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Effects of heat stress on PSII photochemistry in a cyanobacterium Spirulina platensis
Plant Science 175 (2008) 556–564
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Effects of heat stress on PSII photochemistry in a cyanobacterium Spirulina platensis Binbin Zhao a,b, Jia Wang a, Hongmei Gong a, Xiaogang Wen a, Haiyun Ren b, Congming Lu a,* a b
Photosynthesis Research Center, Chinese Academy of Sciences, Beijing 100093, China College of Life Science, Beijing Normal University, Beijing 100875, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 September 2007 Received in revised form 2 June 2008 Accepted 16 June 2008 Available online 26 June 2008
The effects of heat stress (30–50 8C) on photosystem II (PSII) photochemistry in a cyanobacterium Spirulina platensis grown at 30 8C were studied by measuring chlorophyll fluorescence induction kinetics and thermoluminescence. Heat stress inhibited significantly the maximum efficiency of PSII photochemistry. An investigation of the kinetics of flash-induced fluorescence yield decay revealed that heat stress caused a shift of the equilibrium towards QA but showed no effect on the kinetics of flashinduced fluorescence yield decay in the presence of DCMU. An analysis of polyphasic rise of fluorescence transients indicated that heat stress induced a decrease in the total number of PSII active reaction centers. Thermoluminescence measurements demonstrated that the B band in Spirulina cells corresponding to the S2QB state consisted of two different components, the B32 and the B24 bands with the peak temperatures at 32 and 24 8C, respectively. The intensity of the B32 band and the B24 band decreased significantly with increasing temperature but the decrease of the intensity of the B24 band was much greater than that of the B32 band. There were no significant changes in the peak temperatures of the B24 and B32 bands. The intensity of the Q band corresponding to the recombination of the S2QA charge pair decreased significantly with increasing temperature. The period-four oscillation of the B band was significantly damped at temperatures higher than 45 8C. The contents of D1 and PsbO proteins decreased with increasing temperature. The results in this study suggest that heat stress shows no effects on the stability of the S2QA and S2QB states and that the different populations of the active PSII reaction centers show different sensitivity to heat stress in S. platensis cells. ß 2008 Elsevier Ireland Ltd. All rights reserved.
Keywords: Chlorophyll fluorescence Heat stress Photosystem II Spirulina platensis
1. Introduction High temperature stress affects significantly photosynthetic activity [1]. When leaves or algae are exposed to high temperature stress, their oxygen and CO2 assimilation capacities are significantly inhibited. One of the most-sensitive sites in the photosynthetic apparatus is thought to be photosystem II (PSII) and its activity decreases significantly by high temperature stress [1,2]. Numerous studies have shown that high temperature stress has various effects on PSII function. High temperature stress inhibits the function of the oxidizing side of PSII and oxygen evolution is
* Corresponding author at: Photosynthesis Research Center, Institute of Botany, Chinese Academy of Sciences, No. 20, Nanxincun, Beijing 100093, China. Tel.: +86 10 62836554; fax: +86 10 62595516. E-mail address: [email protected] (C. Lu). Abbreviations: Chl, chlorophyll; DCMU, 3-(30 40 -dichlorophenyl)-1,1-dimethylurea; QA, primary acceptor plastoquinone; QB, secondary acceptor plastoquinone; PSI, photosystem I; PSII, photosystem II; TL, thermoluminescence; S-state, oxidation states of the Mn cluster. 0168-9452/$ – see front matter ß 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2008.06.003
significantly decreased [3]. A loss of the oxygen evolving complex activity is thought to be due to the release of manganese atoms and the dissociation of the manganese-stabilizing 33 kDa protein from the PSII reaction center complex [3,4–6]. High temperature stress also results in an inactivation of PSII reaction centers [7–12]. In addition, high temperature results in a shift of the redox equilibrium between the primary acceptor plastoquinone (QA) and the secondary acceptor plastoquinone (QB) [13–19]. Furthermore, high temperature stress induces a dissociation of the peripheral antenna complex of PSII from its core complex [20–25]. Thermoluminescence (TL) is an outburst of light emission occurring at characteristic temperatures from a preilluminated photosynthetic sample. TL is a powerful tool to investigate the properties of the redox components in the photosynthetic apparatus [26,27]. It has thus been extensively used to probe the function of the donor side and the acceptor side of PSII in various mutant cells and under different environmental stress conditions such as photoinhibition, salt stress, and low temperature [26–30]. Since it has been generally considered that high temperature stress induces major structural and functional
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changes of PSII [13,22], TL can be used to estimate more precisely the function of PSII [31]. Spirulina platensis, a filamentous cyanobacterium, isolated from a wide range of habitats has long been cultured for production of health food because of its high content of protein and other nutritional elements, such as phycocyanin, carotenoids and glinolenic acid [32,33]. In cultures grown outdoors in open ponds under arid and semiarid climates, Spirulina cells are subjected to various environmental stresses, such as salinity stress, photoinhibition, and high temperature stress [34]. Many studies have investigated the effects of salinity stress, osmotic stress, and photoinhibition on photosynthesis and PSII photochemistry in S. platensis [35–39]. Recently, we have studied the effects of high temperature on excitation energy transfer from phycobilisomes to photosystem I (PSI) and PSII in S. platensis and found out that high temperature stress induced an inhibition of excitation energy transfer from phycobilisomes to PSII but not to photosystem I (PSI) [40]. However, how high temperature stress affects PSII photochemistry in Spirulina cells remains to be elucidated further. Understandably, a better understanding of heat stress on PSII photochemistry may help optimize the productivity of the algal cultures grown outdoors. In the present study, we have investigated in more detail the responses of the donor and acceptor sides of PSII in a cyanobacterium S. platensis to different high temperatures. 2. Materials and methods 2.1. Alga and growth conditions The cyanobacterium, S. platensis M2, was grown at 30 8C in Zarouk’s medium supplemented with 0.2 M sodium bicarbonate [41]. Illumination of 80 mmol photons m2 s1 was provided by cool daylight tubes (TLD 30W/865, Philips). 2.2. High temperature treatment Exponentially growing cells were harvested and diluted to 10 mg chlorophyll (Chl) ml1 in fresh medium. The cells were placed in the dark for at least 30 min at 30 8C before the heat treatments. Similar to the heat treatments used in algae in the literature [42,43], the glass cuvettes containing the cells were dipped into a water bath and incubated for 5 min at different temperatures. The measurements of Chl fluorescence and thermoluminescence were taken immediately after heat stress treatments. All treatments and measurements were performed in the dark. 2.3. Maximum efficiency of PSII photochemistry The maximum efficiency of PSII photochemistry was determined as Fv/Fm with a portable fluorometer (PAM-2000, Walz, Germany), where Fv = (Fm F0) and Fm and F0 are the maximum and minimal fluorescence yield, respectively, of a dark-adapted suspension. F0 was measured by using modulated measuring light that was of sufficiently low intensity (<0.1 mmol m2 s1) not to induce any significant variable fluorescence and Fm(dark) was determined by applying 0.8 s saturating pulse at 8000 mmol m2 s1 in darkadapted cells. Fm(DCMU) was determined in the presence of 10 mM DCMU (3-3,4-dichlorophenyl-1,1-dimethyl urea) as described previously [44].
