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Photochemical and thermal phases in the short-term chlorophyll fluorescence induction kinetics of Chlorella fusca
J. Photo&em. Photobiol. B: Biol., 12 (1992)
151-159
151
Photochemical and thermal phases in the short-term chlorophyll fluorescence induction kinetics of Chlmella fusca Gabor Laczko and Peter Mar&i Institute of Biophysics, Jksef Attila University, Egyetem tica 2, Szeged H-6722 (Nungary) (Received March 15, 1991; accepted July 17, 1991)
Abstract The yield of chlorophyll fluorescence from photosystem II of ChloreU.uficsca was measured during a short xenon flash (half-width, 2 w) and rectangular laser pulse (duration, 100 ps) of variable intensity. At the highest light intensity (above 1O6W m-2>, the photochemical rise and the decay attributed to the carotenoid triplet quenching could be well separated and a non-photochemical phase was observed which completed within 60 /IS.If the algae are pre-excited with a saturating nitrogen laser pulse, the yield of fluorescence can exceed the maximum value measured routinely (stationary conditions and longer time range). It is argued that the unusually high fluorescence state can arise from a model of the reaction centre of two photoreactions separated by a thermal step. A numerical simulation of the model, based on the freely moving exciton approximation and competition between the carotenoid triplet traps and the different states of the reaction centre for exciton capture, gave reasonably good agreement with the experimental results.
Keywor&: Photosynthesis, chlorophyll a fluorescence, photosystem II centres, quenchers, kinetics, excitons, lake model, Chkn-ells fksca.
1. Introduction The chlorophyll fluorescence yield of photosystem II (PS II) in green plants depends primarily on the photochemical state of the PS II reaction centre (RC); it is small (&,) in the open state and high (4-J in the closed state of the RC [l, 21. Upon illumination (normally under steady state conditions), the RCs are progressively closed and the fluorescence yield increases from & to &_ [3]. The resulting trace, the fluorescence induction curve, is usually measured in the time range of seconds and has become a routine diagnostic tool in studying PS II [4, 51. The fluorescence-photochemistry complementarily, however, fails to describe fluorescence induction kinetics during or immediately after (sub)microsecond saturating flashes. The first such observation was made by Mauzerall [ 61, who reported that the variable fluorescence of dark-adapted loll-1344mB5.00
Q 1992 - Elsevier Sequoia. All rights reserved
152
ChloreUa induced by a 10 ns saturating flash did not reach its maximum value until about 100 ps after the flash. Light-induced drop of the maximum value was observed when Chlm-eUa was fluorescence yield from its 4, subjected to strong flashes [7, 81. Additional studies proved that, in addition to the photochemical quencher (Q), other short-lived fluorescence quenchers were also involved [9 ]. These may include the triplet state of carotenoids, the oxidized state of the RC pigment P+680 and the reduced pheophytin Pheo- in the RC (for a review see ref. 10). The fluorescence quenchers can be induced either by light or by thermal reactions in the RC and the observed fluorescence yield results from their combined effects. Several reports have been published about a non-photochemical phase in addition to the normally observed photochemical rise in the fluorescence induction. They were measured at room temperature in the 100 11stime range either during an actinic electronic flash [ 111 or subsequent to it using delayed analytic sampling flashes [ 121. These findings were interpreted as different types of acceptor (coupled in series or parallel with the primary quinone &A) or as heterogeneities in PS II [ 13, 141. These assumptions violate the analogy between the RCs in PS II of green plants and the RCs of photosynthetic bacteria [2]. This analogy, however, is far from being complete and the complexity of plants compared with bacteria may allow observations in PS II which are different from those in bacterial RCs. Based on our fluorescence yield measurements on Chb-ells during strong xenon flashes and rectangular laser excitation, evidence is presented for the existence of a thermal phase following the light reaction. The assumption of this non-photochemical step in the series of states of the RC enables the interpretation of the peak fluorescence yield measured in our samples preexcited by a saturating nitrogen laser flash that is significantly higher than value determined without pre-excitation. the 4-
2. Materials
and methods
Green algae Chbrellujiuca were cultivated and harvested as described previously [8]. They were used at an equivalent chlorophyll concentration of 30 pg ml-’ in their culture medium. All measurements were made at room temperature. The details of the method of recording the time course of the chlorophyll fluorescence yield (at 685 run) during xenon flashes or rectangular argon laser pulses have been described elsewhere [ 15 1.Fast (25 MHz) data acquisition was achieved by a multichannel analyser speeded up by a charge-coupled device analogue shift register [ 161. The cell suspension was flowed through the quartz cuvette at a constant rate (1 ml s-l) from a light-tight reservoir and returned by pumping. The mean reservoir residence time ensured permanent dark adaptation.
153
The correct determination of the time profIle of the xenon flash and the linear response of the detecting system independent of the shock received during the pre-excitation are of critical importance. The shape of the xenon flash was determined by recording the fluorescence of dilute rhodamine 6G solution excited by the flash (Pig. 1, top). This curve overlaps the time course of the sample fluorescence excited by a flash attenuated to a very low intensity (approximately 0.1% of the full intensity of 2 X lo6 W m-‘). To test the system by optical signal, the fluorescence of the sample was either mimicked or superimposed by a short (4 ps) rectangular pulse of a red-light-emitting diode (HP 5082-4850). These studies proved that the sensitivity of our full detection system remained constant after the nitrogen laser excitation as well as during xenon flashes attenuated to different intensities. The error in determination of the fluorescence yield due to the jitter of the pulse was negligible.
5:
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U-
22 2z
0-J
1 ..,..,......
,,..... ‘.“.“”
..”
,.: ,..’
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. . . . . . . . .. . .. .
‘.... .,,,,
‘...
