Therefore, the higher activity of D1:2 Photosystem II centres may allow more rapid photochemical .... density and pigment measurements (Myers et al. 1980),.
PhotosynthesisResearch 47: 131-144, 1996. (~) 1996KluwerAcademicPublishers. Printedin the Netherlands. Regular paper
Two forms of the Photosystem II D1 protein alter energy dissipation and state transitions in the eyanobaeterium Synechococcussp. PCC 7942 Douglas Campbell 1, Doug Bruce 2, Christene Carpenter 2, Petter Gustafsson 1 & Gunnar Oquist 1 l Department of Plant Physiology, University of Ume~, S-901 87 Ume~t, Sweden; 2Department of Biological Sciences, Brock University, St. Catharine's, Ontario, L2S 3A1, Canada Received 1 December1994;acceptedin revisedform4 December1995
Key words: chlorophyll fluorescence, electron transport, light-acclimation, light-harvesting, photosynthesis, quenching analysis
Abstract
Synechococcus sp. PCC 7942 (Anacystis nidulans R2) contains two forms of the Photosystem II reaction centre protein D1, which differ in 25 of 360 amino acids. DI:I predominates under low light but is transiently replaced by D1:2 upon shifts to higher light. Mutant cells containing only DI:I have lower photochemical energy capture efficiency and decreased resistance to photoinhibition, compared to cells containing Dl:2. We show that when dark-adapted or under low to moderate light, cells with DI:I have higher non-photochemical quenching of PS II fluorescence (higher qN) than do cells with Dl:2. This is reflected in the 77 K chlorophyll emission spectra, with lower Photosystem II fluorescence at 697-698 nm in cells containing DI:I than in cells with Dl:2. This difference in quenching of Photosystem II fluorescence occurs upon excitation of both chlorophyll at 435 nm and phycobilisomes at 570 nm. Measurement of time-resolved room temperature fluorescence shows that Photosystem II fluorescence related to charge stabilization is quenched more rapidly in cells containing DI:I than in those with D1:2. Cells containing D1:1 appear generally shifted towards State II, with PS II down-regulated, while cells with D1:2 tend towards State I. In these cyanobacteria electron transport away from PS II remains non-saturated even under photoinhibitory levels of light. Therefore, the higher activity of D1:2 Photosystem II centres may allow more rapid photochemical dissipation of excess energy into the electron transport chain. D 1:1 confers capacity for extreme State II which may be of benefit under low and variable light. Abbreviations: D 1 - t h e atrazine-binding 32 kDa protein of the PS II reaction centre core; D 1:1 - t h e D 1 protein constitutively expressed during acclimated growth in Synechococcus sp. PCC 7942; D 1:2- an alternate form of the D1 protein induced under excess excitation in Synechococcus sp. PCC 7942; D C M U - 3-(3,4-dichlorophenyl)-1,1dimethyl urea; Fo-minimal fluorescence in the dark-adapted state; Fo'-minimal fluorescence in a light-adapted state; FM- maximum fluorescence with all quenching mechanisms at a minimum, measured in presence of DCMU; FM~- maximal fluorescence in a light-adapted state, measured with a saturating flash; FMdark-maximal fluorescence in the dark-adapted state; Fv ~- variable fluorescence in a light-adapted state (F~ - Fo~); PAM -pulse amplitude modulated fluorometer; qN - non-photochemical quenching of PS II fluorescence; qN(dark)- qN in the dark adapted state; qp -photochemical quenching of fluorescence
132 Introduction
In organisms capable of oxygenic photosynthesis PS II and associated antennae systems are critical to balancing light capture with the dissipation of energy via photosynthesis or controlled heat release. Within the PS II core, a dimer of the related D1 and D2 proteins coordinates the factors essential for light-activated transfer of electrons from water to plastoquinone (Barber and Andersson 1992). The lifetime of the D1 protein is shorter than that of the PS II complex as a whole, indicating that D1 can be replaced without the concomitant degradation of the entire reaction centre. Current models propose that rapid turnover of D1 serves to replace damaged proteins, particularly when the light supply exceeds the level to which the organism is acclimated (Aro et al. 1993). Under excessive excitation the rate of synthesis may fall short of the rate of light inactivation of D1, contributing