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rometer FL200/PS, consisting of Thermoregulator TR2000 (Photon Systems Instruments, Brno, Czech Republic). After 30 min dark adaptation at 30 8C, the cells were cooled to 0 8C and illuminated with one or multiple number of single-turnover flashes with duration of 25 ms. Then the cells were warmed up to 60 8C at a heating rate of 1 8C s1 and the TL light emission was measured during the heating. The volume of the samples for each measurement was 0.4 ml and the Chl concentration of the sample was 10 mg Chl ml1. To detect period-four oscillation of the Bband, cells were illuminated with a series of single-turnover flashes. For S2QA recombination studies, cells were measured in the presence of 3-(30 ,40 -dichlorophenyl)-1,1-dimethylurea (DCMU, 10 mM) before the flash illumination. The nomenclature of Vass and Govindjee [26] was used for characterization of the flashinduced TL glow peaks. 2.5. Decay of Chl fluorescence yield after a single turnover flash The decay of Chl fluorescence yield after a single turnover flash was measured with a double-modulation fluorescence fluorometer (model FL-200, Photon Systems Instruments, Brno, Czech Republic) [45]. The square-shaped, single-turnover, saturated flash was generated by one set of 48 orange LEDs (630 nm) and one set of red LEDs (653 nm) that focus on the opposite sides of the sample cuvette. The duration of the single-turnover flash was 25 ms and its intensity was ca. 200,000 mmol photons m2 s1. The measuring flashes were provided by two sets of 7 orange LEDs with the duration of 2.5 ms. Each measuring flash excited less than 1% of the reaction center of PSII. 2.6. Polyphasic rise of Chl fluorescence transients Chl fluorescence transients were measured by a Plant Efficiency Analyzer (PEA, Hansatech Instruments Ltd., King’s Lynn, Norfolk PE32 1JL, England) by the method described by Strasser et al. [46]. Illumination was provided by an array of 6 high intensity, lightemitting-diodes (LED, a peak at 650 nm), which were focused on the sample surface to provide homogeneous illumination over the exposed area of a sample with a 4 mm diameter. The fluorescence signals were received by a detector, a high performance pin photodiode associated with an amplifier circuit, after passing through a long pass filter. The detector, responding maximally to the longer wavelength fluorescence signal, blocked the reflected shorter wavelength LED light used as the source of illumination. All the fluorescence transients were recorded within a time scan from 10 ms to 1 s with a data acquisition rate of 105 readings per second for the first 2 ms and of 103 per second after 2 ms. According to JIPtest [47–49], Chl transients were analyzed by utilizing the original data from the polyphasic fluorescence transients. The parameter RC/ABS (the total number of active reaction center per absorption) is used for the quantification of PSII behavior referring to time zero. 2.7. SDS-PAGE and immunological analyses Samples were solubilized in the presence of 6 M urea and separated by SDS-PAGE [50] using 15% (w/v) acrylamide gels with 6 M urea. For immunoblotting, polypeptides were electrophoretically transferred to PVDF membranes (Millipore, Saint-Quentin, France) and proteins were detected with antibodies raised against D1 and PsbO proteins.
2.4. Thermoluminescence
3. Results
TL measurements of whole cells were performed with the thermoluminescence extension of the Double-Modulated Fluo-
Many studies have shown that heat stress inhibits the activity of PSII [2–6,23,25]. In this study, the activity of PSII was expressed
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by the maximum efficiency of PSII photochemistry (Fv/Fm). It should be noted that under light conditions or not completely dark adapted conditions, even strong light may not close all reaction centers of PSII. In addition, it has been demonstrated that PSII reaction centers were knocked out under heat stress conditions and a saturating pulse is no longer able to close all PSII reaction centers [11,19]. Therefore, there would be a quenching of Fm under these conditions. The measured Fm would be still lower than the Fm with all reaction centers closed. Therefore, more electrons may have to be provided by cycling of the S states to fill the PQ pool and to reach the maximum fluorescence yield while one electron is sufficient to reduce QA in the presence of DCMU. Probably, the Fm measured in the absence of DCMU is lower than the Fm measured with DCMU, which decreases more slowly with temperature [19]. To obtain the true Fm, Fm in this study was measured in the presence 10 mM DCMU. Fig. 1 shows that Fv/Fm decreased significantly with increasing temperature. We also investigated the effects of heat stress on the effects of Fm and F0. Fm decreased but F0 increased significantly with increasing temperature. TL is the emission of light during re-warming of photosynthetic samples exposed to actinic illumination prior to freezing (for reviews, see Refs. [26,27]). TL is normally due to thermally stimulated recombination of pairs of opposite charges generated by light-induced charge separation in the PSII complex. TL emission peaked at approximately 25 8C (B-band) results largely from the recombination of the S2QB charge pair. At around 10 8C, addition of DCMU to the sample, which blocks oxidation of QA by QB, induces a major emission component (Q-band) from S2QA recombination. The peak temperature of a TL component is an indicator of the energetic stability of a separated charge pair. As a general rule, the higher the peak temperature is, the greater the stabilization is. Thus, these measurements can provide information on the depth of the activation energy wells involved in the stabilization of the S2QA and S2QB charge separated states [51]. Fig. 2 shows the TL glow curves obtained from Spirulina cells excited with a single flash to detect the B-band. TL emission following single flash excitation of dark-adapted cyanobacterial cells results largely from the recombination of S2QB charge pair [52]. Control cells exhibit a TL emission peak at approximately 25 8C and also a shoulder at approximately 32 8C for S2QB charge
recombination. It can be seen from the curves that the B band consists obviously of two components. In order to investigate how heat stress affects the S2QB charge recombination, the curve of control cells was decomposed by two major components: the B32 band and the B24 band with the peaks at 32 and 24 8C, respectively (see the insert of Fig. 2). In addition, we observed a new emission peak occurring at 43 8C when temperature was increased to 45 8C. Fig. 3 shows the changes in the intensity of the B32 band and the B24 band and their peak temperatures during heat stress. The intensity of the B32 and the B24 bands decreased significantly with increasing temperature but the decrease in the intensity of the B24 band was much greater than that in the B32 band. There were no significant changes either in the peak temperature of the B32 band or the B24 band. Since the temperature at which maximum luminescence occurs is a function of the free energy of stabilization of the chargeseparated state, no changes in the peak temperatures of S2QB recombination observed during heat stress indicate that heat stress has no effect on the stability of charge separated state of S2QB. We investigated further how heat stress affects the stability of charge separated state of S2QA. When electron transfer between QA and QB is blocked prior to excitation by DCMU, the TL arises from the recombination of the S2QA charge pair, called as the ‘‘Q band’’. Fig. 4 shows the changes in the glow curves of cells in the presence DCMU during heat stress. The peak temperature of Q band in control cells was observed at about 10 8C. With increasing temperature, the overall TL emission of the Q band also decreased significantly. The peak temperature of the Q band was largely unchanged. Since the temperature at which maximum luminescence occurs is a function of the free energy of stabilization of the charge-separated state, no significant changes in the peak temperature of the S2QA recombination during heat stress indicate that heat stress had no significant effects on the stability of charge separated state of S2QA. We investigated further how heat stress affects the oscillation of the B band intensity by analyzing the TL intensity as a function of flash number of short saturating flashes. When excited by a series
Fig. 1. Effects of high temperature on the maximum fluorescence yield (Fm), the minimal fluorescence yield (F0), and the maximum efficiency of PSII photochemistry (Fv/Fm) in S. platensis cells. The measurements were determined after heat treatments. The values of Fm were measured in the presence of 10 mM DCMU. The data represent the mean S.D. of five independent measurements.
Fig. 2. Effects of high temperature on TL glow curves in S. platensis cells. The cells were excited with a single flash in the absence of DCMU. The curves from the top to bottom were recorded from the control cells and the cells exposed to 35, 40, 45, 47.5 and 50 8C, respectively. The insert shows the decomposition of the curve of the control cells.