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“. . . ... ,, ““... . . . . . . . . . . . . .._
a
Fig. 1. Time profile of the xenon flash (top) and kinetics of the relative yield of chlorophyll fluorescence (& = 1) measured during a xenon flash on green algae ChbmUufusca (bottom). The maximum (100%) light intensity of the xenon flash was 2 X 10’ W rnm2.The yield of fluorescence was measured at low (0.78%) (curves a and c) and high (100%) (curves b and d) xenon flash intensities on dark-adapted samples (curves a and b) or on samples pre-excited by a saturating N, laser flash 60 ps prior to the xenon flash (curves c and d). The full curves represent the numerical solutions of the kinetic equations, eqns. (l)-(4), described in Section 4 using the values of parameters &=SO w-r, &=120 m-l, Icr-0.36 e-r, &,t=SSO ps-l (curves a and b) and 360 q-r (curves c and d), Icr= 1000 w-l, k8 =200 w-‘, k,40 c(s-’ (curves a and b) and 70 JM-’ (curves c and d) and I=20 I.CS-’ (curves b and d) and 0.18 w-’ (curves a and c).
154
3. Results
Depending on the pre-treatment of the sample and the light intensity, a single actinic xenon flash induces different traces of fluorescence induction represented by the experimental points in Fig. 1. At a very low light intensity (0.78%), only a modest rise in the fluorescence yield from its initial level (dark-adapted state, & = 1) can be observed (Fig. 1, curve a). At full intensity (2 X lo6 W mm2), however, a rapid photochemical rise, governed by the rate of photons absorbed from the xenon flash, can be detected which corresponds to the accumulation of reduced QA (Fig. 1, curve b). This photochemical rise is followed by a dip as a result of carotenoid triplet formation. A slight rise at the tail of the xenon flash reflects the decay of the triplets. These observations and analysis of fluorescence rise and the subsequent quenching is generally accepted [7, 9, 171. The curves change dramatically if the sample is pre-excited by a saturating N2 laser pulse 60 ps before the xenon flash. Without causing any induction, the weak analytic xenon flash (0.78%) simply detects the level of fluorescence yield (Fig. 1, curve c). This is the 4,,,_ level because the reduced form of the RC has not yet decayed noticeably (under our experimental circumstances its half-time is more than 600 ps). No carotenoid triplet quenching can be observed as the carotenoid triplets generated by the laser flash have already decayed away and the xenon flash itself is too weak to generate more. If the xenon flash is strong (actinic), then surprisingly the yield does not drop immediately but rises further and reaches a level which is definitely higher than &_ (Fig. 1, curve d). The subsequent drop is due to the carotenoid triplet formation. If the time resolution is not high enough, then only the decay of the fluorescence yield can be observed. That is why earlier studies did not reveal this rising phase [7]. Experiments measuring the fluorescence induction during an actinic single flash (i.e. without pre-illumination) cannot reveal any possible further rise in the fluorescence yield after completion of the photochemical phase as this is buried by the time-dependent carotenoid triplet quenching. Rectangular excitation, however, may eliminate this problem. The results taken with laser phosphoroscope are shown in Fig. 2 (full curves). At the highest excitation intensity (5 X lo4 W rnm2) the photochemical rise is witbin the opening time (about 1 ps) of the rotating sector (Fig. 2, curve c). The triplet formation and its decay reach equilibrium in 10 ps and an additional rise in fluorescence yield can be observed which is completed within 60 ps (Fig. 2, curve c). By decreasing the intensity of the laser flash, the half-time of the thermal rise remains constant and wilI be overlapped by the slower photochemical phase (Fig. 2, curves a and b). At the lowest laser intensity (2 x 1O3Wm-“) used in our experiments, the non-photochemical rise is already completely overlapped by the photochemical phase and cannot be separated at all (Fig. 2, curve a).
155
1.0:
50
100
TIME (pi)
2. Measured (-) and fitted (- - -) kinetic traces of the yield of chlorophyll fluorescence of ChbreUu fu.sca relative to the dark-adapted value (& = 1) during rectangular argon laser excitation at light intensities of 2 X lo3W me2 (curve a), 1 X 10’ W me2 (curve b) and 5 X lo4 W mm2 (curve c). The values of parameters are Z=O.2 ps-l (curve a), I= 1.5 wLs-’ (curve b) andZ=5.0 ps-’ (curve c), kz=0.035 @s-I, k3= 150 PCS-‘,k-=400 ps-'and otherwise identical with those in Fig. 1. Fig.
