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Excitation energy transfer between photosystems in the cyanobacterium Synechocystis 6803
ARTICLE IN PRESS
Journal of Luminescence 128 (2008) 546–548 www.elsevier.com/locate/jlumin
Excitation energy transfer between photosystems in the cyanobacterium Synechocystis 6803 Weimin Ma, Liping Chen, Lanzhen Wei, Quanxi Wang College of Life and Environment Sciences, Shanghai Normal University, Guilin Road 100, Shanghai 200234, PR China Received 27 July 2007; received in revised form 5 October 2007; accepted 11 October 2007 Available online 22 October 2007
Abstract In cyanobacteria, the light-harvesting complex phycobilisomes (PBS) can transfer the harvested energy to photosystem II (PSII) and photosystem I (PSI) and from PSII to PSI. The efficiency of energy transfer from PBS to PSII or PSI, as probed by the ratio of the fluorescence yield of PBS to that of PSII or PSI, was similar in static and aerated cultures of a cyanobacterium Synechocystis sp. strain PCC 6803. In contrast, the fluorescence yield for PSII was notably high while that for PSI was remarkably low in static cultures when compared to that in aerated cultures. Additionally, the level of the efficiency of excitation energy capture by open PSII reaction center was markedly decreased in static cultures in comparison to that in aerated cultures. Thus, the energy transfer from PSII to PSI, but not from PBS to PSII and PSI, was inhibited in static cultures and was most likely caused by the reduction of PSII activity. r 2007 Elsevier B.V. All rights reserved. Keywords: Excitation energy transfer; Phycobilisome; Photosystem II; Photosystem I
1. Introduction In cyanobacteria, phycobilisomes (PBS), photosystem II (PSII), and photosystem I (PSI) closely associate with the photosynthetic yield. PBS plays a light-harvesting role and is linked to the thylakoid membrane via a colored polypeptide (LCM), which also intermediates the energy transfer from PBS to PSII [1]. In addition, a direct energy transfer from PBS to PSI [2–5] and from PSII to PSI has been proposed [6–10]. The inhibition of one of the specific three energy transfer pathways above would result in a reduction in the photosynthetic yield in cyanobacteria. It is well known that the photosynthetic yield of cyanobacterial cells is markedly low in static cultures when compared to that in aerated cultures. Possibly, one or more pathways of energy transfer among PBS, PSII, and PSI are inhibited in static cultures. However, little is known regarding whether and which pathway(s) is (are) inhibited; similarly, the reason for this inhibition remains unknown. Corresponding author. Tel.: +86 21 64321263; fax: +86 21 64322931. Also to be corresponded to.
E-mail addresses: [email protected] (W. Ma), [email protected] (Q. Wang). 0022-2313/$ - see front matter r 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jlumin.2007.10.007
Since PBS, PSII, and PSI are all highly fluorescent pigment–protein complexes, these questions should be solved by measuring their fluorescence changes in static and aerated cultures. In this study, to reveal the inhibition site of the energy transfer among PBS, PSII, and PSI under static culture conditions of a cyanobacterium Synechocystis sp. strain PCC 6803 (hereafter Synechocystis 6803) and the reason for this inhibition, both low-temperature (77 K) emission spectra and chlorophyll fluorescence measurements were conducted in static and aerated cultures of this cyanobacterium.
2. Materials and methods 2.1. Culture conditions Synechocystis 6803, a cyanobacterium was cultured at 30 1C in BG-11 medium [11] buffered with Tris–HCl (5 mM, pH 8.0) with bubble aeration (i.e., aerated culture) or shaken once by hand every 6 h (i.e., static culture) under continuous illumination by fluorescent lamps (40 mE m2 s1).
