Beijing National Laboratory for Molecular Sciences (BNLMS), Photochemistry Laboratory, P. O. Box 101, Institute of Chemistry,Chinese Academy of Sciences, No. 2, 1st North Street, Zhongguancun, Beijing, 100080, P. R. China
Received 24 March 2006; received in revised form 21 August 2006; accepted 22 August 2006Available online 25 August 2006
Keywords: Energy spillover; Mobile phycobilisome; Oligomerization; Photosystem I; Photosystem II; State transition
1. Introduction
Photosynthetic efficiency of the oxygen-evolution organismsdepends on a balanced distribution of the excitation energy fromthe light-harvesting systems to photosystem I (PSI) andphotosystem II (PSII) [1–3]. To respond to various lightconditions, the photosynthetic machine can regulate theexcitation energy distribution into balance between the twophotosystems. This dynamic energy balance process named as“state transition” was firstly observed in 1969 by two researchgroups in red and green alga respectively [4,5]. As an old topic,state transition has been kept continuously interesting for notonly the natural physiological phenomenon itself but also, most
importantly, that the working mechanism of the photosyntheticmachine may be revealed via the spectral responses to aphysical or chemical “stimulus”. Up to now, the regulationmechanism of state transition has been well understood in greenplants [6–8], while in cyanobacteria, whether the state transitionis achieved by “mobile PBS” [9] or by “energy spillover” [4] isstill a question in argument, and the supporting evidences havebeen continuously provided for each [10–14] or both of themodels [15].
Compared to green plants, the most remarkable feature ofcyanobacteria is the light-harvesting phycobilisome (PBS) islocated on outside of the thylakoid membranes while the light-harvesting complex II (LHCII) inside for green plants. This leadsto different architectures of the photosystems in the membranes,i.e., PSII and PSI are closely approximate for the former butseparated far apart for the latter, and the PSI exists mainly astrimers in cyanobacteria while only as monomers in green plants[16]. These different architectures in turn lead to differentstrategies to balance the energy distribution between the two
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photosystems. Thus, for the “energy spillover” model of statetransition in cyanobacteria, it was proposed that the populationproportion of PSI monomers and trimers regulated the energyspillover from PSII to PSI [17,18], i.e., in a probabilistic way—to have a larger or a smaller probability for approximation ofthe two photosystems each other during the state transition.
Now, the topic is focused on following questions: is there acommon mechanism for light-induced and redox-induced statetransitions? If not, then what is it for each case? Is the “energyspillover” regulated by the oligomerization of PSI and is it theonly mechanism? In turn, how does an inducing conditiontrigger a change on the oligomerization of PSI during the statetransition “in vivo”? In the current work, the state transitionmechanisms in cyanobacterial cells of Spirulina platensis wereclarified.
2. Materials and methods
2.1. Culture and growth conditions
Spirulina platensis was obtained from the Institute of Botany, the Chineseacademy of Sciences. Cells were cultured in the Zarrouk medium (pH 9.0) [19]at 28 °C in a 1 l bottle bubbled with air and irradiated with fluorescence lamps(50 μE m−2 s−1) continuously. The cells were harvested from late but stillgrowing batch cultures (10 days) by centrifugation, washed and re-suspended infresh growth medium. To obtain comparable results, cells were harvested fromthe same growth stage for all experiments.
2.2. Preparation of thylakoid membrane
The thylakoid membrane of Spirulina platensis was prepared based on thereport method [19] with minor modifications. 1 g intact cells was suspended in15 ml HEPES–NaOH buffer (25 mM HEPES, 10 mM NaCl pH=7.0) with0.02 g lysozyme in it. After being stirred for 30 min at room temperature, thecells were ultrasonically broken in ice bath for 30 min and then centrifuged at9000×g in a Ti-70 Backman Rotor for 5 min, then the supernatant was againcentrifuged at 50,000×g for 45 min at 4 °C. The pellet was resuspended in theHEPES–NaOH buffer (pH=7.0).
