Photosynthesis Research23: 269-282, 1990. © 1990KluwerAcademicPublishers. Printedin the Netherlands. Regular paper
Luminescence decay kinetics in relation to quenching and stim ation of dark fluorescence from high and low C02 adapted cells of Scenedesmus obliquus and Chlamydomonas reinhardtii Lars-G6ran Sundblad, G6ran Samuelsson, Bosse Wigge & Per Gardestr6m Dept. of Plant Physiol., Univ. of Ume&, S-901 87 Urne~, Sweden Received 21 November 1988; accepted26 July 1989
Key words." luminescence, fluorescence, CO2-accumulation, Clamydomonas obliquus
reinhardtii, Scenedesmus
Abstract
Two green algal species, Chlamydomonas reinhardtii and Scenedesmus obliquus, exhibited a relative maximum during the decay of luminescence, when adapted to low CO2 conditions that was not observed in high CO2 adapted cells. From the kinetics of transient changes in the level of dark fluorescence, after illumination and parallel to the luminescence maxima, it was concluded that the maximum in Scenedesmus was mainly related to a decrease in nonphotochemical quenching, whereas in Chlamydomonas the maximum was mainly related to a dark reduction of the primary PS II acceptor QA. ATP/ADP ratios from low CO2 adapted Seenedesmus showed transient high levels after a dark/light transition that was not observed in high CO2 adapted cells. After 30 s of illumination the ATP/ADP ratios however stabilized at the same steady state level as in high CO2 adapted cells. Dark addition of HCO3 to low CO2 adapted ceils of Chlamydomonas resulted in a rapid transient quenching of luminescence that was not observed in low CO2 adapted cells of neither species. It is concluded that the luminescence maxima present in both low CO2 adapted Scenedesmus and Chlamydomonas reflect adaptation of the cells to low CO2 conditions. It is further suggested that the difference in mechanistic origin of luminescence maxima in the two species reflects differences in adaptation.
Abbreviations; ADP - adenosine-diphosphate, ATP - adenosine-triphosphate, Ci - inorganic carbon, Fo dark fluorescence recorded under dark adapted conditions, F0 - fluorescence with all reaction centers open, FV - variable fluorescence, P S I - photosystem I, PS II - photosystem II, QA - the first quinone acceptor of PS II
Introduction
Chlorophyll a luminescence is the result of the recombination between electrons on the acceptor side of photosystem II (PS II) with positive charges on the donor side (Lavorel 1975). Several factors can affect the intensity, decay kinetics and yield of luminescence. These factors can be classified as affecting one or several of 4 parameters:
- The concentration of electrons on the acceptor side of PS II (Lavorel 1975). - The presence of positive charges on the donor side of PS II (Lavorel 1975). - The activation energy for electrons and positive charges to recombine (Crofts et al. 1971). - The competition from pathways other than light emission for dissipation of excitation energy from charge recombination (Sundblad et al. 1986).
270 Luminescence normally decays asymptotically with the most light emitted during the first few seconds of the decay (Malkin 1977). Secondary kinetics with peaks and shoulders have however been reported by several authors (Bertsch and Azzi 1965, Rubin et al. 1966, Desai et al. 1983, Bj6rn 1971, Palmqvist et al. 1986, Mellvig and Tillberg 1986, Schmidt and Senger 1987a, b). Green algal cells subjected to low CO2 conditions develop a COs concentrating mechanism. This adaptation involves changes both in protein synthesis and in energetics of the cells (Badger et al. 1980). The energetic role of algal chloroplasts was discussed by Spalding (Spalding et al. 1984). In Scenedesmus obliquus a correlation between a transient peak in the minute range decay of luminescence and the ability for active uptake in inorganic carbon (Ci) was shown to be related to the energetic state of the algal chloroplast (Palmqvist et al. 1986, Sundblad et al. 1986a, b). Chlorophyll a fluorescence has been widely used as a probe for the function, induction, intactness and photochemical potential of the photosynthetic apparatus (Krause and Weis 1984, Schreiber et al. 1986). The use of weak, modulated excitation light together with stronger photosynthetically active light and pulses of saturating light in combination with lock in amplifiers have made it possible to discriminate between the two major quenching components in vivo; nonphotochemical quenching (mainly related to the transthylakoid ApH) and photochemical quenching, determined by the redox state of the primary PS II acceptor (QA)(Quick and Horton 1984). The term 'dark fluorescence' was used for the fluorescence resulting from excitation with weak (photosynthetically inactive) light (Schreiber 1986), since it is believed to closely mimic conditions in the dark. Although resulting from excitation incapable of driving photosynthesis, dark fluorescence can work as a probe for changes in photochemical and nonphotochemical quenching, induced by a second stronger and photosynthetically active light, provided that fluorescence is excited with modulated light and the resulting signal is amplified with a lock in amplifier tuned to the frequency of the exciting light. The procedure is based on the assumption that fluorescence unrelated to the function of PS II photochemistry (Fo fluorescence) is not affected by those factors that cause non-
photochemical quenching of variable fluoresence. As shown by Bilger and Schreiber (1986) this assumption does not hold true under conditions of extreme energization of the thylakoid membrane. It was concluded that nonphotochemical quenching of F0 under such conditions (i.e. strong light and low CO2) was a result of state quenching (i.e. quenching due to spillover of excitation energy from PS II to PS I). However, it has also been proposed that nonphotochemical quenching of F0 is caused by the transthylakoid ApH through a decrease in the ratio of, rate of transfer of excitation-energy to open reaction centers/rate of backtransfer, at high ApH (Weis and Berry 1987). In this paper dark fluorescence was measured in parallel with luminescence from high and low COs adapted cells of Scenedesmus obliquus and Chlamydomonas reinhardtii, in order to reveal the mechanistic background to the luminescence maxima observed in low CO2 adapted cells.
Material and methods
Algal material and culturing conditions The unicellular green algae Scenedesmus obliquus, strain WT D3 and Chlamydomonas reinhardtii, strain 137 c ÷ were grown in continuous light (80/~mol m- 2s- ~) in inorganic media (Bishop 1971) at 25°C. Low CO2 adapted cells were obtained by bubbling the growth medium with air for at least 48 h prior to use in experiments. High COs adapted cells were obtained by bubbling with air containing 2% COs. For the experiments illustrated in Fig. 11, high CO2 adapted Chlamydomonas were harvested and centrifuged at 800g for 5 min, whereafter the cells were resuspended in low CO2 medium.
Luminescence measurements The measurements were carried out in a modified Hansatech 02 electrode. White excitation light was provided by a metal halogen lamp (Atlas 24V, 250 W). The photon flux density of the exciting light was 600 pmol m -2 s- ~(except for experiments shown in Fig. 9) and the time of excitation 30 s. Luminescence was detected by a selected Hamamatsu R 374 photomultiplier and the signal ampli-
271 fled and recorded on a strip chart recorder. Excitation light and luminescence emission were guided to the reaction vessel and the photomultiplier by optical fibers. All samples were dark adapted for 3 min before excitation.
