas indicate a higher emission. Phytoluminography can be used to determine the areas of a leaf where lesions in the photosynthetic apparatus occur. Such lesions can be caused by a stress pretreatment or by pathogens (Bj6rn and Forsberg 1979, Ellenson and Amundson 1981)). When a steady state of luminescence has been reached during actinic illumination, the dynamics of the effect of a sudden change in conditions can be followed. For example, Ellenson and co-workers correlated oscillations in luminescence emission with stomatal oscillations and showed that these oc-
curred in patches across the leaf surface (Ellenson and Raba 1983, Ellenson 1985). Steady state luminescence yield during actinic illumination has been found to be minimal over a broad range of temperatures around the normal growth temperature, but it increases when the temperature of the sample is gradually lowered (Havaux and Lannoye 1983, Fork et al. 1985). In chilling sensitive plants the luminescence vs. temperature curves showed a maximum between 8 and 14°C whereas in chilling resistant plants luminescence continued to increase with decreasing the temperature to 0°C (Havaux and Lannoye 1983). The temperature at maximum luminescence has been thought to be associated with the temperature of phase transition in thylakoid membranes (Havaux and Lannoye 1983) although this notion has been recently questioned by Terzaghi et al. (1989). Luminescence also rises as temperature is increased above the growth temperature and reaches a maximum between 40 and 50°C (Dzhanumov et al. 1970, Alexandrov and Dzhanumov 1972, Fork et al. 1985). The decline in luminescence beyond the maximum is irreversible and has been ascribed to a loss of integrity of the thylakoid membrane (Fork et al. 1985). The high- and low-temperature maxima of luminescence vary among plant species according to their origin and optimal growth temperature (Dzhanumov et al. 1970, Havaux and Lannoye 1983, Fork et al. 1985, Terzaghi et al 1989). The critical temperatures of a given species are also dependent on the actual growth temperature in a manner indicative of photosynthetic acclimation (Pukacki et al. 1983, Fork et al 1987). The exact position of the low temperature maximum should be considered with caution, since it has been shown to be affected by the intensity of the actinic light (Fork and Murata 1989). Phytoluminography and measurements of luminescence vs. temperature curves are carried out under continuous illumination. However, luminescence induction curves have also been used to detect stress-induced damage. When a predarkened sample is suddenly illuminated luminescence shows an induction transient similar to that observed in fluorescence measurements. In most cases luminescence yield in induction curves is severely decreased by stress
163 treatment irrespective of whether it involves chilling (Melcarek and Brown 1977, Havaux and Lannoye 1984), heating (Baumann 1970, Yordanov et al. 1987), excessive irradiance (van Hasselt and van Berlo 1980), or rapid desiccation (Schwab et al. 1989). Yordanov et al. (1987) found that the decrease in luminescence yield was correlated with a decrease in the proton gradient across the thylakoid membrane in isolated chloroplasts that had been subjected to different degrees of heat stress. However, a good correlation was only obtained with chloroplasts isolated from plants acclimated to elevated growth temperatures; a poor correlation was obtained with chloroplasts isolated from non-acclimated plants. Many of the studies cited above show that luminescence is a sensitive indicator of stressinduced damage to the photosynthetic apparatus; marked effects are seen at stress levels where other commonly used indicators still showed control values. It was also possible to rank the resistance of different plants using their luminescence response. However, whereas the effects of stress on luminescence were obvious, mechanistic explanations for the observed changes were rarely given. In order to understand which processes are affected by stress, and how plants can adapt to stress, information on the causes of a given inhibition is needed. Since luminescence yield of intact leaves is governed by a complex interaction of several factors, a simple and well-known model system will be used here as a starting point for investigating how stress affects luminescence. The factors governing millisecond-luminescence yield in induction curves from isolated chloroplasts are well understood (Wraight and Crofts 1971, Itoh et al. 1971a,b, Satoh and Katoh 1983). As illustrated in the top panel of Fig. 1 the induction kinetics of broken chloroplasts show a rapid rise and decline during the first second of illumination, followed by a slower rise with a broad maximum at 5 to 10 seconds. The rapid spike (Li) can be selectively inhibited by valinomycin indicating its dependence on the membrane potential, whereas the main maximum (Lm) is selectively inhibited by uncouplers, showing its dependence on the proton gradient (Wraight and Crofts 1971). We, therefore,
choose this system to investigate if heat-induced changes in luminescence are correlated with inhibition of membrane energization and thus, whether luminescence induction curves can indeed be used as an indicator of such a heatstress-induced inhibition.