2.3. Spectral measurements
Absorption spectra were recorded on a UV-1601 ultra-vis spectrophotom-eter (Shimadzu, Japan). In order to detect the long-wavelength fluorescenceemission signal for PSI timer, the raw fluorescence spectra were corrected forthe wavelength-dependent sensitivity of the detection system [11,20,21], andobtained on an F4500 spectrofluorimeter (Hitachi, Japan) with a chlorophyllconcentration 5 μg Chl a ml−1 for intact cells and 10 μg Chl a ml−1 forthylakoid membrane at 77 K. All the emission spectra in this paper arecorrected spectra. The concentrations were estimated based on the absorbanceat 665 nm in methanol extracts [22]. For light-induced state transition, the intactcells were dark-adapted for 15 min and then led to the state 1 by blue light(Ditric optics 460 nm short-pass filter) at 100 μE m−2 s−1 for 5 min or the state2 by orange light (Ditric optics 580 nm long-pass and 600 nm short-pass filter)at 20 μE m−2 s−1 for 5 min. The thylakoid membranes were led to the state 1and the state 2 by the far-red light (Ditric optics 700 nm long-pass) and red light(Ditric optics 680 nm short-pass), respectively [23]. For redox-induced statetransition, the state 2 and the state 1 were achieved by dark-adaptation for15 min and by blue light illumination for 5 min in the presence of 10 μMDCMU respectively [24]. For betaine treatment, the cells were treated by 1 Mbetaine for 1 h before other treatment or spectral measurement. For pH valueinduced spectral changes, the cells were cultured in the medium with the samecomponents to growth medium but at different pH value for 2 h. The excitationand emission slit widths were set in 5 nm and the same capture was used in allthe experiments.
3. Results and discussion
PSI trimers in Spirulina platensis possess the absorption andfluorescence maximum at 735 nm (A735) and 760 nm (F760)respectively while the monomers at about 700 nm (A700) and730 nm (F730) respectively [19–21,25], therefore, the spectralfeatures were taken as the specific probes to monitor theoligomerization of PSI in this work.
3.1. Light-induced state transition depends on “PBS mobility”but not on the oligomerization of PSI
To search for whether the PSI oligomerization changes wereinvolved in the light-induced state transition, both thefluorescence spectra at 77 K for the intact cells and the isolatedthylakoid membranes were measured, as shown in Fig. 1, itdemonstrated that the PSI oligomerization did not changeduring the light-induced state transition.
In Fig. 1, F760 was invariable, suggesting that the PSIoligomerization did not change during the light-induced statetransition whether in the intact cells (Fig. 1A–D) or in themembranes (Fig. 1E and F). Further, as long as the PBSs werefixed on the membranes by betaine [26] (Fig. 1C and D), all thefluorescence fluctuations vanished, just like that observedbefore [27], suggesting that light-induced state transition wascompletely regulated by “PBS mobility”. Furthermore, thelight-induced state transition did not occur for the isolatedmembranes lacking phycobilisomes (Fig. 1E and F), suggestingthat the light-induced state transition is achieved by the mobilityof PBSs only once more.
3.2. PSI oligomerization did change during redox-inducedstate transition
The fluorescence emission and the difference spectra at 77 Kare shown in Fig. 2 for the cells and the membranes undergoingredox-induced state transition, i.e., the state 1 induced by bluelight in the presence of DCMU and the state 2 by darkadaptation.
In Fig. 2, F730 and F760 are apparently variable for both ofthe intact cells (A–D) and the membranes (E, F). At the sametime, the F760 and PSII fluorescence increased synchronouslywith the decrease of F730, clearly demonstrating thataggregation of the PSI monomers into the trimers accompaniedby the state transition from the state 2 to the state 1. Fig. 2C andD also shows that the PBS fluorescence fluctuations vanishedwith PBSs fixed on the membranes by betaine, furthermore, thefluorescence intensities for PSII and PSI decreased while thatfor PSI trimers remained comparable to that in Fig. 1A and B.These results confirm that the redox-induced state transitionoriginated from two contributions, “mobile PBS” and “energyspillover” between the two photosystems [27] and the lattercontribution most likely originated from a change in PSIoligomerization.