Fluorescence measurements
Fluorescence measurements were carried out in the same Hansatech 02 electrode as for the luminescence measurements. Dark fluorescence was excited by weak (0.1 /~molm-2s-1), blue green, modulated (375 Hz) light. The fluorescence signal was detected by a Hamamatsu R 1017 photomultiplier and the signal amplified in a lock in amplifier (PAR mad 124, Princeton, New Jersey, USA) and recorded on a chart strip recorder. The level of dark fluorescence under 'relaxed' dark conditions (here called Fo) was recorded after 3 min dark adaptation and immediately before turning on the strong white preillumination light. The photomultiplier used for fluorescence detection was protected from emission during preillumination since the dynamic properties of the measuring system did not allow
measurements of high fluorescence intensities immediately preceding the dark fluorescence measurements. The fluorescence excitation light was thus modulated and the resulting signal amplified by a lock in amplifier only in order to increase the sensitivity and the signal/noise ratio of the measuring system and to avoid interference of luminescence in the fluorescence signal. For measurements of low intensity modulated fluorescence (not dark fluorescence) with superimposed secondary light, a Heinz Walz fluorometer PAM 101 (Heinz Walz, FRG) was used. The intensity of the excitation light was 2.5 ymol m -2 s-I and the frequency of modulation 100 kHz. Measurements of both fluorescence and luminescence were, except for the experiment illustrated in Fig. 11, performed under CO2 conditions to which the algal cells were adapted. Chlorophyll concentrations were about the same during growth and fluorescence/luminescence measurements and varied between 2.5 and 5 /agml-'. For ATP and ADP measurements, algae were harvested, and pelleted at 1200g for 3min. The cells were then resuspended in growth medium con-
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? steady state reached for low C02 Scen.
Fig. 1. Light/dark sequences used for measurements of luminescence, dark fluorescence and ATP/ADP ratios.
272 taining 0.25 M 4-morpholinepropanesulfonic acid, adjusted to pH 7.0. The chlorophyll concentration during ATP and ADP measurements were 60#g chlorophyll ml ~. To high CO2 grown algae 20 mM NAHCO3 was added. For measurements 0.5ml algae were incubated for 2 min in the dark before light was switched on. Metabolism was stopped by addition of 0.5 ml 6.0 M perchloric acid (3.0 M final concentration) after different times of illumination. 5 min after addition of perchloric acid the extract was neutralised by addition of KOH + 4-(2-hydroxyethyl)-l-piperazin-ethansul-
fonic acid. Concentration of adenine-nucleotides were measured by the luciferase assay as described by Lundin et al. (1976). Figure 1 illustrates the dark adaptation, preillumination, excitation and measuring sequences used for luminescence, dark fluorescence and ATP/ ADP measurements.
Results and discussion
In Figs. 2a and 3a the decay patterns of lumi-
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\ Fig. 2a. Luminescence decay from left, high and right, low CO 2 adapted cells of Scenedesmus obliquus.
273
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High CO 2 Low CO 2
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Fig. 2b. Dark fluorescence from left, high and right, low CO 2 adapted cells of Scenedesmus obliquus after a light/dark transition. Dotted line represents FD level (dark fluorescence during 'relaxed' conditions).
nescence for the two green algae Scenedesmus obliquus and Chlamydomonas reinhardtii are shown when the cells were adpated to high and low CO2 conditions. In low CO 2 adapted cells, a characteristic relative maximum was observed. In Chlamydomonas the maximum appeared ,-~ 7 s after excitation and in Scenedesmus ~ 18s after excitation. The decay from high COz adapted cells was without maxima although a small shoulder was observed in Chlamydomonas. In Sundblad et al. (1986b) the luminescence
maximum from low CO2 adapted Scenedesmus was shown to be correlated to a rapid relaxation of nonphotochemical quenching of maximal fluorescence. It was concluded that the luminescence maximum appeared as a result of a rapid energetic turnover in low CO2 adapted cells and that the luminescence maximum and the rapid relaxation of nonphotochemical quenching reflected a rapid ApH relaxation. This conclusion was further strengthened in the present work by the kinetics of dark fluorescence observed after a light/dark tran-
274 Q
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High C0 2 Low CO2
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Fig. 3a. Luminescence decay from, left high and right, low CO2 adapted cells of Chlamydomonas reinhardtii.
sition similar to the excitation/measurement sequence used for luminescence measurements (Fig. 2b). In low CO2 adapted Scenedesmus, dark fluorescence after illumination was initially quenched to a level substantially lower than the level obtained in dark before illumination. As the quenching occurred below the original dark fluorescence level, the quenching may be concluded to be nonphotochemical since photochemical quenching (resulting from oxidized QA) is likely to be most prominent during relaxed, dark adapted conditions. The steep
increase in dark fluorescence back to the original dark level therefore, at least partially, reflects relaxation of nonphotochemical quenching. This conclusion is strengthened by the results shown in Fig. 4. In the experiment another modulated fluorescence system (Heinz Walz PAM 101) with lower sensitivity but higher dynamic range was applied during the same excitation/'dark' sequence as in Figs. 2 and 3. With this system a higher intensity (2.5/~mol m -2 s -1) modulated light had to be used in order to obtain a good signal because of the low chlorophyll concentration of the algal culture and
275
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Low CO2 m
High CO 2
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m m G. o
Fig. 3b. Dark fluorescence from, left, high and right, low CO2 adapted cells of Chlamydomonas reinhardtii after a light/dark transition. Dotted line represents F D level (dark fluorescence during relaxed conditions),
the imperfect optical geometry of the 02 electrode used during measurements (low chlorophyll concentration was required for optimal growth conditions and concentration of algae during transfer to the electrode avoided in order to minimize disturbance). The modulated light therefore gave rise to some variable fluorescence and also modified the 'dark' levels and kinetics of fluorescence. However, when a saturating flash of white light was given before and after the fast phase of increasing fluorescence a significant relaxation of nonphotochemical quenching was evident, supporting the conclusion
drawn from Fig. 2b. Considering the time scale of the relaxation, we conclude that the major component of the nonphotochemical quenching was related to the transthylakoid ApH. The steep increase in dark fluorescence (observed in Fig. 2b) was correlated to the increase in luminescence that resulted in a maximum, indicating ApH relaxation as the major cause for the appearance of the luminescence maximum in low CO2 adapted Scenedesmus. High CO2 adapted Scenedesmus exhibited neither luminescence maximum, nor any quenching of dark fluorescence below the dark level (Fig. 2b).
276 I
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Fig. 4. Low intensity (2.5/~molm-2s -~ red) modulated fluorescence from low CO 2 adapted cells of Scenedesmus obliquus. Intensity o f continuous white light 600/~molm-2s -~ and of saturating flashes 6000/~mol m - 2s - J.
Another indication of a rapid energetic turnover in low CO2 adapted Scenedesmus was evident when changes in the ATP/ADP ratio after a dark/light transition was compared for high and low CO2 adapted cells (Fig. 5). Low CO2 adapted cells showed a transient very high ATP/ADP ratio, that was not observed in high CO2 adapted cells. After about 30 s of illumination (corresponding to preillumination before luminescence and dark fluorescence measurements) the ratio of both cell types however were stabilized at similar levels, although nonphotochemical quenching suggested higher energization of the thylakoid in the chloroplast of low CO2 adapted cells after 30 s of illumination (Figs. 2b and 4). Since the ATP/ADP ratio depends on both production and consumption of ATP, the low (i.e. normal) steady state level of the ATP/ADP ratio from low CO2 adapted cells, together with high nonphotochemical quenching indicates both high production and consumption of ATP in these cells (and not that ATP/ADP turnover was slowed down by low CO2 conditions). However, the onset of ATP consumption in low CO2 adapted cells must
Fig. 5. ATP/ADP from high • and low [] CO 2 adapted cells of Scenedesmus obliquus after a dark/light transition. Experimental conditions given in text.