Materials and methods
Freshly isolated chloroplasts from greenhouse grown spinach leaves (Spinacia oleracea L., cv. Yates Hybrid 102) were used. The plants received a PFD of 200 to 300/zmol m -2 s -1 (artificial + natural light) with a controlled daylength of 10 h. The daytime air temperature was usually between 18 and 25°C and never exceeded 30°C. The greenhouse was cooled at night to about 10 to 15°C. Intact chloroplasts were isolated following the method of Jensen and Bassham (1966) as modified by Heber (1973), and stored in medium 'C' at a pH of 7.6. The intactness was usually about 80% and chlorophyll concentration approx. 2 mg/cm 3. The chloroplasts were stored on ice in darkness until used for the measurements. In some experiments broken chloroplasts were treated in Tris-HC1 (0.8 M, pH 8.0) for 10 min in the dark. The chloroplasts were heat-treated in the intact state to avoid an artifactual weakening of the heat resistance which occurs in broken chloroplasts. For the heat treatment 60 to 70 mm 3 of a threefold diluted chloroplast suspension (solution 'C', pH 7.6) were placed in a small, thinwalled reaction vessel (4 mm in diameter). Each vessel was kept in the dark in a stirred water bath (MGW Lauda K2, Lauda, FRG) for 5 min at the desired temperature. Subsequently the vessel was cooled in an ice water mixture for 1 min and an aliquot of the heated chloroplast suspension transferred to the measuring cuvette. Before measurement, the heated chloroplasts were broken by suspending them in a solution containing 10 mM MgCI 2 and 5 mM Hepes at pH 7.6. After 30 s double strength medium 'C' was added. All activity measurements were completed during the following 5 min. The temperature in the measuring cuvette was kept at 15°C. The luminescence measurements were con-
164 ducted with a Becquerel type phosphoroscope based on fiberoptics. A chopper wheel driven by a stepper motor interrupted the actinic light beam after 800/~s for a dark period of equal duration. During the dark period the luminescence intensity was measured from 240 to 560/zs following the interruption of the illumination by means of a photomultiplier (Hamamatsu R928) protected by a red plastic filter (Roscolene 821, Edmunds Scientific, Barrington, N J, USA). The photomultiplier signal was amplified and integrated by a custom built lock-in amplifier. The resulting data were registered in a storage oscilloscope (Explorer III, Nicolet, USA). The time constant of the amplifier was set to 10 ms. Actinic light was provided by a halogen light source (Xenophot XLX, Osram, FRG) connected to a stabilized power supply (Model N G R E 30/15, Rohde and Schwarz, FRG). After passing through an optical filter (BG 39 or RG 645, Schott, Mainz, FRG) and a fast shutter (model electronic-m, Compur, FRG) the actinic light was guided by plastic fiber optics to the chopper wheel and the measuring cuvette. With both the bluegreen and the red filter the PFD was 70 to 80/z mol m- z s- 1 The multibranched fiberoptics bundle reaching the measuring cuvette allowed simultaneous measurements of other parameters. Chlorophyll fluorescence was measured by a photodiode (UDT 100) protected by a longpass filter ( R G 695, Schott). To determine light-induced absorbance changes a weak measuring beam (PFD 1 / z m o l m - 2 s -1) at 518nm (BA40 IF 518, DT Cyan, Balzers, Liechtenstein; BG 38, Schott) was provided by a halogen lamp (Osram 64225). After passing the sample, the beam was reflected from a disk shaped mirror at the bottom of the cuvette, which also served as a stirrer. The transmitted measuring light was detected by a photomultiplier (EMI 9558) protected by bluegreen filters (BG 39, Schott, and CS 4-96, Coming). 9-Aminoacridin fluorescence was measured with the measuring beam entering the cuvette from the side. The beam was defined by bluegreen filters (K40, Balzers, BG 39 Schott) and had a PFD of 0.5/zmol m -2 s -1. The 9-AA fluorescence was guided by a branch of the fiberoptics to a photomultiplier (EMI 9558) protected by a suitable filter combination (K50, Balzers, BG 39, Schott). The concentration of 9-AA in