Based on the results above, it can be concluded that thePSI oligomerization change was involved in the redox-induced state transition but not in the light-induced one,
Fig. 2. The 77 K fluorescence emission (A, C, E) and difference (B, D, F) spectra(state 2 minus state 1) for redox-induced state transitions in intact cells without(A, B) or with betaine (C, D) and in isolated thylakoid membranes (E, F). Solidlines: state 1; dashed lines: state 2. The spectra were excited at 580 nm andnormalized at 710 nm for intact cells or excited at 436 nm and normalized at708 nm for isolated membranes.
Fig. 1. The 77 K fluorescence emission (A, C, E) and difference (B, D, F) spectra(state 2 minus state 1) for light-induced state transitions in intact cells without(A, B) or with betaine (C, D) and in isolated thylakoid membranes (E, F). Solidlines: state 1; dashed lines: state 2. The spectra were excited at 580 nm andnormalized at 710 nm for intact cells or excited at 436 nm and normalized at708 nm for isolated membranes.
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therefore it is most likely to be the mechanism for the “energyspillover”. In fact, compared to light-induced state transition,the redox-induced state transition involves two additionalfactors except for light, i.e., DCMU and dark condition. Inthis case, which of the two factors should be responsible forchanges of the PSI oligomerization was systemically studiedin the next section.
3.3. DCMU is not responsible for PSI oligomerization changes
In order to search for the effect of DCMU, the control cellsand betaine-treated cells pre-illuminated by orange or blue lightwere further illuminated by blue light in the presence of DCMU;the fluorescence emission at 77 K and the difference spectrawere shown in Fig. 3.
Fig. 3. The 77 K fluorescence emission (A, C, E, G) and difference (B, D, F, H) spectra (with DCMUminus without) for the transitions in intact cells without (A–D) orwith betaine (E–F). (A, E) Orange light induced state 2 (solid lines) to blue light and DCMU induced state 1 (dashed lines); (C, G) blue light induced state 1 (solidlines) to blue light and DCMU induced state 1 (dashed lines). The spectra were excited at 580 nm and normalized at 710 nm.
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Apparently, the invariable F760 in Fig. 3A–D suggested thatDCMU was not responsible for the change of PSI oligomer-ization, while the fluorescence fluctuations, similar to those forlight-induced state transition (Fig. 1), were originated from thePBSs movement only. After the PBSs were fixed on themembrane by betaine, all the fluorescence fluctuations vanished(Fig. 3E–H). These results suggested that DCMU could inducea further “state 1” but not a change on PSI oligomerization.
3.4. The PSI oligomerization is regulated by a light switchedon and off only
Another condition distinguishing the redox-induced statetransition from the light-induced one is the dark adaptationwhich used to induce state 2 by the respiratory electron flow[24]. In the last section, it was found that DCMU could notinduce any change on PSI oligomerization, however, the PSIoligomerization did change during light (no matter blue light ororange light) to dark transition! As shown in Fig. 4, i.e., the PSItrimers became less under a light to dark transition.
The increase in F730 and decrease in F760 in Fig. 4 suggestedthe dissociation of PSI trimers into the monomers under the casefrom light to dark, suggesting that dark condition is necessary forinducing a change onPSI oligomerization and also for the “energyspillover”. Furthermore, plots of the difference absorption spectra
to time (Fig. 5) demonstrate that the changes in PSI oligomer-ization are reversible, i.e., dissociation of the trimers into themonomers under the case from light to dark while aggregation ofthe monomers into the trimers under the case from dark to light.
Based on these results, it can be known that the statetransition mechanism is not common but depends on theinducing conditions, i.e., the light-induced state transitiondepends on PBS movement completely while the redox-inducedone involves not only the PBS movement but also the “energyspillover” from PSII to PSI which in turn depends on the PSIoligomerization regulated reversibly by light on and off. Buthow does the light-on and -off regulate PSI oligomerization willbe discussed in the next section.
3.5. PSI oligomerization is regulated by the local cytoplasmicH+ concentration
To search for how the PSI oligomerization is regulated, firstof all, it has to know what kind of interaction force is involved inthe regulation of PSI oligomerization. The cytosol subunit PsaDand the inter-membrane subunit PsaL were reported to beessential for formation of a PSI trimer, via both the electrostaticinteraction and hydrophobic interaction [28–31]. As a mem-brane embedded subunit, the hydrophobic interactions of thePsaL between the monomers are shielded by the hydrophobic
Fig. 4. The 77 K fluorescence emission (A, C) and difference (B, D) spectra(dark minus light) for the transition in intact cells. (A) Blue light induced state 1(solid line) to dark adapted induced state 2 (dashed line); (C) orange lightinduced state 2 (solid line) to dark adapted induced state 2 (dashed line). Thespectra were excited at 580 nm and normalized at 710 nm.