to the photoinhibition of PS II centres and photosynthesis (Samuelsson et al. 1987; Wtinschmann and Brand, 1992; Long et al. 1994). The unicellular cyanobacterium Synechococcussp. PCC 7942 (Anacystisnidulans R2) contains three psbA genes for the D1 protein. The psbAI gene encodes the DI:I form of the protein, while the psbAII and psbAIII both encode an identical protein, D 1:2, which differs from DI:I at 25 of 360 amino acids (Golden et al. 1986). Several inactivation mutants have been prepared by Golden et al. (1986). In the R2S2C3 strain the psbAII and the psbAIII genes are interrupted by streptomycin and chloroamphenicol resistance cassettes respectively. Thus the only gene expressed is psbAI and the strain contains only the DI:I form of the protein. Conversely in R2K1 the psbAI gene is interrupted by a kanamycin resistance cassette and the strain contains only D1:2. Studies of gene expression in these and related strains show that transcripts from the psbAI gene predominate during acclimated growth under low to moderate light, but that expression of this gene declines and eventually ceases transiently as light levels increase. Transcripts from psbAIIllII increase under excess excitation, and transiently predominate after shifts to excess excitation (Schaefer and Golden 1989a, b; Bustos et al. 1990; Kulkarni et al. 1992; Campbell et al. 1995). In addition, the levels of the two D1 forms change rapidly during light shifts (Clarke et al. 1993a, 1995; Kulkarni and Golden 1994; reviewed in Golden 1995). In the wild-type a shift from 50 to 200, 500 or 1000/~mol photons m -2 s -1 results in a transient but nearly complete replacement of DI:I by D1:2, with the rate of replacement proportional to light
intensity. After a shift back to 50 #mol photons m -2 s -l the Dl:2 is once again replaced by DI:I. Krupa et al. (1990, 1991) measured photoinhibition in R2S2C3, the wild-type and R2K1. R2S2C3 proved most susceptible to photoinhibition, a finding explained largely by the cessation of D1 synthesis in this strain under high light (Clarke et al. 1993a). The wild-type and R2K1 were progressively more resistant, since D1:2 expression provided a continuing supply of D1 under high light. The question remained as to why the wild-type cyanobacterium rapidly replaces the 'acclimated' DI:I form with D1:2 during a shift to excess excitation. In contrast to this exchange of alternate D 1 forms under increased light, expression of one single form of the related D2 protein increases to compensate for faster turnover under high light (Golden et al. 1989). Unexpectedly, Clarke et al. (1993b) found that R2K1 cells containing only D1:2 have a higher quantum yield of oxygen evolution and PS II electron transport than do R2S2C3 cells containing only DI:I, a functional distinction supported by Krupa et al. (1991) who found that PS II reaction centres containing DI:I were intrinsically more susceptible to photoinhibition than those with Dl:2. This demonstrated a novel system which responds to changing light by modulating the properties of the PS II reaction centre core. These functional distinctions were not caused by increased steady-state photoinhibition in cells with D1:1, since the differences persisted after prolonged low-light incubation, to allow recovery from any putative photoinhibition under growth conditions (Clarke et al. 1993b). In previous studies of oxyphototrophs, the active reaction centres have been considered to be functionally constant (Huner et al. 1993) with light acclimation involving alterations in antenna size and function (for cyanobacteria, reviewed in Tandeau de Marsac and Houmard 1993) or in the stoichiometry of reaction centres and electron transport components (Fujita and Murakami 1987). In the present article we analyze the energy transfer properties of PS II in intact cells of the two strains containing either D I:I or D1:2, to further define the functional distinction between these forms. Room temperature fluorescence induction measurements confirm and extend our initial finding of a functional distinction (Clarke et al. 1993b). The 77 K fluorescence emission spectra were compared under excitation of chlorophyll and of the phycobilisome antenna. Finally, the fluorescence emission lifetimes of the strains were measured
133 using picosecond time-resolved room temperature fluorescence.