the assay was 2 × 10 - 6 M. Methylviologen (10 -4 M) was used as electron acceptor; catalase concentration was 100/xg/cm 3. Photophosphorylation activity was measured in situ using the luciferin-luciferase method (Schreiber and Del Valle Tascon 1982). The assay medium contained 0.33 M sorbitol, 2 m M KH2PO4, 10mM Mg-acetate, 10mM HEPES/ Tris, pH7.6, 5 × 10 -3 M ferricyanide, 10 -3 M DTT, 0.5mg/cm 3 BSA, 0.05 to 0.1cm3/cm 3 luciferin-luciferase (ATP monitoring kit, LKB) and 2.5 x 10 -5 M ADP. ATP (6.5 x 10 - 7 M) was added as internal standard. Luciferin luminescence was measured by a photomultiplier (EMI 9558) protected by bluegreen filters (BG 39, BG 40, Schott). The PFD of the red actinic light (RG 665, Schott) was 8/xmol m -2 s -~ 0 2 evolution was measured in an O z electrode (model DWl, Hansatech, Kings Lynn, UK) in the presence of 5 x 10 - 6 M gramicidin and with a PFD of 1000/zmolm -2 s -~ (RG 610, Schott). Ferricyanide (5 × 10 -3 M) was added as electron acceptor. Chemicals were purchased from Sigma if not stated otherwise.
Results and discussion
Effect of heat stress on luminescence induction kinetics
Induction curves obtained after application of a 5-min heat treatment of the chloroplasts are shown in Fig. 1 (left column, upper curves). The rapid spike and the maximum are labelled Li and Lm, respectively, on the control (20°C) traces. With increasing temperature of the pretreatment, luminescence emission became progressively inhibited. However, above a treatment temperature of about 42°C, L i increased again, especially when luminescence was measured in presence of nigericin and valinomycin, i.e., when membrane energization was totally absent (Fig. 1, left column, lower traces). Henceforth, this type of luminescence emission is termed basal luminescence, or L 0. The difference between the induction curves of total and basal luminescence emission represents that luminescence component which depends on membrane energization and is shown in the right-hand col-
165 i
20 *C
/Lm /Lo
0
3q *C
2
~ o
/
l/
L
38 *C
~ o 42 *C -k-
J
0
48 *C J~-
umn of Fig. 1. The energy dependent component consistently declined with increasing temperature, and after heating to 48°C no response to illumination could be detected. It is also apparent that the heat-induced decline of L'i began and reached completion at lower temperatures than that observed for L ' . This response is more clearly seen in Fig. 2, where L'i, L " and L 0 are plotted against the pretreatment temperature. L I was reduced to about 50% of its control value at 34°C. L " was clearly more heat resistant (50% at about 39°C), whereas L 0 did not respond until the pretreatment temperature exceeded 40°C. The threshold and 50%-inhibition temperatures varied by about I°C among different experiments while the ranking of heat resistance of the measured parameters always remained the same.
L
5
10
15 0 Time, s
5
10
Correlation of L'i with the light-induced membrane potential
15
Fig. 1. Luminescence induction curves of freshly broken spinach chloroplasts after 5 min pretreatment as intact chloroplasts at the indicated temperatures. Left column, upper curves, measured without additions; lower curves, measured in presence of 10-SM valinomycin and 10-7M nigericin. Right column, difference curves between upper and lower curves from left column. The characteristic levels, L~, Lm, and L 0 of the induction curves are indicated. L I = L i - L o ; L~, = L m - L o. Actinic PFD was 70/zmol m -2 s -~ and the sample temperature 20°C. The baseline is indicated by a dashed line.