Fig. 5. The difference absorption spectra at room temperature for the intact cellsduring state transition from the state 1 induced by blue light to the state 2induced by dark adaptation (A) and from the state 2 to the state 1 (B) under aseries of time. 1–10 denoted the dark-adaptation or illumination respectively for0.2, 0.4, 0.6, 1, 1.5, 2, 3, 4, 5 and 6 min. Insets in A and B: the plots of A700(squares) and A735 (open squares) to time. C: reversible changes of A700(squares) and A735 (open squares) for the intact cells induced by light–darkswitches.
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lipid tails so that the PSI oligomerization is mainly determinedby the electrostatic interaction between the cytosol PsaDsubunits [17]. It was reported that the oligomerization of PSIin isolated state or in membranes could be regulated byconcentration of Mg2+, i.e., the PSI mainly exist as trimersunder lower (5 mM) while as monomers under higher (150 mM)concentration of Mg2+ [17,20,21]. These phenomena areunderstandable because a high concentration of Mg2+ wouldchange the electrostatic attraction into repulsion force among thePSI monomers. But in fact, the Mg2+ concentration change "invivo" during light–dark transition is just the opposite direction tothat needed for PSI oligomerization change: the Mg2+ wasmeasured with lower concentration under dark (0.5 mM) buthigher concentration under light condition (2 mM) [32].Therefore, Mg2+ concentration should not be responsible forthe changes in PSI oligomerization during light–dark transition.On the other hand, under light condition, proton transportationfrom the cytosol side to the lumen side builds a trans-membraneproton gradient. Under a light to dark transition, the protontransportation accompanied with photosynthetic electron trans-fer stops immediately while that by ATP synthase in oppositedirection keeps working for several more minutes, which leadsto about 0.5 units increase in the cytoplasmic pH, while the
reverse process takes place during a dark to light transition [33–35]. Based on these analyses above, the reversible cytoplasmicpH changes may be responsible for the PSI oligomerizationchange during the inter-transition between light and dark.
To prove this, the population fluctuations of PSI trimers andthe monomers during a light to dark transition at differentexternal pH were investigated. It was reported that Spirulinaplatensis, a typical alkalophilic organism normally cultured atpH 9.0, could survive at an external pH range from 7.0 to 11.0[36]. Furthermore, it was well estimated that the external pHvalues of 7.0, 8.0, 9.0, 10.0 and 11.0 would produce thecytoplasmic pH values of 7.2, 7.5, 7.8, 8.1 and 8.4 respectivelyunder dark condition [37]. In Fig. 6, the pH-dependentdifference absorption (A and B) and fluorescence (C and D)spectra for the intact cells under a light to dark transitiondemonstrate a dependence of the PSI oligomerization on thecytoplasmic H+ concentrations, i.e., the higher is the H+
concentration, the more the PSI monomers are, vice versa. AtpH 11.0, the PSI oligomerization was invariable under a light todark transition, suggesting that the cytoplasmic H+ concentra-tion was not high enough to induce a dissociation of PSI trimers.
Fig. 6. (A, C) The difference absorption at room temperature and fluorescence at77 K spectra (dark minus light) for intact cells during state transition from thestate 1 induced by blue light to the state 2 induced by dark adaptation at differentpH value. 1–5 denoted the external pH 7.0, 8.0, 9.0, 10.0, 11.0. (B, D) The pH-correlative absorption and fluorescence change at 730 nm (squares) and 760 nm(open squares) derived from A and C, respectively. The fluorescence spectrawere excited at 580 nm and normalized at 710 nm.
Fig. 7. Plot of the absorption change at room temperature for A700 (squares) andA735 (open squares) for the intact cells at pH 9.0 (A) and thylakoid membranesat pH 7.0 (B) to time during dark adaptation after blue-light illumination for5 min. A0 and At defined as the absorbance at the time 0 and t min.