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Fig. 3. Effects of high temperature on the intensity of the B24 and B32 bands (A) and the peak temperatures of the B24 and B32 bands (B) in S. platensis cells. The intensity of the B24 and B32 bands was calculated as areas below the emission spectra. For easy comparison, the intensity of the B24 and B32 bands were expressed as normalization to the values at 30 8C. Values represent mean S.D. of five independent measurements. Error bars are not shown if smaller than symbols.
of saturating flashes, the intensity of the B band exhibits a periodfour oscillation depending on the number of actinic flashes [27]. Fig. 5A shows that the control cells exhibited a typical period-four oscillation depending on the number of flashes used to excite PSII. The intensity of the B band was maximum after the second and sixth flashes and was minimal after the fourth flash. This is the same type of oscillation found for thermophilic cyanobacteria [52] and for whole leaf tissue [53]. The period-four oscillation indicates that the function of the oxygen-evolving complex in the control cells is normal as expected. We observed that heat stress had no significant effect on the period-four oscillation at 35 and 40 8C (data not shown). With increasing temperature, the oscillatory pattern was much more highly damped compared to the controls. When temperature was at 47.5 8C or higher, the period-four oscillation could not be observed. Since the B band consists of two major components, i.e. B32 and B24, we also investigated how heat stress affects the period-four oscillation of the B32 and B24 bands. We observed that the period-four oscillation of the B24 band and the B32 band disappeared totally already at 45 and 50 8C, respectively (Fig. 5B and C).
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Fig. 4. Effects of high temperature on TL glow curves (A) and the intensity of the Q band (B) in S. platensis cells. The cells were excited with a single flash in the presence of 10 mM DCMU. The curves in (A) from the top to bottom were recorded from the control cells and the cells exposed to 35, 40, 45, 47.5 and 50 8C, respectively. The intensity of the Q band was calculated as areas below the emission spectra. Values represent mean S.D. of five independent measurements. Error bars are not shown if smaller than symbols.
The period-four oscillation consists of a mixture of the donor side and the binary oscillations related to the acceptor side which represent the difference in the electron transfer rate between QA to QB and QA to QB. The decrease of the B band emission suggests that heat stress results in the destruction of the oxygen-evolving complex at the donor side. It has been shown that heat stress induces an appearance of the K step in the polyphasic Chl fluorescence transients, which is caused by the destruction of the manganese cluster [19]. We thus investigated how heat stress affects the function of the donor side by analyzing the polyphasic Chl fluorescence transient. It has been demonstrated that when the dark-adapted cells are illuminated suddenly with high intensity actinic light (3000 mmol m2 s1), the fluorescence transient shows a polyphasic rise, including steps O, J, I, and P [46]. Fig. 6A shows that the control cells exhibited a typical polyphasic rise of fluorescence transients, including steps O, J, I, P. There was
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Fig. 6. Effects of high temperature on Chl fluorescence polyphasic rise (A), the Ft F0 kinetics (in the range of 0–0.3 ms time interval) of fluorescence transients (B), and the total number of active reaction centers per absorption (RC/ABS) (C) in S. platensis cells. The curves in (A) and (B) from the top to bottom were recorded from the control cells and the cells exposed to 35, 40, 45, 47.5 and 50 8C, respectively. Values represent mean S.D. of five independent measurements.
Fig. 5. Flash-induced oscillation of thermoluminescence of the B band (A), the B24 band (B), and the B32 band (C) in S. platensis cells. The intensity was calculated as areas below the emission spectra. Values represent mean S.D. of eight independent measurements.
no appearance of the K step with increasing temperature. However, heat stress induced a loss of the JI-phase of the transients on going from 40 to 45 8C and then the differences became minor. Fig. 6B shows the effects of heat stress on the kinetics of the initial rise of the transients. Our results demonstrate that heat stress slowed down the initial rise of the fluorescence transients. We further investigated the effects of heat stress on the
total number of active reaction centers per absorption (RC/ABS) which is calculated from the data derived from the fluorescence transients [47–49]. Heat stress led to a decrease in RC/ABS with increasing temperature (Fig. 6C). We investigated further if the damping of the period-four oscillation was associated with the energetics of the redox components on the donor side. We determined the kinetics of the S2QA recombination reaction during heat stress by measuring Chl fluorescence decay after a saturating flash in the presence of DCMU (Fig. 7). In this case, the decay of fluorescence only corresponded to the reoxidation of QA by positive charges stored on the donor side, when the forward reaction being fully inhibited.
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Table 1 Kinetics parameters of variable fluorescence yield decay after a single turnover flash excitation in the absence of DCMU in Spirulina platensis cells during heat stress Temperature (8C)
Fast phase, t1 (a1)
Middle phase, t2 (a2)
Slow phase, t3 (a3)
30 40 45 47.5 50
123 7 ms (76%) 125 5 ms (74%) 135 7 ms (66%) 140 10 ms (57%) 160 8 ms (52%)
2.1 0.3 ms 2.6 0.4 ms 2.9 0.5 ms 3.3 0.6 ms 4.7 0.8 ms
4.1 0.2 s 3.6 0.3 s 3.3 0.1 s 2.9 0.1 s 2.6 0.2 s
(21%) (16%) (14%) (13%) (12%)
(3%) (10%) (20%) (30%) (35%)
Exponential analyses yielded triphasic kinetics with different half times (t1/2) and amplitudes (a). Mean values S.E. were calculated from 10 measurements.
Fig. 7. Effects of high temperature on Chl fluorescence decay kinetics after singleturnover flash in the presence of 10 mM DCMU in S. platensis cells. (A) The traces are the real data recoded directly from the fluorometer. (B) Traces were calculated relative to the total variable fluorescence measured at the each treatment temperature. The curves are average of 10 measurements.
donor side of PSII, this result indicates that kinetics of the S2QA recombination reaction is not affected in still active reaction centers. We then investigated if the damping of the period-four oscillation is associated with the activity of the acceptor side of PSII. We examined the electron transport reaction from QA to QB by measuring the relaxation kinetics of flash-induced fluorescence in the absence of DCMU. When the control cells were excited with a saturating single turnover flash, the primary acceptor, QA, was rapidly reduced. The decay curves exhibited complex kinetics which could be decomposed into three different decay components (Table 1) similar to other reports [8,54]. The estimated decay half-times for the fast and middle phases of QA re-oxidation in control cells were 123 ms and 2.1 ms, accounting for 76% and 21% of the decay, respectively. The slow phase, which are presumably associated with a back reaction of QA with the S2 states in centers in which QA is poorly connected to QB and the plastoquinone pool [55], exhibited a half time 4.1 s and accounted for the remaining 3% of the decay. Heat stress induced a slow down in the fast and middle components of the QA re-oxidation which was exhibited by an increase in their half times and by a decrease in their amplitudes. For the slow phase, there was a decrease in its half time and an increase in its amplitude with increasing temperature. This result suggests that heat stress caused a shift of the equilibrium towards QA. We further investigated the effects of heat stress on the D1 and PsbO proteins in Spirulina cells. Our results show that heat stress resulted in a significant decrease in the contents of D1 and PsbO proteins and the decrease of D1 protein was much greater than that of PsbO protein (Fig. 8). 4. Discussion
The decay monitors the rate of charge recombination within PSII between S2 and QA. Fig. 7A shows that the most pronounced change induced by heat stress was a significant increase in F0. Heat stress also induced a significant decrease in variable Chl fluorescence yield (Fv). Since only PSII centers contributing to variable Chl fluorescence show a decay of Chl fluorescence yield, all Chl fluorescence decay data refer to the PSII centers that are able to photoreduce QA at the temperature of the treatments. We do stress this point because some PSII centers damaged by high temperature cannot photoreduce QA but make a contribution to the decrease of Fv [6,7]. In order to analyze and compare the reoxidation kinetics of QA in the PSII centers still capable of photoreduction of QA in heattreated and control cells, all amplitudes were calculated relative to the total variable fluorescence measured at each treatment temperature [6,7]. We observed that there were no significant differences in the kinetics of Chl fluorescence decay between control and heat-stressed cells after fluorescence intensity was normalized to the total variable fluorescence (Fig. 7B). Because the kinetics of the S2QA recombination reaction may depend on the concentrations of P680+ and TyrZ+ following equilibration on the
In this study, we have performed a comprehensive study on PSII photochemistry in Spirulina cells by analyzing TL, the fast polyphasic fluorescence rise transient, the kinetics of fluorescence decay after a saturating flash, and the period-four oscillation of the oxygen-evolving complex, and the contents of the D1 and PsbO proteins. The analyses of TL demonstrate that heat stress resulted in a decrease in the intensity of the B band and the Q band. And also the oscillatory pattern of period-four was much more highly
Fig. 8. Effects of high temperature on the contents of D1 and PsbO proteins in Spirulina platensis cells.