Figure 3 shows the results of measurements of light-induced absorbance changes at 518nm (AAs18). These changes in DA are widely used to monitor changes in membrane potential (Vredenberg 1981). The trace in the inset shows an original recording of AA518 measured on a control sample. The dependences on pretreat-
o_.\
o
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Pretreatment temperature, *C
Fig. 2. Pretreatment temperature dependence of LI, L~ and L 0. The values are calculated from the same experiment as shown in Fig. I.
i ~o 40 50 ~d 30
Chlorophyll luminescence as an indicator of stress-induced damage to the photosynthetic apparatus. Effects of heat-stress in isolated chloroplasts Wolfgang Bilger I & Ulrich Schreiber Institut fiir Botanik und Pharmazeutische Biologie der Universitiit Wiirzburg, Mittlerer Dallenbergweg 64, 87 Wiirzburg, FRG; iPresent address: Carnegie Institution of Washington, Department of Plant Biology, 290 Panama Street, Stanford, CA 94305, USA Received 16 August 1989; accepted in revised form 4 May 1990
Key words: Delayed light emission, delayed fluorescence, membrane potential, oxygen evolving system, proton gradient, Spinacia oleracea Abstract
A brief review is given of investigations on stress-induced alterations of ms- to s-luminescence yield of chlorophyll in plants. Three different approaches are considered: phytoluminography, luminescencetemperature curves, and luminescence induction curves. The remainder of this article presents new results of the effect of heat stress on luminescence induction curves of isolated chloroplasts. Three parameters with widely different heat resistances were resolved from induction curves. A fast valinomycin sensitive transient, L'i, with a 50% inhibition temperature of 33 to 34°C was correlated with the magnitude of the light-induced membrane potential after heat pretreatment. A slower nigericin sensitive transient, L ' , with a 50% inhibition temperature of 39 to 40°C was mainly correlated with the light-induced proton gradient. An uncoupler resistant part of the induction curve, L0, was enhanced by heat stress (half maximum after pretreatment at 46°C) and was correlated with the degree of inhibition of oxygen evolution. Since L 0 was also raised by other treatments impairing the oxygen evolving enzyme system, and since this rise was inhibited by DCMU and hydroxylamine, this type of luminescence was ascribed to the intrinsic backreaction. We conclude that luminescence induction curves can serve as an useful indicator of the intactness of the membrane potential, the proton gradient, and the oxygen evolving enzyme system in isolated chloroplasts after heat stress.
Abbreviations: 9 - A A - 9-aminoacridine; CCCP-carbonylcyanide m-chlorophenylhydrazone; AA518 light-induced absorbance change at 518 nm; AAo., A A o , - rapid AA518 upon switching actinic light on or off, respectively; Li, t m , L 0 - i n this order, initial spike, main maximum, uncoupler insensitive transient of luminescence induction curve; L'i = L i - L 0 ; L m = L m - L 0 ; P, P680- primary donor of PS II; P F D - photon flux density (400-700 nm); Q A - primary acceptor of PS II
Introduction
In recent years increased attention has been given to effects on plants of adverse environmental conditions whether natural or caused by man. Non-intrusive techniques play an important role
in the detection of stress effects on photosynthetic activity. A well established technique is the measurement of chlorophyll fluorescence (Schreiber and Bilger 1987). Another non-intrusive probe whose yield is highly dependent on membrane energization is chlorophyll luminesc-
162 ence, or delayed light emission (for reviews see Lavorel 1975, Malkin 1977, Jursinic 1986). This probe provides more direct information on membrane energization, which is considered one of the main targets of heat stress (cf. Weis 1981). Luminescence is observed in the dark following an illumination period. There is general agreement that luminescence is emitted by photosystem II (PS II) and caused by the backreaction of the primary stabilized charged pair in PS II, P680 ÷ and QA. The backreaction can create an excited state whose decay can be observed as light emission. The high activation energy barrier of the backreaction can be lowered by the presence of artificial or light-induced electrical or proton gradients across the thylakoid membrane, which considerably enhance the luminescence yield (Mayne and Clayton 1966, Barber and Kraan 1970, Wraight and Crofts 1971). After turning the light off the luminescence decay is multi-exponential (e.g., Malkin 1977). In stressrelated research, it is primarily the luminescence components decaying in the order of milliseconds and seconds that have been used. In the past, attempts to measure stressinduced changes in luminescence emission include many different approaches depending on the particular stress factor under study (Havaux and Lannoye 1985). In the following three approaches will be considered in more detail. Each approach involves the measurement of luminescence after repetitive illumination of a sample using a phosphoroscope. Phytoluminography is a technique that gives a photographic image of the luminescence emission of whole leaves during actinic illumination (Sundbom and Bj6rn 1977). In such images brighter areas indicate a higher emission. Phytoluminography can be used to determine the areas of a leaf where lesions in the photosynthetic apparatus occur. Such lesions can be caused by a stress pretreatment or by pathogens (Bj6rn and Forsberg 1979, Ellenson and Amundson 1981)). When a steady state of luminescence has been reached during actinic illumination, the dynamics of the effect of a sudden change in conditions can be followed. For example, Ellenson and co-workers correlated oscillations in luminescence emission with stomatal oscillations and showed that these oc-