ARTICLE IN PRESS W. Ma et al. / Journal of Luminescence 128 (2008) 546–548
2.2. Spectral measurement at 77 K Three-day-old cultures were harvested by centrifugation (5000 g for 5 min at 25 1C), washed, and then resuspended in fresh BG-11 medium buffered with Tris–HCl (5 mM, pH 8.0) at a chlorophyll a concentration of 5 mg mL1; the same harvesting procedure was used in all the experiments. The samples for static and aerated cultures were rapidly frozen in liquid nitrogen. Fluorescence emission spectra were obtained at 77 K using an F-4500 spectrofluorimeter (Hitachi, Japan). The excitation wavelengths were 435 and 580 nm, and the fluorescence spectrum was obtained by averaging six spectra obtained for each sample in different tubes. The fluorescence data were analyzed with a Gaussian deconvolution program and the fluorescence areas for all subbands were calculated. 2.3. Chlorophyll fluorescence measurement The efficiency of excitation energy capture by open PSII reaction center, Fv/Fm, was measured at room temperature (i.e., 25 1C) using a Dual-PAM-100 (Walz, Effeltrich, Germany). The modulated nonactinic fluorescencemeasuring light (FML) was switched on to obtain the initial fluorescence (Fo). Maximal fluorescence (Fm) was measured using illumination with a 0.6 s pulse of red saturating light (10,000 mE m2 s1). Fv/Fm was calculated as (FmFo)/Fm.
547
580 nm exhibited changes similar to the abovementioned emission peaks and were compared for static and aerated cultures. In order to quantitatively compare the efficiency of energy transfer among PBS, PSII, and PSI under static and aerated culture conditions, those emissions were fitted to their corresponding components of the photosynthetic apparatus by applying a Gaussian deconvolution program, and the fluorescence yield of every emission subband was calculated as a respective area. A typical Gaussian deconvolution for the 77 K fluorescence emission spectra excited at 580 nm in static cultures is shown in Fig. 2. The ratio of the fluorescence yield of the 725-nm emission (FPSI) in one spectrum excited at 580 nm to that in another spectrum excited at 435 nm, i.e., FPSI (580)/FPSI (435), has been considered to be an indicator of the efficiency of energy transfer from PBS to PSI [16]. Further, the ratio of the fluorescence yields of 647- and 664-nm emissions (FPBS) to those of 685- and 695-nm emission peaks (FPSII), i.e., FPBS/FPSII, demonstrates the efficiency of energy transfer from PBS to PSII. No significant changes in these two ratios (Table 1) indicate a similar efficiency for energy transfer from PBS to PSII and PSI in static and aerated cultures.
3. Results and discussion 3.1. No significant changes in energy transfer from PBS to PSII and PSI in static and aerated cultures It has been previously demonstrated that the 647- and 664-nm emission peaks originate from PBS; the 685- and 695-nm emissions originate from PSII; and the 725-nm band is most effectively produced by PSI [12–15]. As shown in Fig. 1, the fluorescence emission spectra at 435 and
Fig. 2. An example of the Gaussian deconvolution for the 77 K fluorescence spectrum excited at 580 nm in static cultures of Synechocystis 6803. The black line is the full 77 K emission spectrum, and the gray lines are the Gaussian deconvoluted subbands from the full spectrum.
Fig. 1. The 77 K fluorescence emission spectra in static (black line) and aerated (gray line) cultures of Synechocystis 6803. The spectra were excited at 435 (A) and 580 nm (B). Each spectrum was obtained by averaging six spectra of the same sample in different tubes.
ARTICLE IN PRESS W. Ma et al. / Journal of Luminescence 128 (2008) 546–548
548
Table 1 The efficiency of energy transfer from PBS to PSII and PSI, as probed by FPBS/FPSII and FPSI (580)/FPSI (435), in static and aerated cultures of Synechocystis 6803 Culture condition
FPBS/FPSII
FPSI (580)/FPSI (435)
Static Aerated
0.5670.06 0.5570.05
0.2770.02 0.2870.03
not shown). This indicates that the energy transfer from PSII to PSI was inhibited by static culture, implying that this inhibition may be caused by the decrease in PSII activity under this culture condition. To confirm this, Fv/ Fm, a fluorescece parameter that indicates the changes in PSII activity, was measured in static and aerated cultures. 3.3. Inhibition of PSII activity in static cultures
Mean7S.E. from at least six independent experiments.