4. Discussion The observed polyphasicity of the fluorescence yield under flash illumination can be attributed to changes in the states of both the antenna and the reaction centre of PS II [lo]. The kinetics are the results of simultaneous disappearance and formation of various fluorescence quenchers. Under our experimental conditions (light intensity and time range), the contribution of a wide range of factors that could modify the chlorophyll fluorescence yield (exciton-exciton annihilation [ 18, 19 1, membrane potential, spill-over, membrane stacking etc.) can be excluded and only a couple of short-lived fluorescence quenchers should be taken into consideration. The kinetics and the light intensity dependence of the fluorescence yield of dark-adapted Chlorella (Fig. 1, curves a and b) are not surprising as they can be described well by the contribution of the photochemical and the carotenoid triplet quenchers [7, 91. The inductions measured during a xenon flash of full (100%) intensity preceded by a saturatii N2 laser flash (Fig. 1, curve d) or during a rectangular laser flash (Pig. 2, curves b and c) were much more exciting; a further rise in the fluorescence yield was observed after completion of the photochemical rise. Except for our earlier findings (171 a similar effect has not yet been detected probably owing to the insufhcient time resolution [7] or to the differences in the experimental conditions [ 91. These results can be explained assuming that the pre-exciting N2 laser flash and the dark period between the two flashes set the stage for a second photochemical reaction that cannot occur during a single xenon flash. The essence of our interpretation of the high fluorescence yield is a hypothetical dark step between two light reactions in the RC:
156
After the first photon hit, the RC becomes singly reduced (RC-) and needs relaxation via a thermal phase (RC-‘) to prepare for becoming doubly reduced by a second hit (RC’-). Both light reactions can be reversed (omitted from the scheme for simplicity). The net forward rate of the second photoreaction is significantly lower than that of the first. The dark reaction is accomplished within the 60 ~LSdelay between the Na laser pulse and the xenon flash in our experiments (Fig. 1, curve d and Fig. 2, curves b and c). Without going into details of the realization of the different states in terms of chemical species in the RC, the thermal phase could be interpreted by the recently exposed problems of heterogeneity of PS II [ 13, 141. The non-photochemical rise in the yield can be associated alternatively with the slow re-reduction of the (residual) oxidized RC pigment P+680 [ 111, the different acceptors prior to or parallel to QA [2, 12, 20 J or with the various electron (redox) states of the same (primary) quinone [ 2 11. The existence of a low (about 15%) quantum yield side-path acceptor might also be reconciled with the bacterial reaction centre analogy where, in addition to the first bacteriopheophytine on the L branch, a second on the M branch can be reduced with a low yield [22]. For further support of the possibility of double hit in the RC, evidence was published recently about the formation of dianions in bacterial RCs [23]. The induction curves shown in Fig. 1 have been simulated based on the scheme of different states of the RC depicted above using the freely moving exciton approximation and including the carotenoid triplet quenching. In this model the excited singlet state of chlorophyll -denoted by e-is considered to be a freely diffusing exciton. The dynamic behaviour of the system is controlled by the following simple kinetic equation: de dt =r-ICtot~--Ck~[RC]~~-k,[carl~ i
(1)
Excitons produced by light absorption (rate constant r) can be deactivated spontaneously (total rate constant &,J by heat, triplet formation, fluorescence (kn) etc. or captured by different traps (concentrations of which are [RC], and [car J) during their random walk throughout the chlorophyll bed. The different forms of the RC defined by the scheme above are distinguished by the indices i = 1, 2, 3 and 4 (from left to right in the scheme). Together with the carotenoid triplets (car), these are considered to be fluorescence quenchers of different efficiencies as characterized by the kr and k,, secondorder (bimolecular) rate constants. However, after normalization of the quenchers by the RC concentration, they can be treated as first-order rate constants. The time dependences of the concentrations of the quenchers are determined by simple rate equations. Mixing the different states of the RCs can be avoided if the fluorescence yield is monitored either at the onset (without pre-excitation) or after the termination of the thermal phase (60
157
j~s after saturation with N2 laser). In these cases, the time dependence of the concentration of only one RC state should be considered in the third (summed) term on the right-hand side of eqn. (1) and the effect of the rest can be included in the second term by modifying the value of k,. For the oxidized form (of the acceptor side) of the RC:
-WC11 = -kI~[RC]I dt
The carotenoids are produced from the chlorophyll triplets and decay spontaneously with rate constants k,, and kT respectively:
dIc=l
-
dt
=kt,e-kJcar]
The fluorescence yield is (4) As may be seen, the quasi-stationary approximation, i.e. de/dt = 0, involves the use of a Stern-Volmer formalism. The above set of differential equations can be solved numerically. The values of the parameters were chosen based on the following considerations. The lifetime of chlorophyll fluorescence in Chlorellu has been found to be about 2 ns [24] in the &,, state, this translates to kt= 550 ps-‘. The absolute quantum yield of chlorophyll fluorescence in ChZmeZZuwith closed centres is &aT”” = 0.11 and &abs= 0.04 when the centres are open [25]. Thus kfl= kmt&_abs = 60 ps-’ and kl= 1000 ps-r as calculated from the relation (k, + k,+)!k,, = c#~,,“~/cJo*s. The rate constant of carotenoid triplet formation was kw= 120 ps- ’ and that of its decay in air atmosphere was k,=0.35 p-l [8]. To match the photochemical rise, a value of I=20 ps-’ was needed at the peak intensity of the unattenuated (100%) xenon flash. After pre-excitation, the conditions change; thus the values of three parameters need to be modtied in order to obtain an acceptable fit to the experimental data. These modifications will be qualitatively interpreted. Starting from the state RC-’ (as the dark reaction in our scheme is already over), a second photon hit can induce a new charge separation in the RC. This has, however, little chance to be stabilized by the subsequent dark reactions and will be largely dissipated by recombination. As a result, the rate of photochemical trapping reduces to k3 = 200 ps- ‘. Mainly because of the disappearance of the quenching forms of the RC, &,t will decrease to 350 ps-r. No carotenoid triplet quenching can be observed on delayed luminescence, indicating that carotenoids are not present in the neighbourhood of the RCs where the luminescence exciton is emitted from. Hence, the probability of exciton trapping by the carotenoids in the chlorophyll bed will increase if the RCs are already closed. A value of kc, = 70 ps- ’ gave acceptable results. Using the parameters above, the theoretical curve calculated based on the freely moving exciton model fits nicely the fluorescence induction