curred in patches across the leaf surface (Ellenson and Raba 1983, Ellenson 1985). Steady state luminescence yield during actinic illumination has been found to be minimal over a broad range of temperatures around the normal growth temperature, but it increases when the temperature of the sample is gradually lowered (Havaux and Lannoye 1983, Fork et al. 1985). In chilling sensitive plants the luminescence vs. temperature curves showed a maximum between 8 and 14°C whereas in chilling resistant plants luminescence continued to increase with decreasing the temperature to 0°C (Havaux and Lannoye 1983). The temperature at maximum luminescence has been thought to be associated with the temperature of phase transition in thylakoid membranes (Havaux and Lannoye 1983) although this notion has been recently questioned by Terzaghi et al. (1989). Luminescence also rises as temperature is increased above the growth temperature and reaches a maximum between 40 and 50°C (Dzhanumov et al. 1970, Alexandrov and Dzhanumov 1972, Fork et al. 1985). The decline in luminescence beyond the maximum is irreversible and has been ascribed to a loss of integrity of the thylakoid membrane (Fork et al. 1985). The high- and low-temperature maxima of luminescence vary among plant species according to their origin and optimal growth temperature (Dzhanumov et al. 1970, Havaux and Lannoye 1983, Fork et al. 1985, Terzaghi et al 1989). The critical temperatures of a given species are also dependent on the actual growth temperature in a manner indicative of photosynthetic acclimation (Pukacki et al. 1983, Fork et al 1987). The exact position of the low temperature maximum should be considered with caution, since it has been shown to be affected by the intensity of the actinic light (Fork and Murata 1989). Phytoluminography and measurements of luminescence vs. temperature curves are carried out under continuous illumination. However, luminescence induction curves have also been used to detect stress-induced damage. When a predarkened sample is suddenly illuminated luminescence shows an induction transient similar to that observed in fluorescence measurements. In most cases luminescence yield in induction curves is severely decreased by stress
163 treatment irrespective of whether it involves chilling (Melcarek and Brown 1977, Havaux and Lannoye 1984), heating (Baumann 1970, Yordanov et al. 1987), excessive irradiance (van Hasselt and van Berlo 1980), or rapid desiccation (Schwab et al. 1989). Yordanov et al. (1987) found that the decrease in luminescence yield was correlated with a decrease in the proton gradient across the thylakoid membrane in isolated chloroplasts that had been subjected to different degrees of heat stress. However, a good correlation was only obtained with chloroplasts isolated from plants acclimated to elevated growth temperatures; a poor correlation was obtained with chloroplasts isolated from non-acclimated plants. Many of the studies cited above show that luminescence is a sensitive indicator of stressinduced damage to the photosynthetic apparatus; marked effects are seen at stress levels where other commonly used indicators still showed control values. It was also possible to rank the resistance of different plants using their luminescence response. However, whereas the effects of stress on luminescence were obvious, mechanistic explanations for the observed changes were rarely given. In order to understand which processes are affected by stress, and how plants can adapt to stress, information on the causes of a given inhibition is needed. Since luminescence yield of intact leaves is governed by a complex interaction of several factors, a simple and well-known model system will be used here as a starting point for investigating how stress affects luminescence. The factors governing millisecond-luminescence yield in induction curves from isolated chloroplasts are well understood (Wraight and Crofts 1971, Itoh et al. 1971a,b, Satoh and Katoh 1983). As illustrated in the top panel of Fig. 1 the induction kinetics of broken chloroplasts show a rapid rise and decline during the first second of illumination, followed by a slower rise with a broad maximum at 5 to 10 seconds. The rapid spike (Li) can be selectively inhibited by valinomycin indicating its dependence on the membrane potential, whereas the main maximum (Lm) is selectively inhibited by uncouplers, showing its dependence on the proton gradient (Wraight and Crofts 1971). We, therefore,
choose this system to investigate if heat-induced changes in luminescence are correlated with inhibition of membrane energization and thus, whether luminescence induction curves can indeed be used as an indicator of such a heatstress-induced inhibition.