be concluded to lag behind the onset of production as indicated by the high initial ATP/ADP ratio. We suggest that the delay reflects the onset of ATP consumption by active C~ uptake. Low CO2 adapted Chlamydomonas exhibited a relative maximum during the decay of luminescence that was also correlated to a steep increase in dark fluorescence (Figs. 3a and 3b). In Chlamydomonas the steep increase in dark fluorescence however occurred above the dark level. It is therefore less self evident that the steep increase in dark fluorescence, as suggested for Scenedesmus, was related to relaxation of ApH related nonphotochemical quenching, since ApH related nonphotochemical quenching during relaxed conditions must be considered to be minimal. Stimulation of dark fluorescence (and luminescence) by dark reduction of QA must therefore be considered to be a likely cause for the relative maximum observed in dark fluorescence and luminescence from low CO2 adapted Chlamydomonas. Support for this explanation comes from the results shown in Figs. 6 and 7. In Fig. 6 the same experiment as shown in Fig. 4 was performed on Chlamydomonas. As evident from the identical levels of fluorescence obtained at saturating flashes given before and after the phase of increasing fluorescence (correlated to the lumi-
277 I
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Fig. 6. Same experimental conditions as in Fig. 4 but with low CO~ adapted cells of Chlamydomonas reinhardtii.
nescence maximum), no relaxation of nonphotochemical quenching occurred during this phase. In Fig. 7 the effect of superimposed weak far red light after white light pre-illumination is shown. The rapid 'dark' increase of fluorescence was clearly suppressed by far red light as could be expected if the increase was caused by QA reduction since far red illumination causes QA oxidation through dominating P S I excitation. In Fig. 8 the same experiment as in Fig. 6 were performed on
Scenedesmus. The dark increase of fluorescence was in Scenedesmus not affected by weak far red light. The difference between Scenedesmus and Chlamydomonas was also manifested in the different luminescence responses to weak, medium and high white light excitation (Fig. 9). In Scenedesmus high light was required to obtain maximal expression of the luminescence peak, whereas in Chlamydomonas the corresponding kinetics were suppressed by high light. Our interpretation of this result is that in Scenedesmus high energization of the thylakoid membrane is required for the luminescence maximum, whereas in Chlamydomonas the dark reduction of QA will only to a limited extent be expressed as a stimulation of luminescence due to low luminescence substrate concentration on the PS II donor side after some time in darkness following high light excitation (i.e. the positive charges on the donor side of PS II will be rapidly consumed through recombination after high light excitation, leaving little luminescence substrate on the donor side for luminescence stimulation when QA is rereduced after some time in darkness). In Sundblad (1988) it was shown that a lowering of CO2 concentration in the air above an intact barley leaf in the dark, resulted in reduction of QA and a stimulation of both luminescence and dark fluorescence by an unknown mechanism, not re-
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Fig. 7. Same experimental conditions as in Fig. 6butwith 5/~molm-2s ~far red light after white light illumination instead ofsaturating flashes.
278
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Fig. 9a. Luminescence decay from low CO2 adapted cells of Scenedesmus obliquus after excitation with 5,500 and Fig. 8. Same experimental conditions as in Fig. 7 but with low CO2 adapted cells of Scenedesmus obliquus.
lated to the role of CO2 as the primary electron acceptor in photosynthesis. Recently, it was also shown in Synechococcus that an increase in the internal Ci concentration through active uptake, resulted in quenching of variable fluorescence (Miller and Canvin 1987). The quenching was concluded to be photochemical by the authors. Furthermore in low CO2 adapted Anacystis nidulans a sudden light/dark transition was shown to result in a rapid decrease in the internal Ci concentration of the cells (Ogawa et al. 1985). Taking these results into account, the following working hypothesis could be formulated to explain the luminescence maximum (and the corresponding fluorescence transient) in low CO2 adapted Chlamydomonas. During illumination a Ci concentration gradient between the cells and the low CO2 medium is maintained through light dependent active uptake of Ci. When sudden darkness is imposed, this gradient will only sustain as long as the light induced high energetic state providing energy for the uptake will persist. When energy (i.e. ApH, ATP) no longer is
5,000/~mol m-2 s- ~ white light.
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5
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500
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-
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Fig. 9b. Same experimental conditions as in Fig. 9a but with low CO 2 adapted cells of Chlamydomonas reinhardtii.
279
O >
0.5 rain C 0 u 0 C
J
S
/
Fig. 10. The effect of dark addition of HCOj- on the decay kinetics of luminescence. HCOf added as indicated by arrows to a final concentration of 2.5 mM; b, after 5 s in the dark during the first decay phase of luminescence; c, at the relative minimum preceding the relative maximum during the decay of luminescence; d, after the relative luminescence maximum, a, control without addition.
available, the C i concentration within the cells will rapidly decrease when the internal C~concentration equilibrates with the C~ concentration of the medium. As shown in Sundblad (1988) and Miller and Canvin (1987) the decrease will lead to reduction of QA and a concomitant stimulation of luminescence and dark fluorescence. In order to further investigate the above hypothesis, H C O ; was added to low CO2 adapted Chlamydomonas after different times in darkness following illumination (Fig. 10). In all cases addition of HCO~- resulted in rapid quenching of luminescence. However, when the addition was made before the 'naturally occurring' luminescence maximum, the quenching was followed by a secondary stimulation of luminescence (Figs. 10b and c). Assuming that, as suggested in the hypothesis, the luminescence maximum occurring without HCOj- addition (Figs. 3a and 10a, control) is a result of the collapse of a Ci concentration gradient, quenching of luminescence after HCO3 addition made before the luminescence maximum will partly be a result of active uptake of C~ (energy still available for active Ci uptake). Therefore the internal C~ concentration in the cells after HCO3 addition, made before the luminescence maximum, will be transiently higher than in the medium. However,
when the energy for maintaining the gradient is consumed, the internal C i concentration will decrease leading to reduction of QA and stimulation of luminescence. This HCO3 induced quenching/ stimulation of luminescence was more pronounced after 5 s in the dark than after 10 s (cf. Figs. 10b and c). We interpret this quantitative difference in luminescence response, depending on length of dark interval, as a consequence of more energy being available for Ci uptake after shorter dark intervals. At short dark intervals the transient fluctuations in internal Ci concentration could with this interpretation be expected to be more pronounced, giving rise to more pronounced quenching/stimulation of luminescence. The fact that only quenching (and no secondary stimulation) of luminescence occurred upon HCO3 addition after the luminescence maximum further supports the hypothesis that the maximum is a manifestation of ceasing C~ uptake. As (according to the hypothesis) no energy will be available for C~ uptake after the luminescence maximum, the quenching of luminescence at this stage only reflects passive influx of C~ into the cells when the HCO~- concentration in the medium is increased upon addition. No secondary decrease in internal Ci concentration (and no secondary stimulation of
280 luminescence) could therefore be expected since
HCO; addition at this stage will only increase the internal Ci concentration to the concentration of the medium. As discussed above the quenching/stimulation of luminescence by addition of H C 0 3 , could thus be considered as a manifestation of the same phenomenon as the one that gives rise to the luminescence maximum in low C02 adapted Chlamydomonas without H C 0 3 addition, i.e. the collapse of a C~ concentration gradient. The rate of consumption of the post-illumination energy, available for active C~uptake, could in the same way be regarded as the major difference between 'the naturally occurring' luminescence maximum and the H C 0 3 induced quenching/stimulation. This point is supported by the fact that when HCO~- was added before the luminescence maximum only one maximum was observed, i.e. HC03- induced quenching/ stimulation of luminescence replaced the 'naturally occurring' luminescence maximum. The hypothesis that the HCO~- induced quenching/stimulation of luminescence is a phenomenon linked to active uptake of G is further strengthened by the results shown in Fig. 11 where H C 0 3 was added after 5 s in darkness to Chlamydomonas
during adaptation from high to low CO2 conditions. Addition of HCO3 to high CO2 adapted cells had no effect on luminescence. After transfer to a low CO2 medium HCO~- induced quenching/ stimulation of luminescence started to appear after about 15 min, after which the response gradually became more pronounced. In Palmqvist et al. (1988), the physiological characteristics of inorganic carbon uptake in both Chlamydomonas reinhardtff and Scenedesmus obliquus was compared. It was shown that the ability for active uptake of Ci, started to appear about 15 min after transfer from high to low CO2 conditions in both species. However, after 3 h of adaptation the ability for active Ci uptake was shown to be almost twice as efficient in Chlamydomonas as compared to
Scenedesmus. The fact that no quenching appeared in high CO2 adapted cells by passive influx of Ci might be related to lower activity of carbonic anhydrase in these cells (Coleman et al. 1984). The very slow phase of dark fluorescence quenching that was observed in both low CO2 adapted Chlamydomonas and Scenedesmus was possibly related to state 1/state 2 transitions. Differences in protein phosphorylation related to state
u c u
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I 0.5
C
/
rain
J
J
x~
Fig. 11. The effect of dark addition of HCO~- on the decay kinetics of luminescence from Chlamydomonas reinhardtii during the first two hours of adaptation from high to low CO2 conditions. Addition was made to a final concentration of 2.5 mM as indicated by arrows after 5 s in the dark and after; b, 5 min of adaptation; c, 15 min of adaptation; d, 120 min of adaptation, a, control during high CO2 conditions.