158
measured during the xenon flash with or without pre-excitation by a nitrogen laser flash (Fig. 1, full curves). In addition to the xenon flash excitation, the experimental data obtained by rectangular argon laser activation were also quantitatively analysed. Using a simiIar set of parameters as discussed above, we managed to fit the induction curves under this completely different light regime (Fig. 2, broken curves). This further supports the validity of our interpretation. It can be concluded that the further rise in chlorophyll fluorescence yield from the &_ level in PS II measured during a short xenon flash after pre-excitation can be explained by assuming a thermal reaction in the RC that makes a second charge separation possible. This non-photochemical rise is completed within 60 ks. The fluorescence quenchers cannot be eliminated with a single short flash even if it is saturating; this means that the fluorescence yield cannot be maximiz ed with a single flash excitation. Because the thermal phase takes place in the excited RC, only strong and repetitive flashes can msximiie the yield of PS II fluorescence in Chlorella fusca. Acknowledgments The authors thank Dr. L. Szalay for discussions and the Hungarian Ministry of Education (368/86) and the OTKA Foundation (32/6427) for the financial support. References 1 D. C. Fork, Photosynthesis, in K. C. Smith (ed.), The Science of Photobiology, Plenum, New York, 1989, pp. 347390. 2 P. Mathis and A. W. Rutherford, The primary reactions of photosystem I and II of algae and higher plants, in J. Amesz (ed.), Ph.otosynthes~, Now Comprehensive Biochemistry, Vol. 15, Elsevier, Amsterdam, 1987, pp. 63-96. 3 I. Sinclair and S. M. Spence, The analysis of fluorescence induction transients from dichlorophenyldimethylurea-poisoned chloroplasts, B&him. Biophys. Acta, 935 (1988) 184-194. 4 M. Senge and H. Senger, Functional changes in the photosynthetic apparatus during light adaptation of the green alga Chkrrelkzjkca, J. Photuchem. Photobiol., B: Biol., 8 (1990) 63-71. 5 A. Melis, G. E. Guenther, P. J. Morrissey and M. L. Ghirardi, Photsystem II heterogeneity in chloroplasts, in H. K. Lichtenthaler (ed.), Applications of Chlorophyll Flxwrescence in Photosynthesis Research, Stress Physiology, Hydrobiology and Remote Sensing, Khnver, Dordrecht, 1988, pp. 33-43. 6 D. Mauzersll, Light-induced fluorescence changes in ChZoreUu, and the primary photoreactions for the production of oxygen, Proc. Natl. Acad. Sci. U.S.A., 69 (1972) 1358-1362. 7 G. A. den Harm, L. N. M. Duysens and D. J. N. Egberts, Fluorescence yield kinetics in the microsecond-range in Chkrrellu pyrenkka and spinach chloroplasts in the presence of hydroxylamine, Biochim. Biophys. Acta, 368 (1974) 409-421. 8 P. Mar&l and J. Lavorel, Intensity and time-dependence of the csrotenoid triplet quenching under light flashes of rectangular shape in ChloreUu, Photo&em. Photobiol., 29 (1979) 1147-1151.
159 9 A. Sonneveld, H. Rademaker and L. N. M. Duysens,Chlorophyll a fluorescence as a monitor of nanosecond reduction of the photooxidized primary donor P-680+ of photosystem II, B&him. Biophys. Acta, 548 (1979) 536-551. 10 P. Mathis, Primary reactions of photosynthesis: discussion of current issues, in J. Biggens (ed.), Progress in Photosynthesis Research, Vol. I, Martinus Nijhoff, Dordrecht, 1987, pp. 151-160. 11 K. L. Zankel, Rapid fluorescence changes observed in chloroplssts: their relationship to the Oz evolving system, B&him. Biophys Acta, 325 (1973) 138-148. 12 P. Joliot and A. Joliot, Characterization of photosystem II centers by polarographic, spectroscopic and fluorescence methods, in G. Akoyunoglou (ed.), Photosynthesis III. Structure and Molecular Organisation of the Photosynthetic Apparatus, Balaban International Science Services, Philadelphia, PA, 1981, pp. 885-899. 13 Govindjee, Photosystem II heterogeneity: the acceptor side, Photosmh. Rex, 25 (1990) 151-160. 14 M.-J.Delrieu and F. Rosengard,Characterizationof two types of oxygen-evolving photosystem II reaction center by the flash-inducedoxygen and fluorescence yield, B&him. Biophys. Acta, 936. (1988) 39-49. 15 P. Mar&i, G. Laczk6 and L. Szalay, Fast detection of chlorophyll fluorescence yield of green plants, Sci. Instrum., 1 (1986) 3-20. 16 G. Laczko and P. Mar&i, CCD for speeding up multichannel analysers, J. Phys. E, 20 (1987) 691-693. 17 G. Laczk6, P. Mar6ti and L. Szalay, Short-lived fluorescence quenchers in PS II of green plants, in C. Sybesma (ed.), Advances in Photosynthesis Research, Vol. I, MartinusNijhoff, The Hague, 1984, pp. 159-162. 18 A. Dobek, J. Deprez, N. E. Geacintov and J. Breton, Anomalous fluorescence induction on subnanosecond time scales and exciton-exciton annihilationsin PS II, in J. Biggens (ed.), Progress in PhotosynthesisResearch, Vol. I, MartinusNijhoff, Dordrecht, 1987, pp. 103-106. 19 G. Kehrberg, J. Voigt and Th. Bittner, Excited state absorption of strongly interacting cNorophyIls in photosynthetic systems, Stud. Biophys., 137 (1990) 195-206. 20 P. Jursinic and R. Dennenberg, Thylskoid photosystem II activity supported by the nonquinone acceptor Q400 and an ancillary acceptor Aq, Biochim. Biophys. Acta, 935 (1988) 225-235. 21 M.C.W. Evans and R. C. Ford, Evidence for two tightly bound iron quinones in the electron acceptor complex of photosystem II, FEBS I&t., 195 (1986) 290-294. 22 P. Mar&i, Ch. Klrmaier, C. A. Wraight, D. Holten and R. Pearlstein, Photochemistry and electron transfer in borohydride-treatedphotosyntheticreaction centers,B&him. Biophys. Acta, 810 (1985) 132-139. 23 T. Mar and G. Gingras, Evidence for the photoreductive trapping of doubly reduced bacterlopheophytin in photoreaction center of Ectothiorhodospira sp., Biochim Biophys. Acta, 2056 (1991) 190-194. 24 A. Miiller, R. Lumry and M. S. Walker, Light intensitydependence of the in vivo fluorescence lifetime of chlorophyll, Photochem. Photobiol., 9 (1969) 113-126. 25 T. Mar, Govincljee, G. S. Singhal and H. Merkelo, Lifetime of the excited state in vivo, I. Chlorophyll a in algae at room and at liquid nitrogen temperatures; rate constants of radiationless deactivation and trapping, Biophys. J., 12 (1972) 797-808.