Materials and methods
Freshly isolated chloroplasts from greenhouse grown spinach leaves (Spinacia oleracea L., cv. Yates Hybrid 102) were used. The plants received a PFD of 200 to 300/zmol m -2 s -1 (artificial + natural light) with a controlled daylength of 10 h. The daytime air temperature was usually between 18 and 25°C and never exceeded 30°C. The greenhouse was cooled at night to about 10 to 15°C. Intact chloroplasts were isolated following the method of Jensen and Bassham (1966) as modified by Heber (1973), and stored in medium 'C' at a pH of 7.6. The intactness was usually about 80% and chlorophyll concentration approx. 2 mg/cm 3. The chloroplasts were stored on ice in darkness until used for the measurements. In some experiments broken chloroplasts were treated in Tris-HC1 (0.8 M, pH 8.0) for 10 min in the dark. The chloroplasts were heat-treated in the intact state to avoid an artifactual weakening of the heat resistance which occurs in broken chloroplasts. For the heat treatment 60 to 70 mm 3 of a threefold diluted chloroplast suspension (solution 'C', pH 7.6) were placed in a small, thinwalled reaction vessel (4 mm in diameter). Each vessel was kept in the dark in a stirred water bath (MGW Lauda K2, Lauda, FRG) for 5 min at the desired temperature. Subsequently the vessel was cooled in an ice water mixture for 1 min and an aliquot of the heated chloroplast suspension transferred to the measuring cuvette. Before measurement, the heated chloroplasts were broken by suspending them in a solution containing 10 mM MgCI 2 and 5 mM Hepes at pH 7.6. After 30 s double strength medium 'C' was added. All activity measurements were completed during the following 5 min. The temperature in the measuring cuvette was kept at 15°C. The luminescence measurements were con-
164 ducted with a Becquerel type phosphoroscope based on fiberoptics. A chopper wheel driven by a stepper motor interrupted the actinic light beam after 800/~s for a dark period of equal duration. During the dark period the luminescence intensity was measured from 240 to 560/zs following the interruption of the illumination by means of a photomultiplier (Hamamatsu R928) protected by a red plastic filter (Roscolene 821, Edmunds Scientific, Barrington, N J, USA). The photomultiplier signal was amplified and integrated by a custom built lock-in amplifier. The resulting data were registered in a storage oscilloscope (Explorer III, Nicolet, USA). The time constant of the amplifier was set to 10 ms. Actinic light was provided by a halogen light source (Xenophot XLX, Osram, FRG) connected to a stabilized power supply (Model N G R E 30/15, Rohde and Schwarz, FRG). After passing through an optical filter (BG 39 or RG 645, Schott, Mainz, FRG) and a fast shutter (model electronic-m, Compur, FRG) the actinic light was guided by plastic fiber optics to the chopper wheel and the measuring cuvette. With both the bluegreen and the red filter the PFD was 70 to 80/z mol m- z s- 1 The multibranched fiberoptics bundle reaching the measuring cuvette allowed simultaneous measurements of other parameters. Chlorophyll fluorescence was measured by a photodiode (UDT 100) protected by a longpass filter ( R G 695, Schott). To determine light-induced absorbance changes a weak measuring beam (PFD 1 / z m o l m - 2 s -1) at 518nm (BA40 IF 518, DT Cyan, Balzers, Liechtenstein; BG 38, Schott) was provided by a halogen lamp (Osram 64225). After passing the sample, the beam was reflected from a disk shaped mirror at the bottom of the cuvette, which also served as a stirrer. The transmitted measuring light was detected by a photomultiplier (EMI 9558) protected by bluegreen filters (BG 39, Schott, and CS 4-96, Coming). 9-Aminoacridin fluorescence was measured with the measuring beam entering the cuvette from the side. The beam was defined by bluegreen filters (K40, Balzers, BG 39 Schott) and had a PFD of 0.5/zmol m -2 s -1. The 9-AA fluorescence was guided by a branch of the fiberoptics to a photomultiplier (EMI 9558) protected by a suitable filter combination (K50, Balzers, BG 39, Schott). The concentration of 9-AA in