281 1/state 2 transitions between high and low CO2 adapted Scenedesmus obliquus have been reported by Heil and Senger (1986). The slow quenching was reversed after about 10min in darkness (not shown). From the results presented in this paper [and taking into account results from other works, i.e. Sundblad et al. (1986a, b), Sundblad (1988), Miller and Canvin (1987), Ogawa et al. (1985)] we conclude that the luminescence maxima present in low CO2 adapted Scenedesmus obliquus and Chlamydomonas reinhardtii, although similar in appearance, are of a different mechanistic origin. In Scenedesmus the relaxation of nonphotochemical quenching is the dominating luminescence stimulating factor giving rise to the maximum. Addition of HCO3 to low CO2 adapted Scenedesmus in the dark after illumination had therefore no effect on luminescence (not shown). However, dark reduction of QA as a result of lowered internal Ci concentration can not be ruled out as a factor affecting luminescence and being a prerequisite for the luminescence maximum. In fact, the decay of luminescence from Scenedesmus obliquus has been shown to exhibit more than one luminescence maximum (Mellvig and Tillberg 1986, Sundblad et al. 1986a), clearly indicating more than one luminescence stimulating process during the decay. Furthermore, as seen in Fig. 2b the increase in dark fluorescence in low CO2 adapted Scenedesmus was at least biphasic also indicating more than one process stimulating dark fluorescence. Although speculative it is tempting to attribute the slow increase in dark fluorescence above the dark level to QA reduction as a consequence of lowered internal C~ concentration. In Chlamydomonas, the interpretation of luminescence is complicated by the appearance of a small shoulder during the decay in high CO2 adapted cells (Fig. 3a). Also dark fluorescence exhibited small transients in high CO2 adapted Chlamydomonas (as well as in high CO2 adapted Scenedesmus). Although lacking experimental support, we interpret these transients as the result of relaxation of nonphotochemical quenching, although much less pronounced than in low CO2 adapted Scenedesmus. Whatever the exact relative proportions is between stimulation of luminescence (and dark fluorescence) by reduction of QA and/or by relax-
ation of nonphotochemical quenching, in the two species, we conclude that, for the appearance of luminescence maxima in low CO2 adapted cells, QA reduction is more important in Chlamydomonas and relaxation of nonphotochemical quenching more important in Scenedesmus. We furthermore conclude that both phenomena reflect adaptation of the algal chloroplasts to low CO2 environment, but that this adaptation might be different in the two species as indicated by the different mechanistic background to their luminescence maxima. This aspect is currently under investigation from other experimental approaches.
Acknowledgements We are grateful to Prof. Gunnar Oquist for fruitful discussions and for valuable comments on the manuscript. The work was supported by the Swedish Natural Science Research Council, J.C. Kempes Minnes Stipendiefond and Swedish Council for Forestry and Agricultural Research.
References Badger MR, Kaplan A and Berry JA (1980) Internal inorganic carbon pool of Chlamydomonas reinhardtii. Plant Physiol 66: 407-413 Bertsch WF and Azzi JR (1965) A relative maximum in the decay of long term delayed light emission from the photosynthetic apparatus. Biochim Biophys Acta 94:15-26 Bilger W and Schreiber V (1986) Energy-dependent quenching of dark-level chlorophyll fluorescence in intact leaves. Photosynth Res 10:303-308 Bishop NI (1971) Preparations and properties of mutants: Scenedesmus. Methods Enzymol 23:372-408 Bj6rn LO (1971) Far red induced, long lived afterglow from photosynthetic ceils. Size of afterglow unit and paths of energy accumulation and dissipation. Photochem Photobiol 13:5-20 Coleman JR, Berry JA, Togasaki RK and Grossman AR (1984) Identification of extracellular carbonic anhydrase of Chlamydomonas reinhardtii. Plant Physiol 76:472-477 Crofts AR, Wraight CA and Fleischmann DE (1971) Energy conservation in the photochemical reactions of photosynthesis and its relation to delayed fluorescence. FEBS Lett 15 (2): 89-100 Desai TS, Rane SS, Tatake VG and Sane PV (1983) Identification of far-red induced relative increase in the decay of delayed light emission from photosynthetic membranes with thermoluminescence peak V appearing at 321 K. Biochim Biophys Acta 724:485-489
282 Heil WG and Senger H (1986) Thylakoid-protcin phosphorylation during the life cycle of Seenedesmus obliquus in synchronous culture. Planta 167:233-239 Krause GH and Weis E (1984) Chlorophyll fluorescence as a tool in plant physiology. II Interpretation of fluorescence signals. Photosynth Res 5:139-157 Lavorel J (1975) Luminescence. In: Govindjee (¢d) Bioenergetics of Photosynthesis, pp 223-317. New York: Academic Press Lundin A, Rickardsson A and Thore A (1976) Continuous monitoring of ATP-converting reactions by purified firefly luciferase. Anal Biochem 75:611-620 Malkin S (1977) Delayed luminescence. In: J Barber (ed) Primary Processes of Photosynthesis, pp 349-431. North-Holland: Biomedical Press, Elsevier MeUvig S and Tillberg J-E (1986) Transient peaks in the delayed luminescence from Scenedesmus obtusiculus induced by phosphorous starvation and carbon dioxide deficiency. Physiol Plantarum 68:180-188 Miller A G and Canvin DT (1987) The quenching of chlorophyll a fluorescence as a consequence of the transport of inorganic carbon by the cyanobacterium Syneochoceus UTEX 625. Biochim Biophys Acta 894:407-413 Ogawa T, Omata T, Miyano A and Inoue Y (1985) Photosynthetic reactions involved in the CO2-concentration mechanism in the cyanobacterium Anacystis nidulans. In: Lucas WJ and Berry JA (eds), Inorganic Carbon Uptake by Aquatic Photosynthetic Organisms. American Society for Plant Physiologists Palmqvist K, Sj6berg S and Samuelsson G (1988) Induction of inorganic carbon accumulation in the unicellular green algae Scenedesmus obliquus and Chlamydomonas reinhardtii. Plant Physiol 87:437-442 Palmqvist K, Sundblad L-G, Samuelsson G and Sundbom E (1986) A correlation between changes in luminescence decay kinetics and the appearance of a CO2-accumulating mechanism in Scenedesmus obliquus. Photosynth Res 10:113-123 Quick WP and Horton P (1984) Studies on the induction of chlorophyll fluorescence in barley protoplasts. II. Resolution