Table 2 The fluorescence yields of PSII and PSI in static and aerated cultures of Synechocystis 6803 Component
FPSII FPSI
- Ex ¼ 435 nm
- Ex ¼ 580 nm
Static
Aerated
Static
Aerated
132.978.2a 78.976.1b
10076.3a 10072.0b
130.078.1c 75.879.1d
100710.0c 100711.2d
The values obtained in aerated cultures were taken as 100. Mean7S.E. from at least six independent experiments. The same letters indicate significant difference (po0.05) between values.
As shown in Fig. 3, the value of Fv/Fm in static cultures was markedly decreased when compared to that in aerated cultures, indicating that the reduction of PSII activity occurred under static culture conditions, which resulted in the inhibition of energy transfer from PSII to PSI. 4. Conclusion It can be concluded that the energy transfer from PSII to PSI, but not from PBS to PSII and PSI, was remarkably inhibited by the static culture of the cyanobacterium Synechocystis 6803, which was most likely caused by a reduction in PSII activity. Acknowledgments This research was partially supported by the National Natural Science Foundation of China (No. 30770175) and the Key Fundamental Project of Shanghai (No. 06JC14091). The authors are grateful to Prof. J. Zhao (Institute of Chemistry, Chinese Academy of Sciences) for helpful discussion on the fluorescence spectra. References
Fig. 3. The efficiency of excitation energy capture by open PSII reaction center (Fv/Fm) in static and aerated cultures of Synechocystis 6803. The vertical bars indicate standard errors calculated from at least six independent experiments, and ‘‘*’’ represents significant difference compared to the aerated cultures at po0.05.
3.2. Inhibition of energy transfer from PSII to PSI in static cultures The fluorescence yield of PSII, as reflected by the fluorescence levels of FPSII, was remarkably high in static cultures when compared to that in aerated cultures (Table 2). In contrast, the fluorescence yield of PSI, as reflected by the fluorescence area of FPSI, was markedly low in static cultures in comparison with that in aerated cultures (Table 2). The abovementioned results were also observed in those fluorescence emission spectra measured at room temperature, and excited at 435 or 580 nm (data
[1] D.J. Lundell, R.C. Williams, A.N. Glazer, J. Biol. Chem. 256 (1981) 3580. [2] A.N. Glazer, Biochim. Biophys. Acta 768 (1984) 29. [3] X. Su, P.G. Fraenkel, L. Bogorad, J. Biol. Chem. 267 (1992) 22944. [4] A.N. Glazer, Y.M. Gindt, C.F. Chan, K. Sauer, Photosynth. Res. 40 (1994) 167. [5] C.W. Mullineaux, Biochim. Biophys. Acta 1100 (1992) 285. [6] A.C. Ley, W.L. Butler, Biochim. Biophys. Acta 592 (1980) 349. [7] D. Bruce, J. Biggins, T. Steiner, M. Thewalt, Biochim. Biophys. Acta 806 (1985) 237. [8] D. Bruce, S. Brimble, D.A. Bryant, Biochim. Biophys. Acta 974 (1989) 66. [9] J. Biggins, D. Bruce, Photosynth. Res. 20 (1989) 1. [10] J. Olive, G. Ajlani, C. Astier, M. Recouvreur, C. Vernotte, Biochim. Biophys. Acta 1319 (1997) 275. [11] M.M. Allen, J. Phycol. 4 (1968) 1. [12] D. Fork, A. Sen, W.P. Williams, Photosynth. Res. 11 (1997) 71. [13] R. Govindjee, Aust. J. Plant Physiol. 22 (1995) 131. [14] G.H. Krause, E. Weis, Plant Mol. Biol. 42 (1991) 313. [15] N. Murata, K. Satoh, in: J. Govindjee, C. Amez, D.C. Fork (Eds.), Light Emission by Plants and Bacteria, Academic Press, 1986, p. 137. [16] H. Misra, S.K. Mahajan, Biochim. Biophys. Acta 1459 (2000) 139.