151-159
151
Photochemical and thermal phases in the short-term chlorophyll fluorescence induction kinetics of Chlmella fusca Gabor Laczko and Peter Mar&i Institute of Biophysics, Jksef Attila University, Egyetem tica 2, Szeged H-6722 (Nungary) (Received March 15, 1991; accepted July 17, 1991)
Abstract The yield of chlorophyll fluorescence from photosystem II of ChloreU.uficsca was measured during a short xenon flash (half-width, 2 w) and rectangular laser pulse (duration, 100 ps) of variable intensity. At the highest light intensity (above 1O6W m-2>, the photochemical rise and the decay attributed to the carotenoid triplet quenching could be well separated and a non-photochemical phase was observed which completed within 60 /IS.If the algae are pre-excited with a saturating nitrogen laser pulse, the yield of fluorescence can exceed the maximum value measured routinely (stationary conditions and longer time range). It is argued that the unusually high fluorescence state can arise from a model of the reaction centre of two photoreactions separated by a thermal step. A numerical simulation of the model, based on the freely moving exciton approximation and competition between the carotenoid triplet traps and the different states of the reaction centre for exciton capture, gave reasonably good agreement with the experimental results.
Keywor&: Photosynthesis, chlorophyll a fluorescence, photosystem II centres, quenchers, kinetics, excitons, lake model, Chkn-ells fksca.
1. Introduction The chlorophyll fluorescence yield of photosystem II (PS II) in green plants depends primarily on the photochemical state of the PS II reaction centre (RC); it is small (&,) in the open state and high (4-J in the closed state of the RC [l, 21. Upon illumination (normally under steady state conditions), the RCs are progressively closed and the fluorescence yield increases from & to &_ [3]. The resulting trace, the fluorescence induction curve, is usually measured in the time range of seconds and has become a routine diagnostic tool in studying PS II [4, 51. The fluorescence-photochemistry complementarily, however, fails to describe fluorescence induction kinetics during or immediately after (sub)microsecond saturating flashes. The first such observation was made by Mauzerall [ 61, who reported that the variable fluorescence of dark-adapted loll-1344mB5.00
Q 1992 - Elsevier Sequoia. All rights reserved
152
ChloreUa induced by a 10 ns saturating flash did not reach its maximum value until about 100 ps after the flash. Light-induced drop of the maximum value was observed when Chlm-eUa was fluorescence yield from its 4, subjected to strong flashes [7, 81. Additional studies proved that, in addition to the photochemical quencher (Q), other short-lived fluorescence quenchers were also involved [9 ]. These may include the triplet state of carotenoids, the oxidized state of the RC pigment P+680 and the reduced pheophytin Pheo- in the RC (for a review see ref. 10). The fluorescence quenchers can be induced either by light or by thermal reactions in the RC and the observed fluorescence yield results from their combined effects. Several reports have been published about a non-photochemical phase in addition to the normally observed photochemical rise in the fluorescence induction. They were measured at room temperature in the 100 11stime range either during an actinic electronic flash [ 111 or subsequent to it using delayed analytic sampling flashes [ 121. These findings were interpreted as different types of acceptor (coupled in series or parallel with the primary quinone &A) or as heterogeneities in PS II [ 13, 141. These assumptions violate the analogy between the RCs in PS II of green plants and the RCs of photosynthetic bacteria [2]. This analogy, however, is far from being complete and the complexity of plants compared with bacteria may allow observations in PS II which are different from those in bacterial RCs. Based on our fluorescence yield measurements on Chb-ells during strong xenon flashes and rectangular laser excitation, evidence is presented for the existence of a thermal phase following the light reaction. The assumption of this non-photochemical step in the series of states of the RC enables the interpretation of the peak fluorescence yield measured in our samples preexcited by a saturating nitrogen laser flash that is significantly higher than value determined without pre-excitation. the 4-
2. Materials
and methods
Green algae Chbrellujiuca were cultivated and harvested as described previously [8]. They were used at an equivalent chlorophyll concentration of 30 pg ml-’ in their culture medium. All measurements were made at room temperature. The details of the method of recording the time course of the chlorophyll fluorescence yield (at 685 run) during xenon flashes or rectangular argon laser pulses have been described elsewhere [ 15 1.Fast (25 MHz) data acquisition was achieved by a multichannel analyser speeded up by a charge-coupled device analogue shift register [ 161. The cell suspension was flowed through the quartz cuvette at a constant rate (1 ml s-l) from a light-tight reservoir and returned by pumping. The mean reservoir residence time ensured permanent dark adaptation.
153
The correct determination of the time profIle of the xenon flash and the linear response of the detecting system independent of the shock received during the pre-excitation are of critical importance. The shape of the xenon flash was determined by recording the fluorescence of dilute rhodamine 6G solution excited by the flash (Pig. 1, top). This curve overlaps the time course of the sample fluorescence excited by a flash attenuated to a very low intensity (approximately 0.1% of the full intensity of 2 X lo6 W m-‘). To test the system by optical signal, the fluorescence of the sample was either mimicked or superimposed by a short (4 ps) rectangular pulse of a red-light-emitting diode (HP 5082-4850). These studies proved that the sensitivity of our full detection system remained constant after the nitrogen laser excitation as well as during xenon flashes attenuated to different intensities. The error in determination of the fluorescence yield due to the jitter of the pulse was negligible.
5:
‘.O
U-
22 2z
0-J
1 ..,..,......
,,..... ‘.“.“”
..”
,.: ,..’
...”
(.,..‘.
;..:
. . . . . . . . .. . .. .
‘.... .,,,,
‘...
‘%.