of fluorescence quenching by redox state and transthylakoid pH gradient. Proc R Soc Lond B220:371-382 Rubin AB, Fokht AS and Venediktov PS (1966) Investigation of the kinetics of attenuation of the afterglow of photosynthesizing organism. Biofizika 11:299-305 Schmidt W and Senger H (1987a) Long term delayed luminescence in Scenedesmus obliquus. I. Spectral and kinetic properties. Biochim Biophys Acta 890:15-22 Schmidt W and Senger H (1987b) Long-term delayed luminescence in Scenedesmus obliquus. II. Infuence of exogenous factors. Biochim Biophys Acta 891:22-27 Schreibcr U (1986) Detection of rapid induction kinetics with a new type of high frequency modulated chlorophyll fluorometer. Photosynth Res 9:261-272 Schreibcr U, Schliwa U and Bilger W (1986) Continuous recording of photochemical and non-photochemical chlorophyll fluorescence quenching with a new type of modulation fluorometer. Photosynth Res 10:51-62 Spalding MH, Critchley C, Govindjee and Ogren WL (1984) Influence of carbon dioxide concentration during growth on fluorescence induction characteristics of the green algae Chlamydomonas reinhardtii. Photosynth Res 5:169-176 Sundblad L-G (1988) Dark reduction of QA in intact barley leaves, as an effect of lowered CO 2 concentration, monitored by chorophyll a luminescence and chlorophyll a F 0 dark fluorescence. Biochim Biophys Acta 936:429-434 Sundblad L-G, Palmqvist K and Samuelsson G (1986a)An energy dependent, transient peak in the minute range decay of luminescence, present in CO2 accumulating aells of Scenedesmus obliquus. FEBS Lett 199:75-79 Sundblad L-G, Palmqvist K and Samuelsson G (1986b) Luminescence decay kinetics in relation to the relaxation of the transthylakoid ApH from high and low CO2 adapted cells of Scenedesmus obliquus. FEBS Lett 209:28-32 Weis E and Berry JA (1987) Quantum et~ciency of Photosystem II in relation to energy-dependent quenching of chlorophyll fluorescence. Biochim Biophys Acta 894:198-208
Luminescence decay kinetics in relation to quenching and stim ation of dark fluorescence from high and low C02 adapted cells of Scenedesmus obliquus and Chlamydomonas reinhardtii Lars-G6ran Sundblad, G6ran Samuelsson, Bosse Wigge & Per Gardestr6m Dept. of Plant Physiol., Univ. of Ume&, S-901 87 Urne~, Sweden Received 21 November 1988; accepted26 July 1989
Key words." luminescence, fluorescence, CO2-accumulation, Clamydomonas obliquus
reinhardtii, Scenedesmus
Abstract
Two green algal species, Chlamydomonas reinhardtii and Scenedesmus obliquus, exhibited a relative maximum during the decay of luminescence, when adapted to low CO2 conditions that was not observed in high CO2 adapted cells. From the kinetics of transient changes in the level of dark fluorescence, after illumination and parallel to the luminescence maxima, it was concluded that the maximum in Scenedesmus was mainly related to a decrease in nonphotochemical quenching, whereas in Chlamydomonas the maximum was mainly related to a dark reduction of the primary PS II acceptor QA. ATP/ADP ratios from low CO2 adapted Seenedesmus showed transient high levels after a dark/light transition that was not observed in high CO2 adapted cells. After 30 s of illumination the ATP/ADP ratios however stabilized at the same steady state level as in high CO2 adapted cells. Dark addition of HCO3 to low CO2 adapted ceils of Chlamydomonas resulted in a rapid transient quenching of luminescence that was not observed in low CO2 adapted cells of neither species. It is concluded that the luminescence maxima present in both low CO2 adapted Scenedesmus and Chlamydomonas reflect adaptation of the cells to low CO2 conditions. It is further suggested that the difference in mechanistic origin of luminescence maxima in the two species reflects differences in adaptation.
Abbreviations; ADP - adenosine-diphosphate, ATP - adenosine-triphosphate, Ci - inorganic carbon, Fo dark fluorescence recorded under dark adapted conditions, F0 - fluorescence with all reaction centers open, FV - variable fluorescence, P S I - photosystem I, PS II - photosystem II, QA - the first quinone acceptor of PS II
Introduction
Chlorophyll a luminescence is the result of the recombination between electrons on the acceptor side of photosystem II (PS II) with positive charges on the donor side (Lavorel 1975). Several factors can affect the intensity, decay kinetics and yield of luminescence. These factors can be classified as affecting one or several of 4 parameters:
- The concentration of electrons on the acceptor side of PS II (Lavorel 1975). - The presence of positive charges on the donor side of PS II (Lavorel 1975). - The activation energy for electrons and positive charges to recombine (Crofts et al. 1971). - The competition from pathways other than light emission for dissipation of excitation energy from charge recombination (Sundblad et al. 1986).
270 Luminescence normally decays asymptotically with the most light emitted during the first few seconds of the decay (Malkin 1977). Secondary kinetics with peaks and shoulders have however been reported by several authors (Bertsch and Azzi 1965, Rubin et al. 1966, Desai et al. 1983, Bj6rn 1971, Palmqvist et al. 1986, Mellvig and Tillberg 1986, Schmidt and Senger 1987a, b). Green algal cells subjected to low CO2 conditions develop a COs concentrating mechanism. This adaptation involves changes both in protein synthesis and in energetics of the cells (Badger et al. 1980). The energetic role of algal chloroplasts was discussed by Spalding (Spalding et al. 1984). In Scenedesmus obliquus a correlation between a transient peak in the minute range decay of luminescence and the ability for active uptake in inorganic carbon (Ci) was shown to be related to the energetic state of the algal chloroplast (Palmqvist et al. 1986, Sundblad et al. 1986a, b). Chlorophyll a fluorescence has been widely used as a probe for the function, induction, intactness and photochemical potential of the photosynthetic apparatus (Krause and Weis 1984, Schreiber et al. 1986). The use of weak, modulated excitation light together with stronger photosynthetically active light and pulses of saturating light in combination with lock in amplifiers have made it possible to discriminate between the two major quenching components in vivo; nonphotochemical quenching (mainly related to the transthylakoid ApH) and photochemical quenching, determined by the redox state of the primary PS II acceptor (QA)(Quick and Horton 1984). The term 'dark fluorescence' was used for the fluorescence resulting from excitation with weak (photosynthetically inactive) light (Schreiber 1986), since it is believed to closely mimic conditions in the dark. Although resulting from excitation incapable of driving photosynthesis, dark fluorescence can work as a probe for changes in photochemical and nonphotochemical quenching, induced by a second stronger and photosynthetically active light, provided that fluorescence is excited with modulated light and the resulting signal is amplified with a lock in amplifier tuned to the frequency of the exciting light. The procedure is based on the assumption that fluorescence unrelated to the function of PS II photochemistry (Fo fluorescence) is not affected by those factors that cause non-
photochemical quenching of variable fluoresence. As shown by Bilger and Schreiber (1986) this assumption does not hold true under conditions of extreme energization of the thylakoid membrane. It was concluded that nonphotochemical quenching of F0 under such conditions (i.e. strong light and low CO2) was a result of state quenching (i.e. quenching due to spillover of excitation energy from PS II to PS I). However, it has also been proposed that nonphotochemical quenching of F0 is caused by the transthylakoid ApH through a decrease in the ratio of, rate of transfer of excitation-energy to open reaction centers/rate of backtransfer, at high ApH (Weis and Berry 1987). In this paper dark fluorescence was measured in parallel with luminescence from high and low COs adapted cells of Scenedesmus obliquus and Chlamydomonas reinhardtii, in order to reveal the mechanistic background to the luminescence maxima observed in low CO2 adapted cells.