Journal of Luminescence 128 (2008) 546–548 www.elsevier.com/locate/jlumin
Excitation energy transfer between photosystems in the cyanobacterium Synechocystis 6803 Weimin Ma, Liping Chen, Lanzhen Wei, Quanxi Wang College of Life and Environment Sciences, Shanghai Normal University, Guilin Road 100, Shanghai 200234, PR China Received 27 July 2007; received in revised form 5 October 2007; accepted 11 October 2007 Available online 22 October 2007
Abstract In cyanobacteria, the light-harvesting complex phycobilisomes (PBS) can transfer the harvested energy to photosystem II (PSII) and photosystem I (PSI) and from PSII to PSI. The efficiency of energy transfer from PBS to PSII or PSI, as probed by the ratio of the fluorescence yield of PBS to that of PSII or PSI, was similar in static and aerated cultures of a cyanobacterium Synechocystis sp. strain PCC 6803. In contrast, the fluorescence yield for PSII was notably high while that for PSI was remarkably low in static cultures when compared to that in aerated cultures. Additionally, the level of the efficiency of excitation energy capture by open PSII reaction center was markedly decreased in static cultures in comparison to that in aerated cultures. Thus, the energy transfer from PSII to PSI, but not from PBS to PSII and PSI, was inhibited in static cultures and was most likely caused by the reduction of PSII activity. r 2007 Elsevier B.V. All rights reserved. Keywords: Excitation energy transfer; Phycobilisome; Photosystem II; Photosystem I
1. Introduction In cyanobacteria, phycobilisomes (PBS), photosystem II (PSII), and photosystem I (PSI) closely associate with the photosynthetic yield. PBS plays a light-harvesting role and is linked to the thylakoid membrane via a colored polypeptide (LCM), which also intermediates the energy transfer from PBS to PSII [1]. In addition, a direct energy transfer from PBS to PSI [2–5] and from PSII to PSI has been proposed [6–10]. The inhibition of one of the specific three energy transfer pathways above would result in a reduction in the photosynthetic yield in cyanobacteria. It is well known that the photosynthetic yield of cyanobacterial cells is markedly low in static cultures when compared to that in aerated cultures. Possibly, one or more pathways of energy transfer among PBS, PSII, and PSI are inhibited in static cultures. However, little is known regarding whether and which pathway(s) is (are) inhibited; similarly, the reason for this inhibition remains unknown. Corresponding author. Tel.: +86 21 64321263; fax: +86 21 64322931. Also to be corresponded to.
E-mail addresses: [email protected] (W. Ma), [email protected] (Q. Wang). 0022-2313/$ - see front matter r 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jlumin.2007.10.007
Since PBS, PSII, and PSI are all highly fluorescent pigment–protein complexes, these questions should be solved by measuring their fluorescence changes in static and aerated cultures. In this study, to reveal the inhibition site of the energy transfer among PBS, PSII, and PSI under static culture conditions of a cyanobacterium Synechocystis sp. strain PCC 6803 (hereafter Synechocystis 6803) and the reason for this inhibition, both low-temperature (77 K) emission spectra and chlorophyll fluorescence measurements were conducted in static and aerated cultures of this cyanobacterium.
2. Materials and methods 2.1. Culture conditions Synechocystis 6803, a cyanobacterium was cultured at 30 1C in BG-11 medium [11] buffered with Tris–HCl (5 mM, pH 8.0) with bubble aeration (i.e., aerated culture) or shaken once by hand every 6 h (i.e., static culture) under continuous illumination by fluorescent lamps (40 mE m2 s1).