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damped compared to the controls when temperature was at 47.5 8C (Figs. 2–4), indicating that heat stress led to the destruction of the oxygen-evolving complex. In addition, our results show that there was a loss of the JI-phase of the transients on going from 40 to 45 8C (Fig. 6A). As was shown by Schreiber and Neubauer [56], an inactivation of the PSII-donor side has a specific effect on the JIphase. The JI-rise parallels the reduction of the PQ-pool and for this quite a few electrons are needed. These electrons can only be produced if there is an active and intact manganese cluster. A loss of the JI-phase at higher temperatures suggests that heat stress resulted in the destruction of the oxygen-evolving complex which may be due to a loss of the manganese cluster activity. The loss of the manganese cluster activity might be due to the dissociation of the manganese-stabilizing PsbO protein from PSII as we observed that heat stress resulted in a decrease in the content of the PsbO protein in the thylakoid membranes (Fig. 8), which has also been reported by previous studies [5,6]. Thus, the destruction of the oxygen-evolving complex in heat-stressed cells resulted in damped period-four oscillation at the temperatures higher than 45 8C (Fig. 5). It has been reported that the loss of the oxygen-evolving function of PSII is associated with an appearance of the K step in the Chl fluorescence transient (OJIP) [19]. The K step is thought to be resulting from the only stable charge separation possible when the oxygen-evolving complex is completely destroyed [57]. However, we did not observe an obvious appearance of the K step after Spirulina cell were treated with different high temperatures (Fig. 6A). It has been reported that the disappearing of the K step was observed after a few hours light treatment following a heat pulse, which is suggested to a further damage or degradation of the PSII reaction centers [11]. In the present study, we observed that the decrease in the content of D1 protein was in parallel to the decrease in the content of PsbO protein and moreover, the decrease in the content of D1 protein was greater than that in the content of PsbO protein in heat-stressed cells (Fig. 8). Thus, no appearance of the K step in Spirulina cells under high temperature conditions in this study would suggest that the degradation of heat-damaged PSII reaction centers is faster in Spirulina than in higher plants. It is also possible that there may be a difference in an appearance of K step between higher plants and cyanobacteria under heat stress conditions. The results in this study show that heat stress had an effect on the very beginning of the fluorescence transient. There was a slow down in the rise of the very beginning of the fluorescence transient in heat-stressed cells (Fig. 6B). This result suggests that heat stress resulted in a decrease in the effective cross-section of PSII (sPSII) in Spirulina cells [58]. Such a decrease is associated with the structural changes within the phycobilisome core but not to a detachment of phycobilisomes from PSII as indicated in our previous study [40]. However, heat stress induces a dissociation of the peripheral antenna complex of PSII from its core complex in higher plants [20–25]. Our results indicate that there may be a difference in the antenna system in response to heat stress between higher plants and cyanobacteria. TL intensity after a single flash is a measure of the extent of recombination from the single state S2QB [59,60]. TL emission following single flash excitation of dark-adapted cyanobacterial cells results largely from the recombination of S2QB charge pair [52]. In cyanobacteria, the TL glow curves normally exhibit only an emission peak at around 24–35 8C after excited with a single flash [61–63]. Recently, it has been demonstrated that there is only an emission peak for the TL curves in Spirulina cells [64]. In the present study, we observed that Spirulina cells exhibit a TL emission peak at approximately 25 8C and also a shoulder at approximately 32 8C for S2QB charge recombination. It can be seen from the curves that
the B band consists obviously of two components (Fig. 2). In order to check if the two bands result from the double hits, we have examined the effects of flash duration on the TL curves. We observed that the two bands still existed even if duration of flash was shortened to 5 ms from 25 ms (data not shown). Therefore, our results suggest that the two bands were not resulting from the double hits due to long duration of flash. The B band in the Spirulina cells obviously consists of two major components, i.e. the B32 band and the B24 band with their peak temperatures at 32 and 24 8C, respectively (Figs. 2 and 3). This result suggests that there are two populations of the PSII reaction centers in Spirulina cells, one with a characteristic temperature at 24 8C and the other at 32 8C. Moreover, the decrease in the intensity of the B24 band was much greater than in the intensity of the B32 band during heat stress (Fig. 3), suggesting that the PSII reaction centers with characteristic temperature at 24 8C were much sensitive to heat stress than these with the characteristics temperature at 32 8C. These results indicate that the different subpopulations of PSII reaction centers showed different sensitivity to heat stress. Two populations of the PSII reaction centers in Spirulina cells may be explained by different D1 isoforms as observed in cyanobacteria. It has been shown that the critical D1 protein is encoded by the psbA gene present as a single chloroplastic gene in eukaryotic photoautrophs. On the other hand, the D1 protein in known cyanobacteria is encoded by a small gene family with one to five members (http://www.kazusa.or.jp/cyano/, http://genome.jgi-psf.org/mic_home.html). These psbA genes encode two distinct D1 protein isoforms [65–68], which are changed by different environmental conditions. When exposed to high light, low temperature, and UV, Synechococcus alters psbA expression to selectively exchange the D1:1 isoform encoded by psbAI with the D1:2 isoform encoded by psbAII and psbAIII [68–70]. Similar results have also been observed in Anabaena, Synechocystis, and Gloeobacter violaceus PCC 7421 [71,72]. The different sensitivity of two subpopulations of PSII reaction centers to heat stress observed in this study may be explained by the possible exchange between different D1 isoforms. We observed that there were no changes in their peak temperatures during heat stress but their intensity decreased significantly with increasing temperature (Fig. 3). These results indicate that heat stress induced a significant decrease in the PSII reaction centers which have charge recombination ability of S2 and QA. Our results indeed show that heat stress resulted in a decrease in the content of D1 protein, indicating that heat stress led to a decrease in the PSII reaction centers (Fig. 8). In addition, our results show that heat stress resulted in a decrease in RC/ABS (Fig. 6), suggesting further a decrease in the total number of active reaction center. It should be pointed out that the expression RC/ABS refers to QA reducing reaction centers of PSII [47–49]. The decrease in RC/ ABS in heat-stressed Spirulina cells also indicates a decrease in the total number of QA reducing reaction centers of PSII. Our results further indicate that heat stress caused a shift of the equilibrium towards QA, making the electrons tend to stay longer on QA (Table 1), suggesting that heat stress induced an increase in the QBnon-reducing reaction centers of PSII. The analyses of fluorescence yield decay kinetics after a single turnover flash excitation in the absence of DCMU demonstrated that there was a decrease in the half time and an increase in the amplitude for the slow phase in heat-stressed Spirulina cells (Table 1). Heat stress has also no significant effects on fluorescence decay after a saturating flash in the presence of DCMU (Fig. 7), suggesting that the S2QA is not changed by heat stress. Moreover, a shift of the equilibrium towards QA induced by heat stress suggests a slower electron transfer to empty (non-QB-reducing)
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centers which could be due to a lower binding constant of the QBsite for plastoquinone (PQ). Since the slow phase is presumably associated with a back reaction of QA with the S2 states in centers in which QA is poorly connected to QB and the plastoquinone pool [55], an increase in the amplitude of the slow phase also means that some QB secondary acceptors has been destroyed by heat stress.