“. . . ... ,, ““... . . . . . . . . . . . . .._
a
Fig. 1. Time profile of the xenon flash (top) and kinetics of the relative yield of chlorophyll fluorescence (& = 1) measured during a xenon flash on green algae ChbmUufusca (bottom). The maximum (100%) light intensity of the xenon flash was 2 X 10’ W rnm2.The yield of fluorescence was measured at low (0.78%) (curves a and c) and high (100%) (curves b and d) xenon flash intensities on dark-adapted samples (curves a and b) or on samples pre-excited by a saturating N, laser flash 60 ps prior to the xenon flash (curves c and d). The full curves represent the numerical solutions of the kinetic equations, eqns. (l)-(4), described in Section 4 using the values of parameters &=SO w-r, &=120 m-l, Icr-0.36 e-r, &,t=SSO ps-l (curves a and b) and 360 q-r (curves c and d), Icr= 1000 w-l, k8 =200 w-‘, k,40 c(s-’ (curves a and b) and 70 JM-’ (curves c and d) and I=20 I.CS-’ (curves b and d) and 0.18 w-’ (curves a and c).
154
3. Results
Depending on the pre-treatment of the sample and the light intensity, a single actinic xenon flash induces different traces of fluorescence induction represented by the experimental points in Fig. 1. At a very low light intensity (0.78%), only a modest rise in the fluorescence yield from its initial level (dark-adapted state, & = 1) can be observed (Fig. 1, curve a). At full intensity (2 X lo6 W mm2), however, a rapid photochemical rise, governed by the rate of photons absorbed from the xenon flash, can be detected which corresponds to the accumulation of reduced QA (Fig. 1, curve b). This photochemical rise is followed by a dip as a result of carotenoid triplet formation. A slight rise at the tail of the xenon flash reflects the decay of the triplets. These observations and analysis of fluorescence rise and the subsequent quenching is generally accepted [7, 9, 171. The curves change dramatically if the sample is pre-excited by a saturating N2 laser pulse 60 ps before the xenon flash. Without causing any induction, the weak analytic xenon flash (0.78%) simply detects the level of fluorescence yield (Fig. 1, curve c). This is the 4,,,_ level because the reduced form of the RC has not yet decayed noticeably (under our experimental circumstances its half-time is more than 600 ps). No carotenoid triplet quenching can be observed as the carotenoid triplets generated by the laser flash have already decayed away and the xenon flash itself is too weak to generate more. If the xenon flash is strong (actinic), then surprisingly the yield does not drop immediately but rises further and reaches a level which is definitely higher than &_ (Fig. 1, curve d). The subsequent drop is due to the carotenoid triplet formation. If the time resolution is not high enough, then only the decay of the fluorescence yield can be observed. That is why earlier studies did not reveal this rising phase [7]. Experiments measuring the fluorescence induction during an actinic single flash (i.e. without pre-illumination) cannot reveal any possible further rise in the fluorescence yield after completion of the photochemical phase as this is buried by the time-dependent carotenoid triplet quenching. Rectangular excitation, however, may eliminate this problem. The results taken with laser phosphoroscope are shown in Fig. 2 (full curves). At the highest excitation intensity (5 X lo4 W rnm2) the photochemical rise is witbin the opening time (about 1 ps) of the rotating sector (Fig. 2, curve c). The triplet formation and its decay reach equilibrium in 10 ps and an additional rise in fluorescence yield can be observed which is completed within 60 ps (Fig. 2, curve c). By decreasing the intensity of the laser flash, the half-time of the thermal rise remains constant and wilI be overlapped by the slower photochemical phase (Fig. 2, curves a and b). At the lowest laser intensity (2 x 1O3Wm-“) used in our experiments, the non-photochemical rise is already completely overlapped by the photochemical phase and cannot be separated at all (Fig. 2, curve a).
155
1.0:
50
100
TIME (pi)
2. Measured (-) and fitted (- - -) kinetic traces of the yield of chlorophyll fluorescence of ChbreUu fu.sca relative to the dark-adapted value (& = 1) during rectangular argon laser excitation at light intensities of 2 X lo3W me2 (curve a), 1 X 10’ W me2 (curve b) and 5 X lo4 W mm2 (curve c). The values of parameters are Z=O.2 ps-l (curve a), I= 1.5 wLs-’ (curve b) andZ=5.0 ps-’ (curve c), kz=0.035 @s-I, k3= 150 PCS-‘,k-=400 ps-'and otherwise identical with those in Fig. 1. Fig.