Fig. 8. The 77 K fluorescence emission (A, B, C) and difference spectra (D) forthe intact cells during the transition from dark-induced state 2 (solid lines inA–C)to blue-light induced state 1 (dashed lines in A–C). A and solid line in D: controlcells in pH 9.0 growthmedium; B and dashed line inD: betaine treated cells in pH9.0 growth medium; C and thick solid line in D: betaine treated cells in pH 11.0medium. The spectra were excited at 580 nm and normalized at 710 nm.
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It should be indicated that the non-zero value for F730 at pH11.0 in the Fig. 6C and D is due to a contribution of “mobilePBS” but not that of the PSI oligomerization.
Based on the results above, it can be deduced that theisolated thylakoid membranes, in which the cytoplasmic H+
would exchange with those in the solvent, would possessdistinct response to a light to dark transition from the intactcells. As shown in Fig. 7, the PSI monomerization induced by alight to dark transition for the membranes was exactly the sameto that for the intact cells at first several minutes. However, afterthat the PSI monomers would be re-aggregated until reaching anequilibrium for the membranes (Fig. 7B) while keep invariablefor the intact cells (Fig. 7A) under further dark adaptation. Thedifference could be ascribed to that the cytosol side wasprotected by the cell membranes in the intact cells while thatexposed to the solvent in the isolated membranes for a faster H+
equilibrium. Therefore, this could be further evidence for that itis the cytoplasmic H+ concentration that regulates the PSIoligomerization under light–dark transitions.
3.6. PSI monomerization is the only mechanism of the “energyspillover”
Now, a final question is whether the PSI oligomerizationchange is the only mechanism for the “energy spillover” fromPSII to PSI. Fig. 8 shows the fluorescence emission anddifference spectra for the state transition from a dark-induced
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state 2 to a blue-light induced state 1 for the cells at differentconditions. In Fig. 8A and the solid line of Fig. 8D, thefluorescence fluctuations for PBS rods (640 nm), PBS core(660 nm), PSII (693 nm) and PSI monomers (730 nm) wellsuggested the state transition to state 1, while the synchronousincrease of PSI-trimer fluorescence (760 nm) suggested anaggregation of the PSI monomers into trimers during the dark tolight transition. As shown in Fig. 8B and the dashed line in Fig.8D, as soon as PBSs were fixed on the membranes by betaine,the fluorescence fluctuations of the PBS components vanish butthose of the photosystems remain, suggesting that thecontribution of “PBS mobility” was eliminated while that ofthe “energy spillover” was left for the state transition. Further,besides adding betaine, adjusting the external pH to 11.0, thefluorescence fluctuations for not only the PBSs but also PSIIand PSI (including the trimers) all disappeared (Fig. 8C andthick solid line in D). As demonstrated previously, at pH 11.0,light-to-dark transition could not induce a PSI monomerization,therefore, it can be deduced that the PSI monomerization,depending on a rise of the local H+ concentration on the cytosolside, is the only mechanism for the “energy spillover”.
4. Conclusions
Based on the results above, a clear picture can be drawn forthe state transitions in cyanobacteria. First of all, the redox-induced state transition involves different mechanism from thelight-induced one, i.e., the latter completely depends on “PBSmobility” while the former depends on not only the “mobilePBS” but also on “energy spillover” from PSII to PSI. Secondly,it is the PSI oligomerization change that regulates the “energyspillover”, i.e., a dissociation of PSI trimers into the monomerswould lead to a larger probability for approximation of the twophotosystems so an “energy spillover” from PSII to PSI for thelatter is a more efficient energy trap than the former [16].Thirdly, the PSI oligomerization depends on the local H+
concentration on the cytosol side of the thylakoid membraneswhich is in turn regulated by light-on and -off. Specifically, alight to dark transition would produce a higher H+ concentrationon the cytosol side leading to a dissociation of PSI trimers intothe monomers while in a dark to light transition a lower H+
concentration would result the re-aggregation of the monomersinto the trimers. Furthermore, it was proved that the change onthe PSI oligomerization was the only mechanism for the“energy spillover” in redox-induced state transitions.
Acknowledgement
The project was supported by the National Natural ScienceFoundation of China (NSFC). (No. 30570422, 50221201, and90306013).
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