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Effects of heat stress on PSII photochemistry in a cyanobacterium Spirulina platensis Binbin Zhao a,b, Jia Wang a, Hongmei Gong a, Xiaogang Wen a, Haiyun Ren b, Congming Lu a,* a b
Photosynthesis Research Center, Chinese Academy of Sciences, Beijing 100093, China College of Life Science, Beijing Normal University, Beijing 100875, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 September 2007 Received in revised form 2 June 2008 Accepted 16 June 2008 Available online 26 June 2008
The effects of heat stress (30–50 8C) on photosystem II (PSII) photochemistry in a cyanobacterium Spirulina platensis grown at 30 8C were studied by measuring chlorophyll fluorescence induction kinetics and thermoluminescence. Heat stress inhibited significantly the maximum efficiency of PSII photochemistry. An investigation of the kinetics of flash-induced fluorescence yield decay revealed that heat stress caused a shift of the equilibrium towards QA but showed no effect on the kinetics of flashinduced fluorescence yield decay in the presence of DCMU. An analysis of polyphasic rise of fluorescence transients indicated that heat stress induced a decrease in the total number of PSII active reaction centers. Thermoluminescence measurements demonstrated that the B band in Spirulina cells corresponding to the S2QB state consisted of two different components, the B32 and the B24 bands with the peak temperatures at 32 and 24 8C, respectively. The intensity of the B32 band and the B24 band decreased significantly with increasing temperature but the decrease of the intensity of the B24 band was much greater than that of the B32 band. There were no significant changes in the peak temperatures of the B24 and B32 bands. The intensity of the Q band corresponding to the recombination of the S2QA charge pair decreased significantly with increasing temperature. The period-four oscillation of the B band was significantly damped at temperatures higher than 45 8C. The contents of D1 and PsbO proteins decreased with increasing temperature. The results in this study suggest that heat stress shows no effects on the stability of the S2QA and S2QB states and that the different populations of the active PSII reaction centers show different sensitivity to heat stress in S. platensis cells. ß 2008 Elsevier Ireland Ltd. All rights reserved.
Keywords: Chlorophyll fluorescence Heat stress Photosystem II Spirulina platensis
1. Introduction High temperature stress affects significantly photosynthetic activity [1]. When leaves or algae are exposed to high temperature stress, their oxygen and CO2 assimilation capacities are significantly inhibited. One of the most-sensitive sites in the photosynthetic apparatus is thought to be photosystem II (PSII) and its activity decreases significantly by high temperature stress [1,2]. Numerous studies have shown that high temperature stress has various effects on PSII function. High temperature stress inhibits the function of the oxidizing side of PSII and oxygen evolution is
* Corresponding author at: Photosynthesis Research Center, Institute of Botany, Chinese Academy of Sciences, No. 20, Nanxincun, Beijing 100093, China. Tel.: +86 10 62836554; fax: +86 10 62595516. E-mail address: [email protected] (C. Lu). Abbreviations: Chl, chlorophyll; DCMU, 3-(30 40 -dichlorophenyl)-1,1-dimethylurea; QA, primary acceptor plastoquinone; QB, secondary acceptor plastoquinone; PSI, photosystem I; PSII, photosystem II; TL, thermoluminescence; S-state, oxidation states of the Mn cluster. 0168-9452/$ – see front matter ß 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2008.06.003
significantly decreased [3]. A loss of the oxygen evolving complex activity is thought to be due to the release of manganese atoms and the dissociation of the manganese-stabilizing 33 kDa protein from the PSII reaction center complex [3,4–6]. High temperature stress also results in an inactivation of PSII reaction centers [7–12]. In addition, high temperature results in a shift of the redox equilibrium between the primary acceptor plastoquinone (QA) and the secondary acceptor plastoquinone (QB) [13–19]. Furthermore, high temperature stress induces a dissociation of the peripheral antenna complex of PSII from its core complex [20–25]. Thermoluminescence (TL) is an outburst of light emission occurring at characteristic temperatures from a preilluminated photosynthetic sample. TL is a powerful tool to investigate the properties of the redox components in the photosynthetic apparatus [26,27]. It has thus been extensively used to probe the function of the donor side and the acceptor side of PSII in various mutant cells and under different environmental stress conditions such as photoinhibition, salt stress, and low temperature [26–30]. Since it has been generally considered that high temperature stress induces major structural and functional
B. Zhao et al. / Plant Science 175 (2008) 556–564
changes of PSII [13,22], TL can be used to estimate more precisely the function of PSII [31]. Spirulina platensis, a filamentous cyanobacterium, isolated from a wide range of habitats has long been cultured for production of health food because of its high content of protein and other nutritional elements, such as phycocyanin, carotenoids and glinolenic acid [32,33]. In cultures grown outdoors in open ponds under arid and semiarid climates, Spirulina cells are subjected to various environmental stresses, such as salinity stress, photoinhibition, and high temperature stress [34]. Many studies have investigated the effects of salinity stress, osmotic stress, and photoinhibition on photosynthesis and PSII photochemistry in S. platensis [35–39]. Recently, we have studied the effects of high temperature on excitation energy transfer from phycobilisomes to photosystem I (PSI) and PSII in S. platensis and found out that high temperature stress induced an inhibition of excitation energy transfer from phycobilisomes to PSII but not to photosystem I (PSI) [40]. However, how high temperature stress affects PSII photochemistry in Spirulina cells remains to be elucidated further. Understandably, a better understanding of heat stress on PSII photochemistry may help optimize the productivity of the algal cultures grown outdoors. In the present study, we have investigated in more detail the responses of the donor and acceptor sides of PSII in a cyanobacterium S. platensis to different high temperatures. 2. Materials and methods 2.1. Alga and growth conditions The cyanobacterium, S. platensis M2, was grown at 30 8C in Zarouk’s medium supplemented with 0.2 M sodium bicarbonate [41]. Illumination of 80 mmol photons m2 s1 was provided by cool daylight tubes (TLD 30W/865, Philips). 2.2. High temperature treatment Exponentially growing cells were harvested and diluted to 10 mg chlorophyll (Chl) ml1 in fresh medium. The cells were placed in the dark for at least 30 min at 30 8C before the heat treatments. Similar to the heat treatments used in algae in the literature [42,43], the glass cuvettes containing the cells were dipped into a water bath and incubated for 5 min at different temperatures. The measurements of Chl fluorescence and thermoluminescence were taken immediately after heat stress treatments. All treatments and measurements were performed in the dark. 2.3. Maximum efficiency of PSII photochemistry The maximum efficiency of PSII photochemistry was determined as Fv/Fm with a portable fluorometer (PAM-2000, Walz, Germany), where Fv = (Fm F0) and Fm and F0 are the maximum and minimal fluorescence yield, respectively, of a dark-adapted suspension. F0 was measured by using modulated measuring light that was of sufficiently low intensity (<0.1 mmol m2 s1) not to induce any significant variable fluorescence and Fm(dark) was determined by applying 0.8 s saturating pulse at 8000 mmol m2 s1 in darkadapted cells. Fm(DCMU) was determined in the presence of 10 mM DCMU (3-3,4-dichlorophenyl-1,1-dimethyl urea) as described previously [44].