Material and methods
Algal material and culturing conditions The unicellular green algae Scenedesmus obliquus, strain WT D3 and Chlamydomonas reinhardtii, strain 137 c ÷ were grown in continuous light (80/~mol m- 2s- ~) in inorganic media (Bishop 1971) at 25°C. Low CO2 adapted cells were obtained by bubbling the growth medium with air for at least 48 h prior to use in experiments. High COs adapted cells were obtained by bubbling with air containing 2% COs. For the experiments illustrated in Fig. 11, high CO2 adapted Chlamydomonas were harvested and centrifuged at 800g for 5 min, whereafter the cells were resuspended in low CO2 medium.
Luminescence measurements The measurements were carried out in a modified Hansatech 02 electrode. White excitation light was provided by a metal halogen lamp (Atlas 24V, 250 W). The photon flux density of the exciting light was 600 pmol m -2 s- ~(except for experiments shown in Fig. 9) and the time of excitation 30 s. Luminescence was detected by a selected Hamamatsu R 374 photomultiplier and the signal ampli-
271 fled and recorded on a strip chart recorder. Excitation light and luminescence emission were guided to the reaction vessel and the photomultiplier by optical fibers. All samples were dark adapted for 3 min before excitation.
Fluorescence measurements
Fluorescence measurements were carried out in the same Hansatech 02 electrode as for the luminescence measurements. Dark fluorescence was excited by weak (0.1 /~molm-2s-1), blue green, modulated (375 Hz) light. The fluorescence signal was detected by a Hamamatsu R 1017 photomultiplier and the signal amplified in a lock in amplifier (PAR mad 124, Princeton, New Jersey, USA) and recorded on a chart strip recorder. The level of dark fluorescence under 'relaxed' dark conditions (here called Fo) was recorded after 3 min dark adaptation and immediately before turning on the strong white preillumination light. The photomultiplier used for fluorescence detection was protected from emission during preillumination since the dynamic properties of the measuring system did not allow
measurements of high fluorescence intensities immediately preceding the dark fluorescence measurements. The fluorescence excitation light was thus modulated and the resulting signal amplified by a lock in amplifier only in order to increase the sensitivity and the signal/noise ratio of the measuring system and to avoid interference of luminescence in the fluorescence signal. For measurements of low intensity modulated fluorescence (not dark fluorescence) with superimposed secondary light, a Heinz Walz fluorometer PAM 101 (Heinz Walz, FRG) was used. The intensity of the excitation light was 2.5 ymol m -2 s-I and the frequency of modulation 100 kHz. Measurements of both fluorescence and luminescence were, except for the experiment illustrated in Fig. 11, performed under CO2 conditions to which the algal cells were adapted. Chlorophyll concentrations were about the same during growth and fluorescence/luminescence measurements and varied between 2.5 and 5 /agml-'. For ATP and ADP measurements, algae were harvested, and pelleted at 1200g for 3min. The cells were then resuspended in growth medium con-
I
lure m e a s
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I
dark
measurement of F D
growth conditions 80 u real m - 2 s-1
dark adaptation
pre ill 600 u real m - 2 s- I
dark fluo. m e a s 0 I u molto-2 s-1
centrifugation
dark adaptation
ATP/ADP
meas
? steady state reached for low C02 Scen.
Fig. 1. Light/dark sequences used for measurements of luminescence, dark fluorescence and ATP/ADP ratios.
272 taining 0.25 M 4-morpholinepropanesulfonic acid, adjusted to pH 7.0. The chlorophyll concentration during ATP and ADP measurements were 60#g chlorophyll ml ~. To high CO2 grown algae 20 mM NAHCO3 was added. For measurements 0.5ml algae were incubated for 2 min in the dark before light was switched on. Metabolism was stopped by addition of 0.5 ml 6.0 M perchloric acid (3.0 M final concentration) after different times of illumination. 5 min after addition of perchloric acid the extract was neutralised by addition of KOH + 4-(2-hydroxyethyl)-l-piperazin-ethansul-
fonic acid. Concentration of adenine-nucleotides were measured by the luciferase assay as described by Lundin et al. (1976). Figure 1 illustrates the dark adaptation, preillumination, excitation and measuring sequences used for luminescence, dark fluorescence and ATP/ ADP measurements.
Results and discussion
In Figs. 2a and 3a the decay patterns of lumi-
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Low CO 2 High CO 2
e-
C
\ Fig. 2a. Luminescence decay from left, high and right, low CO 2 adapted cells of Scenedesmus obliquus.
273
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t
L. U
High CO 2 Low CO 2
0 J
Fig. 2b. Dark fluorescence from left, high and right, low CO 2 adapted cells of Scenedesmus obliquus after a light/dark transition. Dotted line represents FD level (dark fluorescence during 'relaxed' conditions).
nescence for the two green algae Scenedesmus obliquus and Chlamydomonas reinhardtii are shown when the cells were adpated to high and low CO2 conditions. In low CO 2 adapted cells, a characteristic relative maximum was observed. In Chlamydomonas the maximum appeared ,-~ 7 s after excitation and in Scenedesmus ~ 18s after excitation. The decay from high COz adapted cells was without maxima although a small shoulder was observed in Chlamydomonas. In Sundblad et al. (1986b) the luminescence
maximum from low CO2 adapted Scenedesmus was shown to be correlated to a rapid relaxation of nonphotochemical quenching of maximal fluorescence. It was concluded that the luminescence maximum appeared as a result of a rapid energetic turnover in low CO2 adapted cells and that the luminescence maximum and the rapid relaxation of nonphotochemical quenching reflected a rapid ApH relaxation. This conclusion was further strengthened in the present work by the kinetics of dark fluorescence observed after a light/dark tran-
274 Q
I O m
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I L O U G
High C0 2 Low CO2
C
.=J
Fig. 3a. Luminescence decay from, left high and right, low CO2 adapted cells of Chlamydomonas reinhardtii.