the assay was 2 × 10 - 6 M. Methylviologen (10 -4 M) was used as electron acceptor; catalase concentration was 100/xg/cm 3. Photophosphorylation activity was measured in situ using the luciferin-luciferase method (Schreiber and Del Valle Tascon 1982). The assay medium contained 0.33 M sorbitol, 2 m M KH2PO4, 10mM Mg-acetate, 10mM HEPES/ Tris, pH7.6, 5 × 10 -3 M ferricyanide, 10 -3 M DTT, 0.5mg/cm 3 BSA, 0.05 to 0.1cm3/cm 3 luciferin-luciferase (ATP monitoring kit, LKB) and 2.5 x 10 -5 M ADP. ATP (6.5 x 10 - 7 M) was added as internal standard. Luciferin luminescence was measured by a photomultiplier (EMI 9558) protected by bluegreen filters (BG 39, BG 40, Schott). The PFD of the red actinic light (RG 665, Schott) was 8/xmol m -2 s -~ 0 2 evolution was measured in an O z electrode (model DWl, Hansatech, Kings Lynn, UK) in the presence of 5 x 10 - 6 M gramicidin and with a PFD of 1000/zmolm -2 s -~ (RG 610, Schott). Ferricyanide (5 × 10 -3 M) was added as electron acceptor. Chemicals were purchased from Sigma if not stated otherwise.
Results and discussion
Effect of heat stress on luminescence induction kinetics
Induction curves obtained after application of a 5-min heat treatment of the chloroplasts are shown in Fig. 1 (left column, upper curves). The rapid spike and the maximum are labelled Li and Lm, respectively, on the control (20°C) traces. With increasing temperature of the pretreatment, luminescence emission became progressively inhibited. However, above a treatment temperature of about 42°C, L i increased again, especially when luminescence was measured in presence of nigericin and valinomycin, i.e., when membrane energization was totally absent (Fig. 1, left column, lower traces). Henceforth, this type of luminescence emission is termed basal luminescence, or L 0. The difference between the induction curves of total and basal luminescence emission represents that luminescence component which depends on membrane energization and is shown in the right-hand col-
165 i
20 *C
/Lm /Lo
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umn of Fig. 1. The energy dependent component consistently declined with increasing temperature, and after heating to 48°C no response to illumination could be detected. It is also apparent that the heat-induced decline of L'i began and reached completion at lower temperatures than that observed for L ' . This response is more clearly seen in Fig. 2, where L'i, L " and L 0 are plotted against the pretreatment temperature. L I was reduced to about 50% of its control value at 34°C. L " was clearly more heat resistant (50% at about 39°C), whereas L 0 did not respond until the pretreatment temperature exceeded 40°C. The threshold and 50%-inhibition temperatures varied by about I°C among different experiments while the ranking of heat resistance of the measured parameters always remained the same.
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Correlation of L'i with the light-induced membrane potential
15
Fig. 1. Luminescence induction curves of freshly broken spinach chloroplasts after 5 min pretreatment as intact chloroplasts at the indicated temperatures. Left column, upper curves, measured without additions; lower curves, measured in presence of 10-SM valinomycin and 10-7M nigericin. Right column, difference curves between upper and lower curves from left column. The characteristic levels, L~, Lm, and L 0 of the induction curves are indicated. L I = L i - L o ; L~, = L m - L o. Actinic PFD was 70/zmol m -2 s -~ and the sample temperature 20°C. The baseline is indicated by a dashed line.
Figure 3 shows the results of measurements of light-induced absorbance changes at 518nm (AAs18). These changes in DA are widely used to monitor changes in membrane potential (Vredenberg 1981). The trace in the inset shows an original recording of AA518 measured on a control sample. The dependences on pretreat-
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Fig. 2. Pretreatment temperature dependence of LI, L~ and L 0. The values are calculated from the same experiment as shown in Fig. I.
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