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rometer FL200/PS, consisting of Thermoregulator TR2000 (Photon Systems Instruments, Brno, Czech Republic). After 30 min dark adaptation at 30 8C, the cells were cooled to 0 8C and illuminated with one or multiple number of single-turnover flashes with duration of 25 ms. Then the cells were warmed up to 60 8C at a heating rate of 1 8C s1 and the TL light emission was measured during the heating. The volume of the samples for each measurement was 0.4 ml and the Chl concentration of the sample was 10 mg Chl ml1. To detect period-four oscillation of the Bband, cells were illuminated with a series of single-turnover flashes. For S2QA recombination studies, cells were measured in the presence of 3-(30 ,40 -dichlorophenyl)-1,1-dimethylurea (DCMU, 10 mM) before the flash illumination. The nomenclature of Vass and Govindjee [26] was used for characterization of the flashinduced TL glow peaks. 2.5. Decay of Chl fluorescence yield after a single turnover flash The decay of Chl fluorescence yield after a single turnover flash was measured with a double-modulation fluorescence fluorometer (model FL-200, Photon Systems Instruments, Brno, Czech Republic) [45]. The square-shaped, single-turnover, saturated flash was generated by one set of 48 orange LEDs (630 nm) and one set of red LEDs (653 nm) that focus on the opposite sides of the sample cuvette. The duration of the single-turnover flash was 25 ms and its intensity was ca. 200,000 mmol photons m2 s1. The measuring flashes were provided by two sets of 7 orange LEDs with the duration of 2.5 ms. Each measuring flash excited less than 1% of the reaction center of PSII. 2.6. Polyphasic rise of Chl fluorescence transients Chl fluorescence transients were measured by a Plant Efficiency Analyzer (PEA, Hansatech Instruments Ltd., King’s Lynn, Norfolk PE32 1JL, England) by the method described by Strasser et al. [46]. Illumination was provided by an array of 6 high intensity, lightemitting-diodes (LED, a peak at 650 nm), which were focused on the sample surface to provide homogeneous illumination over the exposed area of a sample with a 4 mm diameter. The fluorescence signals were received by a detector, a high performance pin photodiode associated with an amplifier circuit, after passing through a long pass filter. The detector, responding maximally to the longer wavelength fluorescence signal, blocked the reflected shorter wavelength LED light used as the source of illumination. All the fluorescence transients were recorded within a time scan from 10 ms to 1 s with a data acquisition rate of 105 readings per second for the first 2 ms and of 103 per second after 2 ms. According to JIPtest [47–49], Chl transients were analyzed by utilizing the original data from the polyphasic fluorescence transients. The parameter RC/ABS (the total number of active reaction center per absorption) is used for the quantification of PSII behavior referring to time zero. 2.7. SDS-PAGE and immunological analyses Samples were solubilized in the presence of 6 M urea and separated by SDS-PAGE [50] using 15% (w/v) acrylamide gels with 6 M urea. For immunoblotting, polypeptides were electrophoretically transferred to PVDF membranes (Millipore, Saint-Quentin, France) and proteins were detected with antibodies raised against D1 and PsbO proteins.
2.4. Thermoluminescence
3. Results
TL measurements of whole cells were performed with the thermoluminescence extension of the Double-Modulated Fluo-
Many studies have shown that heat stress inhibits the activity of PSII [2–6,23,25]. In this study, the activity of PSII was expressed
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by the maximum efficiency of PSII photochemistry (Fv/Fm). It should be noted that under light conditions or not completely dark adapted conditions, even strong light may not close all reaction centers of PSII. In addition, it has been demonstrated that PSII reaction centers were knocked out under heat stress conditions and a saturating pulse is no longer able to close all PSII reaction centers [11,19]. Therefore, there would be a quenching of Fm under these conditions. The measured Fm would be still lower than the Fm with all reaction centers closed. Therefore, more electrons may have to be provided by cycling of the S states to fill the PQ pool and to reach the maximum fluorescence yield while one electron is sufficient to reduce QA in the presence of DCMU. Probably, the Fm measured in the absence of DCMU is lower than the Fm measured with DCMU, which decreases more slowly with temperature [19]. To obtain the true Fm, Fm in this study was measured in the presence 10 mM DCMU. Fig. 1 shows that Fv/Fm decreased significantly with increasing temperature. We also investigated the effects of heat stress on the effects of Fm and F0. Fm decreased but F0 increased significantly with increasing temperature. TL is the emission of light during re-warming of photosynthetic samples exposed to actinic illumination prior to freezing (for reviews, see Refs. [26,27]). TL is normally due to thermally stimulated recombination of pairs of opposite charges generated by light-induced charge separation in the PSII complex. TL emission peaked at approximately 25 8C (B-band) results largely from the recombination of the S2QB charge pair. At around 10 8C, addition of DCMU to the sample, which blocks oxidation of QA by QB, induces a major emission component (Q-band) from S2QA recombination. The peak temperature of a TL component is an indicator of the energetic stability of a separated charge pair. As a general rule, the higher the peak temperature is, the greater the stabilization is. Thus, these measurements can provide information on the depth of the activation energy wells involved in the stabilization of the S2QA and S2QB charge separated states [51]. Fig. 2 shows the TL glow curves obtained from Spirulina cells excited with a single flash to detect the B-band. TL emission following single flash excitation of dark-adapted cyanobacterial cells results largely from the recombination of S2QB charge pair [52]. Control cells exhibit a TL emission peak at approximately 25 8C and also a shoulder at approximately 32 8C for S2QB charge
recombination. It can be seen from the curves that the B band consists obviously of two components. In order to investigate how heat stress affects the S2QB charge recombination, the curve of control cells was decomposed by two major components: the B32 band and the B24 band with the peaks at 32 and 24 8C, respectively (see the insert of Fig. 2). In addition, we observed a new emission peak occurring at 43 8C when temperature was increased to 45 8C. Fig. 3 shows the changes in the intensity of the B32 band and the B24 band and their peak temperatures during heat stress. The intensity of the B32 and the B24 bands decreased significantly with increasing temperature but the decrease in the intensity of the B24 band was much greater than that in the B32 band. There were no significant changes either in the peak temperature of the B32 band or the B24 band. Since the temperature at which maximum luminescence occurs is a function of the free energy of stabilization of the chargeseparated state, no changes in the peak temperatures of S2QB recombination observed during heat stress indicate that heat stress has no effect on the stability of charge separated state of S2QB. We investigated further how heat stress affects the stability of charge separated state of S2QA. When electron transfer between QA and QB is blocked prior to excitation by DCMU, the TL arises from the recombination of the S2QA charge pair, called as the ‘‘Q band’’. Fig. 4 shows the changes in the glow curves of cells in the presence DCMU during heat stress. The peak temperature of Q band in control cells was observed at about 10 8C. With increasing temperature, the overall TL emission of the Q band also decreased significantly. The peak temperature of the Q band was largely unchanged. Since the temperature at which maximum luminescence occurs is a function of the free energy of stabilization of the charge-separated state, no significant changes in the peak temperature of the S2QA recombination during heat stress indicate that heat stress had no significant effects on the stability of charge separated state of S2QA. We investigated further how heat stress affects the oscillation of the B band intensity by analyzing the TL intensity as a function of flash number of short saturating flashes. When excited by a series
Fig. 1. Effects of high temperature on the maximum fluorescence yield (Fm), the minimal fluorescence yield (F0), and the maximum efficiency of PSII photochemistry (Fv/Fm) in S. platensis cells. The measurements were determined after heat treatments. The values of Fm were measured in the presence of 10 mM DCMU. The data represent the mean S.D. of five independent measurements.
Fig. 2. Effects of high temperature on TL glow curves in S. platensis cells. The cells were excited with a single flash in the absence of DCMU. The curves from the top to bottom were recorded from the control cells and the cells exposed to 35, 40, 45, 47.5 and 50 8C, respectively. The insert shows the decomposition of the curve of the control cells.