ARTICLE IN PRESS W. Ma et al. / Journal of Luminescence 128 (2008) 546–548
2.2. Spectral measurement at 77 K Three-day-old cultures were harvested by centrifugation (5000 g for 5 min at 25 1C), washed, and then resuspended in fresh BG-11 medium buffered with Tris–HCl (5 mM, pH 8.0) at a chlorophyll a concentration of 5 mg mL1; the same harvesting procedure was used in all the experiments. The samples for static and aerated cultures were rapidly frozen in liquid nitrogen. Fluorescence emission spectra were obtained at 77 K using an F-4500 spectrofluorimeter (Hitachi, Japan). The excitation wavelengths were 435 and 580 nm, and the fluorescence spectrum was obtained by averaging six spectra obtained for each sample in different tubes. The fluorescence data were analyzed with a Gaussian deconvolution program and the fluorescence areas for all subbands were calculated. 2.3. Chlorophyll fluorescence measurement The efficiency of excitation energy capture by open PSII reaction center, Fv/Fm, was measured at room temperature (i.e., 25 1C) using a Dual-PAM-100 (Walz, Effeltrich, Germany). The modulated nonactinic fluorescencemeasuring light (FML) was switched on to obtain the initial fluorescence (Fo). Maximal fluorescence (Fm) was measured using illumination with a 0.6 s pulse of red saturating light (10,000 mE m2 s1). Fv/Fm was calculated as (FmFo)/Fm.
547
580 nm exhibited changes similar to the abovementioned emission peaks and were compared for static and aerated cultures. In order to quantitatively compare the efficiency of energy transfer among PBS, PSII, and PSI under static and aerated culture conditions, those emissions were fitted to their corresponding components of the photosynthetic apparatus by applying a Gaussian deconvolution program, and the fluorescence yield of every emission subband was calculated as a respective area. A typical Gaussian deconvolution for the 77 K fluorescence emission spectra excited at 580 nm in static cultures is shown in Fig. 2. The ratio of the fluorescence yield of the 725-nm emission (FPSI) in one spectrum excited at 580 nm to that in another spectrum excited at 435 nm, i.e., FPSI (580)/FPSI (435), has been considered to be an indicator of the efficiency of energy transfer from PBS to PSI [16]. Further, the ratio of the fluorescence yields of 647- and 664-nm emissions (FPBS) to those of 685- and 695-nm emission peaks (FPSII), i.e., FPBS/FPSII, demonstrates the efficiency of energy transfer from PBS to PSII. No significant changes in these two ratios (Table 1) indicate a similar efficiency for energy transfer from PBS to PSII and PSI in static and aerated cultures.
3. Results and discussion 3.1. No significant changes in energy transfer from PBS to PSII and PSI in static and aerated cultures It has been previously demonstrated that the 647- and 664-nm emission peaks originate from PBS; the 685- and 695-nm emissions originate from PSII; and the 725-nm band is most effectively produced by PSI [12–15]. As shown in Fig. 1, the fluorescence emission spectra at 435 and
Fig. 2. An example of the Gaussian deconvolution for the 77 K fluorescence spectrum excited at 580 nm in static cultures of Synechocystis 6803. The black line is the full 77 K emission spectrum, and the gray lines are the Gaussian deconvoluted subbands from the full spectrum.
Fig. 1. The 77 K fluorescence emission spectra in static (black line) and aerated (gray line) cultures of Synechocystis 6803. The spectra were excited at 435 (A) and 580 nm (B). Each spectrum was obtained by averaging six spectra of the same sample in different tubes.
ARTICLE IN PRESS W. Ma et al. / Journal of Luminescence 128 (2008) 546–548
548
Table 1 The efficiency of energy transfer from PBS to PSII and PSI, as probed by FPBS/FPSII and FPSI (580)/FPSI (435), in static and aerated cultures of Synechocystis 6803 Culture condition
FPBS/FPSII
FPSI (580)/FPSI (435)
Static Aerated
0.5670.06 0.5570.05
0.2770.02 0.2870.03
not shown). This indicates that the energy transfer from PSII to PSI was inhibited by static culture, implying that this inhibition may be caused by the decrease in PSII activity under this culture condition. To confirm this, Fv/ Fm, a fluorescece parameter that indicates the changes in PSII activity, was measured in static and aerated cultures. 3.3. Inhibition of PSII activity in static cultures
Mean7S.E. from at least six independent experiments.