sition similar to the excitation/measurement sequence used for luminescence measurements (Fig. 2b). In low CO2 adapted Scenedesmus, dark fluorescence after illumination was initially quenched to a level substantially lower than the level obtained in dark before illumination. As the quenching occurred below the original dark fluorescence level, the quenching may be concluded to be nonphotochemical since photochemical quenching (resulting from oxidized QA) is likely to be most prominent during relaxed, dark adapted conditions. The steep
increase in dark fluorescence back to the original dark level therefore, at least partially, reflects relaxation of nonphotochemical quenching. This conclusion is strengthened by the results shown in Fig. 4. In the experiment another modulated fluorescence system (Heinz Walz PAM 101) with lower sensitivity but higher dynamic range was applied during the same excitation/'dark' sequence as in Figs. 2 and 3. With this system a higher intensity (2.5/~mol m -2 s -1) modulated light had to be used in order to obtain a good signal because of the low chlorophyll concentration of the algal culture and
275
I ,m
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Low CO2 m
High CO 2
U
m m G. o
Fig. 3b. Dark fluorescence from, left, high and right, low CO2 adapted cells of Chlamydomonas reinhardtii after a light/dark transition. Dotted line represents F D level (dark fluorescence during relaxed conditions),
the imperfect optical geometry of the 02 electrode used during measurements (low chlorophyll concentration was required for optimal growth conditions and concentration of algae during transfer to the electrode avoided in order to minimize disturbance). The modulated light therefore gave rise to some variable fluorescence and also modified the 'dark' levels and kinetics of fluorescence. However, when a saturating flash of white light was given before and after the fast phase of increasing fluorescence a significant relaxation of nonphotochemical quenching was evident, supporting the conclusion
drawn from Fig. 2b. Considering the time scale of the relaxation, we conclude that the major component of the nonphotochemical quenching was related to the transthylakoid ApH. The steep increase in dark fluorescence (observed in Fig. 2b) was correlated to the increase in luminescence that resulted in a maximum, indicating ApH relaxation as the major cause for the appearance of the luminescence maximum in low CO2 adapted Scenedesmus. High CO2 adapted Scenedesmus exhibited neither luminescence maximum, nor any quenching of dark fluorescence below the dark level (Fig. 2b).
276 I
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L 11 u eu L. 0 -q J h
.
.
.
.
.
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.
.
.
.
.
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r
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150
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Fig. 4. Low intensity (2.5/~molm-2s -~ red) modulated fluorescence from low CO 2 adapted cells of Scenedesmus obliquus. Intensity o f continuous white light 600/~molm-2s -~ and of saturating flashes 6000/~mol m - 2s - J.
Another indication of a rapid energetic turnover in low CO2 adapted Scenedesmus was evident when changes in the ATP/ADP ratio after a dark/light transition was compared for high and low CO2 adapted cells (Fig. 5). Low CO2 adapted cells showed a transient very high ATP/ADP ratio, that was not observed in high CO2 adapted cells. After about 30 s of illumination (corresponding to preillumination before luminescence and dark fluorescence measurements) the ratio of both cell types however were stabilized at similar levels, although nonphotochemical quenching suggested higher energization of the thylakoid in the chloroplast of low CO2 adapted cells after 30 s of illumination (Figs. 2b and 4). Since the ATP/ADP ratio depends on both production and consumption of ATP, the low (i.e. normal) steady state level of the ATP/ADP ratio from low CO2 adapted cells, together with high nonphotochemical quenching indicates both high production and consumption of ATP in these cells (and not that ATP/ADP turnover was slowed down by low CO2 conditions). However, the onset of ATP consumption in low CO2 adapted cells must
Fig. 5. ATP/ADP from high • and low [] CO 2 adapted cells of Scenedesmus obliquus after a dark/light transition. Experimental conditions given in text.
be concluded to lag behind the onset of production as indicated by the high initial ATP/ADP ratio. We suggest that the delay reflects the onset of ATP consumption by active C~ uptake. Low CO2 adapted Chlamydomonas exhibited a relative maximum during the decay of luminescence that was also correlated to a steep increase in dark fluorescence (Figs. 3a and 3b). In Chlamydomonas the steep increase in dark fluorescence however occurred above the dark level. It is therefore less self evident that the steep increase in dark fluorescence, as suggested for Scenedesmus, was related to relaxation of ApH related nonphotochemical quenching, since ApH related nonphotochemical quenching during relaxed conditions must be considered to be minimal. Stimulation of dark fluorescence (and luminescence) by dark reduction of QA must therefore be considered to be a likely cause for the relative maximum observed in dark fluorescence and luminescence from low CO2 adapted Chlamydomonas. Support for this explanation comes from the results shown in Figs. 6 and 7. In Fig. 6 the same experiment as shown in Fig. 4 was performed on Chlamydomonas. As evident from the identical levels of fluorescence obtained at saturating flashes given before and after the phase of increasing fluorescence (correlated to the lumi-
277 I
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8
i I.
o white on
T
white on
flesh
T flesh
Fig. 6. Same experimental conditions as in Fig. 4 but with low CO~ adapted cells of Chlamydomonas reinhardtii.
nescence maximum), no relaxation of nonphotochemical quenching occurred during this phase. In Fig. 7 the effect of superimposed weak far red light after white light pre-illumination is shown. The rapid 'dark' increase of fluorescence was clearly suppressed by far red light as could be expected if the increase was caused by QA reduction since far red illumination causes QA oxidation through dominating P S I excitation. In Fig. 8 the same experiment as in Fig. 6 were performed on
Scenedesmus. The dark increase of fluorescence was in Scenedesmus not affected by weak far red light. The difference between Scenedesmus and Chlamydomonas was also manifested in the different luminescence responses to weak, medium and high white light excitation (Fig. 9). In Scenedesmus high light was required to obtain maximal expression of the luminescence peak, whereas in Chlamydomonas the corresponding kinetics were suppressed by high light. Our interpretation of this result is that in Scenedesmus high energization of the thylakoid membrane is required for the luminescence maximum, whereas in Chlamydomonas the dark reduction of QA will only to a limited extent be expressed as a stimulation of luminescence due to low luminescence substrate concentration on the PS II donor side after some time in darkness following high light excitation (i.e. the positive charges on the donor side of PS II will be rapidly consumed through recombination after high light excitation, leaving little luminescence substrate on the donor side for luminescence stimulation when QA is rereduced after some time in darkness). In Sundblad (1988) it was shown that a lowering of CO2 concentration in the air above an intact barley leaf in the dark, resulted in reduction of QA and a stimulation of both luminescence and dark fluorescence by an unknown mechanism, not re-
I
I I min
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~
hite off
whi~e off
,
8
m
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Fig. 7. Same experimental conditions as in Fig. 6butwith 5/~molm-2s ~far red light after white light illumination instead ofsaturating flashes.
278
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white off
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Fig. 9a. Luminescence decay from low CO2 adapted cells of Scenedesmus obliquus after excitation with 5,500 and Fig. 8. Same experimental conditions as in Fig. 7 but with low CO2 adapted cells of Scenedesmus obliquus.
lated to the role of CO2 as the primary electron acceptor in photosynthesis. Recently, it was also shown in Synechococcus that an increase in the internal Ci concentration through active uptake, resulted in quenching of variable fluorescence (Miller and Canvin 1987). The quenching was concluded to be photochemical by the authors. Furthermore in low CO2 adapted Anacystis nidulans a sudden light/dark transition was shown to result in a rapid decrease in the internal Ci concentration of the cells (Ogawa et al. 1985). Taking these results into account, the following working hypothesis could be formulated to explain the luminescence maximum (and the corresponding fluorescence transient) in low CO2 adapted Chlamydomonas. During illumination a Ci concentration gradient between the cells and the low CO2 medium is maintained through light dependent active uptake of Ci. When sudden darkness is imposed, this gradient will only sustain as long as the light induced high energetic state providing energy for the uptake will persist. When energy (i.e. ApH, ATP) no longer is
5,000/~mol m-2 s- ~ white light.
I
....
I
I min
I,,.
° U r" °U
5
/ /
500
~
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-
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Fig. 9b. Same experimental conditions as in Fig. 9a but with low CO 2 adapted cells of Chlamydomonas reinhardtii.