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Fig. 3. Effects of high temperature on the intensity of the B24 and B32 bands (A) and the peak temperatures of the B24 and B32 bands (B) in S. platensis cells. The intensity of the B24 and B32 bands was calculated as areas below the emission spectra. For easy comparison, the intensity of the B24 and B32 bands were expressed as normalization to the values at 30 8C. Values represent mean S.D. of five independent measurements. Error bars are not shown if smaller than symbols.
of saturating flashes, the intensity of the B band exhibits a periodfour oscillation depending on the number of actinic flashes [27]. Fig. 5A shows that the control cells exhibited a typical period-four oscillation depending on the number of flashes used to excite PSII. The intensity of the B band was maximum after the second and sixth flashes and was minimal after the fourth flash. This is the same type of oscillation found for thermophilic cyanobacteria [52] and for whole leaf tissue [53]. The period-four oscillation indicates that the function of the oxygen-evolving complex in the control cells is normal as expected. We observed that heat stress had no significant effect on the period-four oscillation at 35 and 40 8C (data not shown). With increasing temperature, the oscillatory pattern was much more highly damped compared to the controls. When temperature was at 47.5 8C or higher, the period-four oscillation could not be observed. Since the B band consists of two major components, i.e. B32 and B24, we also investigated how heat stress affects the period-four oscillation of the B32 and B24 bands. We observed that the period-four oscillation of the B24 band and the B32 band disappeared totally already at 45 and 50 8C, respectively (Fig. 5B and C).
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Fig. 4. Effects of high temperature on TL glow curves (A) and the intensity of the Q band (B) in S. platensis cells. The cells were excited with a single flash in the presence of 10 mM DCMU. The curves in (A) from the top to bottom were recorded from the control cells and the cells exposed to 35, 40, 45, 47.5 and 50 8C, respectively. The intensity of the Q band was calculated as areas below the emission spectra. Values represent mean S.D. of five independent measurements. Error bars are not shown if smaller than symbols.
The period-four oscillation consists of a mixture of the donor side and the binary oscillations related to the acceptor side which represent the difference in the electron transfer rate between QA to QB and QA to QB. The decrease of the B band emission suggests that heat stress results in the destruction of the oxygen-evolving complex at the donor side. It has been shown that heat stress induces an appearance of the K step in the polyphasic Chl fluorescence transients, which is caused by the destruction of the manganese cluster [19]. We thus investigated how heat stress affects the function of the donor side by analyzing the polyphasic Chl fluorescence transient. It has been demonstrated that when the dark-adapted cells are illuminated suddenly with high intensity actinic light (3000 mmol m2 s1), the fluorescence transient shows a polyphasic rise, including steps O, J, I, and P [46]. Fig. 6A shows that the control cells exhibited a typical polyphasic rise of fluorescence transients, including steps O, J, I, P. There was
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Fig. 6. Effects of high temperature on Chl fluorescence polyphasic rise (A), the Ft F0 kinetics (in the range of 0–0.3 ms time interval) of fluorescence transients (B), and the total number of active reaction centers per absorption (RC/ABS) (C) in S. platensis cells. The curves in (A) and (B) from the top to bottom were recorded from the control cells and the cells exposed to 35, 40, 45, 47.5 and 50 8C, respectively. Values represent mean S.D. of five independent measurements.
Fig. 5. Flash-induced oscillation of thermoluminescence of the B band (A), the B24 band (B), and the B32 band (C) in S. platensis cells. The intensity was calculated as areas below the emission spectra. Values represent mean S.D. of eight independent measurements.
no appearance of the K step with increasing temperature. However, heat stress induced a loss of the JI-phase of the transients on going from 40 to 45 8C and then the differences became minor. Fig. 6B shows the effects of heat stress on the kinetics of the initial rise of the transients. Our results demonstrate that heat stress slowed down the initial rise of the fluorescence transients. We further investigated the effects of heat stress on the
total number of active reaction centers per absorption (RC/ABS) which is calculated from the data derived from the fluorescence transients [47–49]. Heat stress led to a decrease in RC/ABS with increasing temperature (Fig. 6C). We investigated further if the damping of the period-four oscillation was associated with the energetics of the redox components on the donor side. We determined the kinetics of the S2QA recombination reaction during heat stress by measuring Chl fluorescence decay after a saturating flash in the presence of DCMU (Fig. 7). In this case, the decay of fluorescence only corresponded to the reoxidation of QA by positive charges stored on the donor side, when the forward reaction being fully inhibited.
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Table 1 Kinetics parameters of variable fluorescence yield decay after a single turnover flash excitation in the absence of DCMU in Spirulina platensis cells during heat stress Temperature (8C)
Fast phase, t1 (a1)
Middle phase, t2 (a2)
Slow phase, t3 (a3)
30 40 45 47.5 50
123 7 ms (76%) 125 5 ms (74%) 135 7 ms (66%) 140 10 ms (57%) 160 8 ms (52%)
2.1 0.3 ms 2.6 0.4 ms 2.9 0.5 ms 3.3 0.6 ms 4.7 0.8 ms
4.1 0.2 s 3.6 0.3 s 3.3 0.1 s 2.9 0.1 s 2.6 0.2 s
(21%) (16%) (14%) (13%) (12%)
(3%) (10%) (20%) (30%) (35%)
Exponential analyses yielded triphasic kinetics with different half times (t1/2) and amplitudes (a). Mean values S.E. were calculated from 10 measurements.
Fig. 7. Effects of high temperature on Chl fluorescence decay kinetics after singleturnover flash in the presence of 10 mM DCMU in S. platensis cells. (A) The traces are the real data recoded directly from the fluorometer. (B) Traces were calculated relative to the total variable fluorescence measured at the each treatment temperature. The curves are average of 10 measurements.
donor side of PSII, this result indicates that kinetics of the S2QA recombination reaction is not affected in still active reaction centers. We then investigated if the damping of the period-four oscillation is associated with the activity of the acceptor side of PSII. We examined the electron transport reaction from QA to QB by measuring the relaxation kinetics of flash-induced fluorescence in the absence of DCMU. When the control cells were excited with a saturating single turnover flash, the primary acceptor, QA, was rapidly reduced. The decay curves exhibited complex kinetics which could be decomposed into three different decay components (Table 1) similar to other reports [8,54]. The estimated decay half-times for the fast and middle phases of QA re-oxidation in control cells were 123 ms and 2.1 ms, accounting for 76% and 21% of the decay, respectively. The slow phase, which are presumably associated with a back reaction of QA with the S2 states in centers in which QA is poorly connected to QB and the plastoquinone pool [55], exhibited a half time 4.1 s and accounted for the remaining 3% of the decay. Heat stress induced a slow down in the fast and middle components of the QA re-oxidation which was exhibited by an increase in their half times and by a decrease in their amplitudes. For the slow phase, there was a decrease in its half time and an increase in its amplitude with increasing temperature. This result suggests that heat stress caused a shift of the equilibrium towards QA. We further investigated the effects of heat stress on the D1 and PsbO proteins in Spirulina cells. Our results show that heat stress resulted in a significant decrease in the contents of D1 and PsbO proteins and the decrease of D1 protein was much greater than that of PsbO protein (Fig. 8). 4. Discussion
The decay monitors the rate of charge recombination within PSII between S2 and QA. Fig. 7A shows that the most pronounced change induced by heat stress was a significant increase in F0. Heat stress also induced a significant decrease in variable Chl fluorescence yield (Fv). Since only PSII centers contributing to variable Chl fluorescence show a decay of Chl fluorescence yield, all Chl fluorescence decay data refer to the PSII centers that are able to photoreduce QA at the temperature of the treatments. We do stress this point because some PSII centers damaged by high temperature cannot photoreduce QA but make a contribution to the decrease of Fv [6,7]. In order to analyze and compare the reoxidation kinetics of QA in the PSII centers still capable of photoreduction of QA in heattreated and control cells, all amplitudes were calculated relative to the total variable fluorescence measured at each treatment temperature [6,7]. We observed that there were no significant differences in the kinetics of Chl fluorescence decay between control and heat-stressed cells after fluorescence intensity was normalized to the total variable fluorescence (Fig. 7B). Because the kinetics of the S2QA recombination reaction may depend on the concentrations of P680+ and TyrZ+ following equilibration on the
In this study, we have performed a comprehensive study on PSII photochemistry in Spirulina cells by analyzing TL, the fast polyphasic fluorescence rise transient, the kinetics of fluorescence decay after a saturating flash, and the period-four oscillation of the oxygen-evolving complex, and the contents of the D1 and PsbO proteins. The analyses of TL demonstrate that heat stress resulted in a decrease in the intensity of the B band and the Q band. And also the oscillatory pattern of period-four was much more highly
Fig. 8. Effects of high temperature on the contents of D1 and PsbO proteins in Spirulina platensis cells.