Table 2 The fluorescence yields of PSII and PSI in static and aerated cultures of Synechocystis 6803 Component
FPSII FPSI
- Ex ¼ 435 nm
- Ex ¼ 580 nm
Static
Aerated
Static
Aerated
132.978.2a 78.976.1b
10076.3a 10072.0b
130.078.1c 75.879.1d
100710.0c 100711.2d
The values obtained in aerated cultures were taken as 100. Mean7S.E. from at least six independent experiments. The same letters indicate significant difference (po0.05) between values.
As shown in Fig. 3, the value of Fv/Fm in static cultures was markedly decreased when compared to that in aerated cultures, indicating that the reduction of PSII activity occurred under static culture conditions, which resulted in the inhibition of energy transfer from PSII to PSI. 4. Conclusion It can be concluded that the energy transfer from PSII to PSI, but not from PBS to PSII and PSI, was remarkably inhibited by the static culture of the cyanobacterium Synechocystis 6803, which was most likely caused by a reduction in PSII activity. Acknowledgments This research was partially supported by the National Natural Science Foundation of China (No. 30770175) and the Key Fundamental Project of Shanghai (No. 06JC14091). The authors are grateful to Prof. J. Zhao (Institute of Chemistry, Chinese Academy of Sciences) for helpful discussion on the fluorescence spectra. References
Fig. 3. The efficiency of excitation energy capture by open PSII reaction center (Fv/Fm) in static and aerated cultures of Synechocystis 6803. The vertical bars indicate standard errors calculated from at least six independent experiments, and ‘‘*’’ represents significant difference compared to the aerated cultures at po0.05.
3.2. Inhibition of energy transfer from PSII to PSI in static cultures The fluorescence yield of PSII, as reflected by the fluorescence levels of FPSII, was remarkably high in static cultures when compared to that in aerated cultures (Table 2). In contrast, the fluorescence yield of PSI, as reflected by the fluorescence area of FPSI, was markedly low in static cultures in comparison with that in aerated cultures (Table 2). The abovementioned results were also observed in those fluorescence emission spectra measured at room temperature, and excited at 435 or 580 nm (data
[1] D.J. Lundell, R.C. Williams, A.N. Glazer, J. Biol. Chem. 256 (1981) 3580. [2] A.N. Glazer, Biochim. Biophys. Acta 768 (1984) 29. [3] X. Su, P.G. Fraenkel, L. Bogorad, J. Biol. Chem. 267 (1992) 22944. [4] A.N. Glazer, Y.M. Gindt, C.F. Chan, K. Sauer, Photosynth. Res. 40 (1994) 167. [5] C.W. Mullineaux, Biochim. Biophys. Acta 1100 (1992) 285. [6] A.C. Ley, W.L. Butler, Biochim. Biophys. Acta 592 (1980) 349. [7] D. Bruce, J. Biggins, T. Steiner, M. Thewalt, Biochim. Biophys. Acta 806 (1985) 237. [8] D. Bruce, S. Brimble, D.A. Bryant, Biochim. Biophys. Acta 974 (1989) 66. [9] J. Biggins, D. Bruce, Photosynth. Res. 20 (1989) 1. [10] J. Olive, G. Ajlani, C. Astier, M. Recouvreur, C. Vernotte, Biochim. Biophys. Acta 1319 (1997) 275. [11] M.M. Allen, J. Phycol. 4 (1968) 1. [12] D. Fork, A. Sen, W.P. Williams, Photosynth. Res. 11 (1997) 71. [13] R. Govindjee, Aust. J. Plant Physiol. 22 (1995) 131. [14] G.H. Krause, E. Weis, Plant Mol. Biol. 42 (1991) 313. [15] N. Murata, K. Satoh, in: J. Govindjee, C. Amez, D.C. Fork (Eds.), Light Emission by Plants and Bacteria, Academic Press, 1986, p. 137. [16] H. Misra, S.K. Mahajan, Biochim. Biophys. Acta 1459 (2000) 139.