279
O >
0.5 rain C 0 u 0 C
J
S
/
Fig. 10. The effect of dark addition of HCOj- on the decay kinetics of luminescence. HCOf added as indicated by arrows to a final concentration of 2.5 mM; b, after 5 s in the dark during the first decay phase of luminescence; c, at the relative minimum preceding the relative maximum during the decay of luminescence; d, after the relative luminescence maximum, a, control without addition.
available, the C i concentration within the cells will rapidly decrease when the internal C~concentration equilibrates with the C~ concentration of the medium. As shown in Sundblad (1988) and Miller and Canvin (1987) the decrease will lead to reduction of QA and a concomitant stimulation of luminescence and dark fluorescence. In order to further investigate the above hypothesis, H C O ; was added to low CO2 adapted Chlamydomonas after different times in darkness following illumination (Fig. 10). In all cases addition of HCO~- resulted in rapid quenching of luminescence. However, when the addition was made before the 'naturally occurring' luminescence maximum, the quenching was followed by a secondary stimulation of luminescence (Figs. 10b and c). Assuming that, as suggested in the hypothesis, the luminescence maximum occurring without HCOj- addition (Figs. 3a and 10a, control) is a result of the collapse of a Ci concentration gradient, quenching of luminescence after HCO3 addition made before the luminescence maximum will partly be a result of active uptake of C~ (energy still available for active Ci uptake). Therefore the internal C~ concentration in the cells after HCO3 addition, made before the luminescence maximum, will be transiently higher than in the medium. However,
when the energy for maintaining the gradient is consumed, the internal C i concentration will decrease leading to reduction of QA and stimulation of luminescence. This HCO3 induced quenching/ stimulation of luminescence was more pronounced after 5 s in the dark than after 10 s (cf. Figs. 10b and c). We interpret this quantitative difference in luminescence response, depending on length of dark interval, as a consequence of more energy being available for Ci uptake after shorter dark intervals. At short dark intervals the transient fluctuations in internal Ci concentration could with this interpretation be expected to be more pronounced, giving rise to more pronounced quenching/stimulation of luminescence. The fact that only quenching (and no secondary stimulation) of luminescence occurred upon HCO3 addition after the luminescence maximum further supports the hypothesis that the maximum is a manifestation of ceasing C~ uptake. As (according to the hypothesis) no energy will be available for C~ uptake after the luminescence maximum, the quenching of luminescence at this stage only reflects passive influx of C~ into the cells when the HCO~- concentration in the medium is increased upon addition. No secondary decrease in internal Ci concentration (and no secondary stimulation of
280 luminescence) could therefore be expected since
HCO; addition at this stage will only increase the internal Ci concentration to the concentration of the medium. As discussed above the quenching/stimulation of luminescence by addition of H C 0 3 , could thus be considered as a manifestation of the same phenomenon as the one that gives rise to the luminescence maximum in low C02 adapted Chlamydomonas without H C 0 3 addition, i.e. the collapse of a C~ concentration gradient. The rate of consumption of the post-illumination energy, available for active C~uptake, could in the same way be regarded as the major difference between 'the naturally occurring' luminescence maximum and the H C 0 3 induced quenching/stimulation. This point is supported by the fact that when HCO~- was added before the luminescence maximum only one maximum was observed, i.e. HC03- induced quenching/ stimulation of luminescence replaced the 'naturally occurring' luminescence maximum. The hypothesis that the HCO~- induced quenching/stimulation of luminescence is a phenomenon linked to active uptake of G is further strengthened by the results shown in Fig. 11 where H C 0 3 was added after 5 s in darkness to Chlamydomonas
during adaptation from high to low CO2 conditions. Addition of HCO3 to high CO2 adapted cells had no effect on luminescence. After transfer to a low CO2 medium HCO~- induced quenching/ stimulation of luminescence started to appear after about 15 min, after which the response gradually became more pronounced. In Palmqvist et al. (1988), the physiological characteristics of inorganic carbon uptake in both Chlamydomonas reinhardtff and Scenedesmus obliquus was compared. It was shown that the ability for active uptake of Ci, started to appear about 15 min after transfer from high to low CO2 conditions in both species. However, after 3 h of adaptation the ability for active Ci uptake was shown to be almost twice as efficient in Chlamydomonas as compared to
Scenedesmus. The fact that no quenching appeared in high CO2 adapted cells by passive influx of Ci might be related to lower activity of carbonic anhydrase in these cells (Coleman et al. 1984). The very slow phase of dark fluorescence quenching that was observed in both low CO2 adapted Chlamydomonas and Scenedesmus was possibly related to state 1/state 2 transitions. Differences in protein phosphorylation related to state
u c u
I
I 0.5
C
/
rain
J
J
x~
Fig. 11. The effect of dark addition of HCO~- on the decay kinetics of luminescence from Chlamydomonas reinhardtii during the first two hours of adaptation from high to low CO2 conditions. Addition was made to a final concentration of 2.5 mM as indicated by arrows after 5 s in the dark and after; b, 5 min of adaptation; c, 15 min of adaptation; d, 120 min of adaptation, a, control during high CO2 conditions.
281 1/state 2 transitions between high and low CO2 adapted Scenedesmus obliquus have been reported by Heil and Senger (1986). The slow quenching was reversed after about 10min in darkness (not shown). From the results presented in this paper [and taking into account results from other works, i.e. Sundblad et al. (1986a, b), Sundblad (1988), Miller and Canvin (1987), Ogawa et al. (1985)] we conclude that the luminescence maxima present in low CO2 adapted Scenedesmus obliquus and Chlamydomonas reinhardtii, although similar in appearance, are of a different mechanistic origin. In Scenedesmus the relaxation of nonphotochemical quenching is the dominating luminescence stimulating factor giving rise to the maximum. Addition of HCO3 to low CO2 adapted Scenedesmus in the dark after illumination had therefore no effect on luminescence (not shown). However, dark reduction of QA as a result of lowered internal Ci concentration can not be ruled out as a factor affecting luminescence and being a prerequisite for the luminescence maximum. In fact, the decay of luminescence from Scenedesmus obliquus has been shown to exhibit more than one luminescence maximum (Mellvig and Tillberg 1986, Sundblad et al. 1986a), clearly indicating more than one luminescence stimulating process during the decay. Furthermore, as seen in Fig. 2b the increase in dark fluorescence in low CO2 adapted Scenedesmus was at least biphasic also indicating more than one process stimulating dark fluorescence. Although speculative it is tempting to attribute the slow increase in dark fluorescence above the dark level to QA reduction as a consequence of lowered internal C~ concentration. In Chlamydomonas, the interpretation of luminescence is complicated by the appearance of a small shoulder during the decay in high CO2 adapted cells (Fig. 3a). Also dark fluorescence exhibited small transients in high CO2 adapted Chlamydomonas (as well as in high CO2 adapted Scenedesmus). Although lacking experimental support, we interpret these transients as the result of relaxation of nonphotochemical quenching, although much less pronounced than in low CO2 adapted Scenedesmus. Whatever the exact relative proportions is between stimulation of luminescence (and dark fluorescence) by reduction of QA and/or by relax-
ation of nonphotochemical quenching, in the two species, we conclude that, for the appearance of luminescence maxima in low CO2 adapted cells, QA reduction is more important in Chlamydomonas and relaxation of nonphotochemical quenching more important in Scenedesmus. We furthermore conclude that both phenomena reflect adaptation of the algal chloroplasts to low CO2 environment, but that this adaptation might be different in the two species as indicated by the different mechanistic background to their luminescence maxima. This aspect is currently under investigation from other experimental approaches.
Acknowledgements We are grateful to Prof. Gunnar Oquist for fruitful discussions and for valuable comments on the manuscript. The work was supported by the Swedish Natural Science Research Council, J.C. Kempes Minnes Stipendiefond and Swedish Council for Forestry and Agricultural Research.
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