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damped compared to the controls when temperature was at 47.5 8C (Figs. 2–4), indicating that heat stress led to the destruction of the oxygen-evolving complex. In addition, our results show that there was a loss of the JI-phase of the transients on going from 40 to 45 8C (Fig. 6A). As was shown by Schreiber and Neubauer [56], an inactivation of the PSII-donor side has a specific effect on the JIphase. The JI-rise parallels the reduction of the PQ-pool and for this quite a few electrons are needed. These electrons can only be produced if there is an active and intact manganese cluster. A loss of the JI-phase at higher temperatures suggests that heat stress resulted in the destruction of the oxygen-evolving complex which may be due to a loss of the manganese cluster activity. The loss of the manganese cluster activity might be due to the dissociation of the manganese-stabilizing PsbO protein from PSII as we observed that heat stress resulted in a decrease in the content of the PsbO protein in the thylakoid membranes (Fig. 8), which has also been reported by previous studies [5,6]. Thus, the destruction of the oxygen-evolving complex in heat-stressed cells resulted in damped period-four oscillation at the temperatures higher than 45 8C (Fig. 5). It has been reported that the loss of the oxygen-evolving function of PSII is associated with an appearance of the K step in the Chl fluorescence transient (OJIP) [19]. The K step is thought to be resulting from the only stable charge separation possible when the oxygen-evolving complex is completely destroyed [57]. However, we did not observe an obvious appearance of the K step after Spirulina cell were treated with different high temperatures (Fig. 6A). It has been reported that the disappearing of the K step was observed after a few hours light treatment following a heat pulse, which is suggested to a further damage or degradation of the PSII reaction centers [11]. In the present study, we observed that the decrease in the content of D1 protein was in parallel to the decrease in the content of PsbO protein and moreover, the decrease in the content of D1 protein was greater than that in the content of PsbO protein in heat-stressed cells (Fig. 8). Thus, no appearance of the K step in Spirulina cells under high temperature conditions in this study would suggest that the degradation of heat-damaged PSII reaction centers is faster in Spirulina than in higher plants. It is also possible that there may be a difference in an appearance of K step between higher plants and cyanobacteria under heat stress conditions. The results in this study show that heat stress had an effect on the very beginning of the fluorescence transient. There was a slow down in the rise of the very beginning of the fluorescence transient in heat-stressed cells (Fig. 6B). This result suggests that heat stress resulted in a decrease in the effective cross-section of PSII (sPSII) in Spirulina cells [58]. Such a decrease is associated with the structural changes within the phycobilisome core but not to a detachment of phycobilisomes from PSII as indicated in our previous study [40]. However, heat stress induces a dissociation of the peripheral antenna complex of PSII from its core complex in higher plants [20–25]. Our results indicate that there may be a difference in the antenna system in response to heat stress between higher plants and cyanobacteria. TL intensity after a single flash is a measure of the extent of recombination from the single state S2QB [59,60]. TL emission following single flash excitation of dark-adapted cyanobacterial cells results largely from the recombination of S2QB charge pair [52]. In cyanobacteria, the TL glow curves normally exhibit only an emission peak at around 24–35 8C after excited with a single flash [61–63]. Recently, it has been demonstrated that there is only an emission peak for the TL curves in Spirulina cells [64]. In the present study, we observed that Spirulina cells exhibit a TL emission peak at approximately 25 8C and also a shoulder at approximately 32 8C for S2QB charge recombination. It can be seen from the curves that
the B band consists obviously of two components (Fig. 2). In order to check if the two bands result from the double hits, we have examined the effects of flash duration on the TL curves. We observed that the two bands still existed even if duration of flash was shortened to 5 ms from 25 ms (data not shown). Therefore, our results suggest that the two bands were not resulting from the double hits due to long duration of flash. The B band in the Spirulina cells obviously consists of two major components, i.e. the B32 band and the B24 band with their peak temperatures at 32 and 24 8C, respectively (Figs. 2 and 3). This result suggests that there are two populations of the PSII reaction centers in Spirulina cells, one with a characteristic temperature at 24 8C and the other at 32 8C. Moreover, the decrease in the intensity of the B24 band was much greater than in the intensity of the B32 band during heat stress (Fig. 3), suggesting that the PSII reaction centers with characteristic temperature at 24 8C were much sensitive to heat stress than these with the characteristics temperature at 32 8C. These results indicate that the different subpopulations of PSII reaction centers showed different sensitivity to heat stress. Two populations of the PSII reaction centers in Spirulina cells may be explained by different D1 isoforms as observed in cyanobacteria. It has been shown that the critical D1 protein is encoded by the psbA gene present as a single chloroplastic gene in eukaryotic photoautrophs. On the other hand, the D1 protein in known cyanobacteria is encoded by a small gene family with one to five members (http://www.kazusa.or.jp/cyano/, http://genome.jgi-psf.org/mic_home.html). These psbA genes encode two distinct D1 protein isoforms [65–68], which are changed by different environmental conditions. When exposed to high light, low temperature, and UV, Synechococcus alters psbA expression to selectively exchange the D1:1 isoform encoded by psbAI with the D1:2 isoform encoded by psbAII and psbAIII [68–70]. Similar results have also been observed in Anabaena, Synechocystis, and Gloeobacter violaceus PCC 7421 [71,72]. The different sensitivity of two subpopulations of PSII reaction centers to heat stress observed in this study may be explained by the possible exchange between different D1 isoforms. We observed that there were no changes in their peak temperatures during heat stress but their intensity decreased significantly with increasing temperature (Fig. 3). These results indicate that heat stress induced a significant decrease in the PSII reaction centers which have charge recombination ability of S2 and QA. Our results indeed show that heat stress resulted in a decrease in the content of D1 protein, indicating that heat stress led to a decrease in the PSII reaction centers (Fig. 8). In addition, our results show that heat stress resulted in a decrease in RC/ABS (Fig. 6), suggesting further a decrease in the total number of active reaction center. It should be pointed out that the expression RC/ABS refers to QA reducing reaction centers of PSII [47–49]. The decrease in RC/ ABS in heat-stressed Spirulina cells also indicates a decrease in the total number of QA reducing reaction centers of PSII. Our results further indicate that heat stress caused a shift of the equilibrium towards QA, making the electrons tend to stay longer on QA (Table 1), suggesting that heat stress induced an increase in the QBnon-reducing reaction centers of PSII. The analyses of fluorescence yield decay kinetics after a single turnover flash excitation in the absence of DCMU demonstrated that there was a decrease in the half time and an increase in the amplitude for the slow phase in heat-stressed Spirulina cells (Table 1). Heat stress has also no significant effects on fluorescence decay after a saturating flash in the presence of DCMU (Fig. 7), suggesting that the S2QA is not changed by heat stress. Moreover, a shift of the equilibrium towards QA induced by heat stress suggests a slower electron transfer to empty (non-QB-reducing)
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centers which could be due to a lower binding constant of the QBsite for plastoquinone (PQ). Since the slow phase is presumably associated with a back reaction of QA with the S2 states in centers in which QA is poorly connected to QB and the plastoquinone pool [55], an increase in the amplitude of the slow phase also means that some QB secondary acceptors has been destroyed by heat stress.
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