4. Discussion The observed polyphasicity of the fluorescence yield under flash illumination can be attributed to changes in the states of both the antenna and the reaction centre of PS II [lo]. The kinetics are the results of simultaneous disappearance and formation of various fluorescence quenchers. Under our experimental conditions (light intensity and time range), the contribution of a wide range of factors that could modify the chlorophyll fluorescence yield (exciton-exciton annihilation [ 18, 19 1, membrane potential, spill-over, membrane stacking etc.) can be excluded and only a couple of short-lived fluorescence quenchers should be taken into consideration. The kinetics and the light intensity dependence of the fluorescence yield of dark-adapted Chlorella (Fig. 1, curves a and b) are not surprising as they can be described well by the contribution of the photochemical and the carotenoid triplet quenchers [7, 91. The inductions measured during a xenon flash of full (100%) intensity preceded by a saturatii N2 laser flash (Fig. 1, curve d) or during a rectangular laser flash (Pig. 2, curves b and c) were much more exciting; a further rise in the fluorescence yield was observed after completion of the photochemical rise. Except for our earlier findings (171 a similar effect has not yet been detected probably owing to the insufhcient time resolution [7] or to the differences in the experimental conditions [ 91. These results can be explained assuming that the pre-exciting N2 laser flash and the dark period between the two flashes set the stage for a second photochemical reaction that cannot occur during a single xenon flash. The essence of our interpretation of the high fluorescence yield is a hypothetical dark step between two light reactions in the RC:
156
After the first photon hit, the RC becomes singly reduced (RC-) and needs relaxation via a thermal phase (RC-‘) to prepare for becoming doubly reduced by a second hit (RC’-). Both light reactions can be reversed (omitted from the scheme for simplicity). The net forward rate of the second photoreaction is significantly lower than that of the first. The dark reaction is accomplished within the 60 ~LSdelay between the Na laser pulse and the xenon flash in our experiments (Fig. 1, curve d and Fig. 2, curves b and c). Without going into details of the realization of the different states in terms of chemical species in the RC, the thermal phase could be interpreted by the recently exposed problems of heterogeneity of PS II [ 13, 141. The non-photochemical rise in the yield can be associated alternatively with the slow re-reduction of the (residual) oxidized RC pigment P+680 [ 111, the different acceptors prior to or parallel to QA [2, 12, 20 J or with the various electron (redox) states of the same (primary) quinone [ 2 11. The existence of a low (about 15%) quantum yield side-path acceptor might also be reconciled with the bacterial reaction centre analogy where, in addition to the first bacteriopheophytine on the L branch, a second on the M branch can be reduced with a low yield [22]. For further support of the possibility of double hit in the RC, evidence was published recently about the formation of dianions in bacterial RCs [23]. The induction curves shown in Fig. 1 have been simulated based on the scheme of different states of the RC depicted above using the freely moving exciton approximation and including the carotenoid triplet quenching. In this model the excited singlet state of chlorophyll -denoted by e-is considered to be a freely diffusing exciton. The dynamic behaviour of the system is controlled by the following simple kinetic equation: de dt =r-ICtot~--Ck~[RC]~~-k,[carl~ i
(1)
Excitons produced by light absorption (rate constant r) can be deactivated spontaneously (total rate constant &,J by heat, triplet formation, fluorescence (kn) etc. or captured by different traps (concentrations of which are [RC], and [car J) during their random walk throughout the chlorophyll bed. The different forms of the RC defined by the scheme above are distinguished by the indices i = 1, 2, 3 and 4 (from left to right in the scheme). Together with the carotenoid triplets (car), these are considered to be fluorescence quenchers of different efficiencies as characterized by the kr and k,, secondorder (bimolecular) rate constants. However, after normalization of the quenchers by the RC concentration, they can be treated as first-order rate constants. The time dependences of the concentrations of the quenchers are determined by simple rate equations. Mixing the different states of the RCs can be avoided if the fluorescence yield is monitored either at the onset (without pre-excitation) or after the termination of the thermal phase (60
157
j~s after saturation with N2 laser). In these cases, the time dependence of the concentration of only one RC state should be considered in the third (summed) term on the right-hand side of eqn. (1) and the effect of the rest can be included in the second term by modifying the value of k,. For the oxidized form (of the acceptor side) of the RC:
-WC11 = -kI~[RC]I dt
The carotenoids are produced from the chlorophyll triplets and decay spontaneously with rate constants k,, and kT respectively:
dIc=l
-
dt
=kt,e-kJcar]
The fluorescence yield is (4) As may be seen, the quasi-stationary approximation, i.e. de/dt = 0, involves the use of a Stern-Volmer formalism. The above set of differential equations can be solved numerically. The values of the parameters were chosen based on the following considerations. The lifetime of chlorophyll fluorescence in Chlorellu has been found to be about 2 ns [24] in the &,, state, this translates to kt= 550 ps-‘. The absolute quantum yield of chlorophyll fluorescence in ChZmeZZuwith closed centres is &aT”” = 0.11 and &abs= 0.04 when the centres are open [25]. Thus kfl= kmt&_abs = 60 ps-’ and kl= 1000 ps-r as calculated from the relation (k, + k,+)!k,, = c#~,,“~/cJo*s. The rate constant of carotenoid triplet formation was kw= 120 ps- ’ and that of its decay in air atmosphere was k,=0.35 p-l [8]. To match the photochemical rise, a value of I=20 ps-’ was needed at the peak intensity of the unattenuated (100%) xenon flash. After pre-excitation, the conditions change; thus the values of three parameters need to be modtied in order to obtain an acceptable fit to the experimental data. These modifications will be qualitatively interpreted. Starting from the state RC-’ (as the dark reaction in our scheme is already over), a second photon hit can induce a new charge separation in the RC. This has, however, little chance to be stabilized by the subsequent dark reactions and will be largely dissipated by recombination. As a result, the rate of photochemical trapping reduces to k3 = 200 ps- ‘. Mainly because of the disappearance of the quenching forms of the RC, &,t will decrease to 350 ps-r. No carotenoid triplet quenching can be observed on delayed luminescence, indicating that carotenoids are not present in the neighbourhood of the RCs where the luminescence exciton is emitted from. Hence, the probability of exciton trapping by the carotenoids in the chlorophyll bed will increase if the RCs are already closed. A value of kc, = 70 ps- ’ gave acceptable results. Using the parameters above, the theoretical curve calculated based on the freely moving exciton model fits nicely the fluorescence induction
158
measured during the xenon flash with or without pre-excitation by a nitrogen laser flash (Fig. 1, full curves). In addition to the xenon flash excitation, the experimental data obtained by rectangular argon laser activation were also quantitatively analysed. Using a simiIar set of parameters as discussed above, we managed to fit the induction curves under this completely different light regime (Fig. 2, broken curves). This further supports the validity of our interpretation. It can be concluded that the further rise in chlorophyll fluorescence yield from the &_ level in PS II measured during a short xenon flash after pre-excitation can be explained by assuming a thermal reaction in the RC that makes a second charge separation possible. This non-photochemical rise is completed within 60 ks. The fluorescence quenchers cannot be eliminated with a single short flash even if it is saturating; this means that the fluorescence yield cannot be maximiz ed with a single flash excitation. Because the thermal phase takes place in the excited RC, only strong and repetitive flashes can msximiie the yield of PS II fluorescence in Chlorella fusca. Acknowledgments The authors thank Dr. L. Szalay for discussions and the Hungarian Ministry of Education (368/86) and the OTKA Foundation (32/6427) for the financial support. References 1 D. C. Fork, Photosynthesis, in K. C. Smith (ed.), The Science of Photobiology, Plenum, New York, 1989, pp. 347390. 2 P. Mathis and A. W. Rutherford, The primary reactions of photosystem I and II of algae and higher plants, in J. Amesz (ed.), Ph.otosynthes~, Now Comprehensive Biochemistry, Vol. 15, Elsevier, Amsterdam, 1987, pp. 63-96. 3 I. Sinclair and S. M. Spence, The analysis of fluorescence induction transients from dichlorophenyldimethylurea-poisoned chloroplasts, B&him. Biophys. Acta, 935 (1988) 184-194. 4 M. Senge and H. Senger, Functional changes in the photosynthetic apparatus during light adaptation of the green alga Chkrrelkzjkca, J. Photuchem. Photobiol., B: Biol., 8 (1990) 63-71. 5 A. Melis, G. E. Guenther, P. J. Morrissey and M. L. Ghirardi, Photsystem II heterogeneity in chloroplasts, in H. K. Lichtenthaler (ed.), Applications of Chlorophyll Flxwrescence in Photosynthesis Research, Stress Physiology, Hydrobiology and Remote Sensing, Khnver, Dordrecht, 1988, pp. 33-43. 6 D. Mauzersll, Light-induced fluorescence changes in ChZoreUu, and the primary photoreactions for the production of oxygen, Proc. Natl. Acad. Sci. U.S.A., 69 (1972) 1358-1362. 7 G. A. den Harm, L. N. M. Duysens and D. J. N. Egberts, Fluorescence yield kinetics in the microsecond-range in Chkrrellu pyrenkka and spinach chloroplasts in the presence of hydroxylamine, Biochim. Biophys. Acta, 368 (1974) 409-421. 8 P. Mar&l and J. Lavorel, Intensity and time-dependence of the csrotenoid triplet quenching under light flashes of rectangular shape in ChloreUu, Photo&em. Photobiol., 29 (1979) 1147-1151.
159 9 A. Sonneveld, H. Rademaker and L. N. M. Duysens,Chlorophyll a fluorescence as a monitor of nanosecond reduction of the photooxidized primary donor P-680+ of photosystem II, B&him. Biophys. Acta, 548 (1979) 536-551. 10 P. Mathis, Primary reactions of photosynthesis: discussion of current issues, in J. Biggens (ed.), Progress in Photosynthesis Research, Vol. I, Martinus Nijhoff, Dordrecht, 1987, pp. 151-160. 11 K. L. Zankel, Rapid fluorescence changes observed in chloroplssts: their relationship to the Oz evolving system, B&him. Biophys Acta, 325 (1973) 138-148. 12 P. Joliot and A. Joliot, Characterization of photosystem II centers by polarographic, spectroscopic and fluorescence methods, in G. Akoyunoglou (ed.), Photosynthesis III. Structure and Molecular Organisation of the Photosynthetic Apparatus, Balaban International Science Services, Philadelphia, PA, 1981, pp. 885-899. 13 Govindjee, Photosystem II heterogeneity: the acceptor side, Photosmh. Rex, 25 (1990) 151-160. 14 M.-J.Delrieu and F. Rosengard,Characterizationof two types of oxygen-evolving photosystem II reaction center by the flash-inducedoxygen and fluorescence yield, B&him. Biophys. Acta, 936. (1988) 39-49. 15 P. Mar&i, G. Laczk6 and L. Szalay, Fast detection of chlorophyll fluorescence yield of green plants, Sci. Instrum., 1 (1986) 3-20. 16 G. Laczko and P. Mar&i, CCD for speeding up multichannel analysers, J. Phys. E, 20 (1987) 691-693. 17 G. Laczk6, P. Mar6ti and L. Szalay, Short-lived fluorescence quenchers in PS II of green plants, in C. Sybesma (ed.), Advances in Photosynthesis Research, Vol. I, MartinusNijhoff, The Hague, 1984, pp. 159-162. 18 A. Dobek, J. Deprez, N. E. Geacintov and J. Breton, Anomalous fluorescence induction on subnanosecond time scales and exciton-exciton annihilationsin PS II, in J. Biggens (ed.), Progress in PhotosynthesisResearch, Vol. I, MartinusNijhoff, Dordrecht, 1987, pp. 103-106. 19 G. Kehrberg, J. Voigt and Th. Bittner, Excited state absorption of strongly interacting cNorophyIls in photosynthetic systems, Stud. Biophys., 137 (1990) 195-206. 20 P. Jursinic and R. Dennenberg, Thylskoid photosystem II activity supported by the nonquinone acceptor Q400 and an ancillary acceptor Aq, Biochim. Biophys. Acta, 935 (1988) 225-235. 21 M.C.W. Evans and R. C. Ford, Evidence for two tightly bound iron quinones in the electron acceptor complex of photosystem II, FEBS I&t., 195 (1986) 290-294. 22 P. Mar&i, Ch. Klrmaier, C. A. Wraight, D. Holten and R. Pearlstein, Photochemistry and electron transfer in borohydride-treatedphotosyntheticreaction centers,B&him. Biophys. Acta, 810 (1985) 132-139. 23 T. Mar and G. Gingras, Evidence for the photoreductive trapping of doubly reduced bacterlopheophytin in photoreaction center of Ectothiorhodospira sp., Biochim Biophys. Acta, 2056 (1991) 190-194. 24 A. Miiller, R. Lumry and M. S. Walker, Light intensitydependence of the in vivo fluorescence lifetime of chlorophyll, Photochem. Photobiol., 9 (1969) 113-126. 25 T. Mar, Govincljee, G. S. Singhal and H. Merkelo, Lifetime of the excited state in vivo, I. Chlorophyll a in algae at room and at liquid nitrogen temperatures; rate constants of radiationless deactivation and trapping, Biophys. J., 12 (1972) 797-808.