Materials and methods
Cell strains and growth conditions Synechococcus sp. PCC 7942 and two psbA geneinactivated mutants (strains R2S2C3 and R2K1) (Golden et al. 1986) were grown in BG-11 inorganic medium (Rippka et al. 1979), supplementally buffered with 10 mM 3-(N-morpholino) propanesulfonic acid (MOPS), pH 7.5. For chlorophyll fluorescence quenching analysis and PS II content determinations 300 ml cultures were grown in flat flasks, bubbled with 5% CO2 in air (about 1 ml s -1) at 37°C with continuous, even illumination of 50/amol photons m -2 s -l as measured by a Li-Cor quantum radiometer (Lambda Instruments, Lincoln, NB). For the 77 K fluorescence and time-resolved fluorescence measurements, growth was under ambient CO2 and 33 °C, conditions which allowed precise standardization of the phycocyanin/chlorophyll ratio. 77 K fluorescence spectra were also performed on cells growing with 5% CO2 in air at 37 °C and 50 #mol photons m -2 s -1 . For each experiment, parallel cultures of the mutant strains were grown and actively growing cells, as determined by cell density and pigment measurements (Myers et al. 1980), were harvested for all experiments. These growth light intensities were too low to cause steady-state photoinhibition in either strain (Clarke et al. 1993b). Chlorophyll fluorescence and quenching analysis Chlorophyll a fluorescence induction was measured at 37°C using a pulse amplitude modulated fluorometer (PAM chlorophyll fluorometer; Walz, Effeltrich, Germany) with the PAM 103 accessory and a Schott KL 1500 lamp (Schott, Mainz, Germany) to provide saturating flashes. A PAM-compatible system of cuvette, magnetic stirrer, oxygen electrode and Bjorkmann type actinic lamp were used for the simultaneous measurement of fluorescence and oxygen evolution (Hansatech, King's Lynn, England) (Walker 1987). Culture samples were made up to about 2/zg chlorophyll ml-1 in growth medium and dark-adapted for 5 min in the Hansatech cuvette. The analysis procedure is outlined in Figure 1A. Minimum fluorescence, Fo, was determined by illuminating the dark adapted cells with a low-intensity light modulated at 1.6 kHz (aver-
age intensity of 0.14 #mol photons m -2 s -1) from a light-emitting diode (peak emission 650 nm). A 1 s flash of saturating white light (8000 #mol photons m -2 s -1) was then given to determine maximal fluorescence in the dark-adapted state, FM(dark). After a further 30 s, the actinic light was activated at 17 #mol photons m -2 s-I and the modulated light was switched to 100 kHz for a better signal to noise ratio. Steady state fluorescence, Fs, was achieved within 2 minutes, and minimum fluorescence in the light adapted state, Fo', was measured by briefly interrupting the actinic beam. After Fs was re-established a saturating light pulse was given to determine maximal fluorescence in the light-adapted state, FM~. The actinic light was then increased and the process was repeated in steps up to an actinic light of 272 #mol photons m -2 s -1, when oxygen evolution was light saturated. The cells did not become CO2 limited, as supplementation with 10 mM NaH2CO3 had no effect on oxygen evolution nor the fluorescence signal. Finally, 3-(3,4dichlorophenyl)- 1,1-dimethyl urea (DCMU) (0.5 #M final) was injected into the cuvette to stop electron transport, for determination of the true maximal fluorescence, FM. The parameters Fo, FM(dark), Fs, Fo', FM' and FM were used for calculation of photochemical (qv) and non-photochemical (qN) quenching (van Kooten and Snel 1990) and the efficiency of excitation energy capture by open PS II reaction centres (Fv'/FM') (Genty et al. 1989). See the results section for further explanation of these procedures.
Determination of PS H content by oxygen flash yield The cuvette system described above was used to measure oxygen evolution resulting from a train of saturating, 2.5 #s single turnover flashes of white light, supplied at a frequency of 10 Hz. The resulting rate of oxygen evolution was converted to active PS II content per 1000 chl molecules by assuming that every active PS II centre produces 1 02 molecule for every 4 flashes (Myers et al. 1980; Park et al. 1995). PS I/PS II ratios are derived from this value by assuming 52 chl/PS II and 118 chl/PS I, as determined for the very closely related strain Synechococcus sp. PCC 6301 under a variety of conditions (Myers et al. 1980).
77 K chlorophyllfluorescence spectra The 77 K fluorescence emission spectra were collected with a laboratory built diode array based fluorimeter (Brimble and Bruce 1989). Excitation was at 570 nm
134 A: Fluorescence Quenching Analysis 1 rain
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Figure 1. PAM chlorophyll fluorescence induction A: Typical experiment redrawn from the chart recorder trace and annotated. B: Dependence of Fo fluorescence on cellular phycocyanin content in Synechococcus sp. PCC 7942. Fo fluorescence is normalized to the chlorophyll content to correct for different cell densities. Each point is a single determination on an independently grown culture. Linear regression line of slope 0.245 and R 2 of 0.77.
in the phycocyanin absorption region or at 435 nm in the chlorophyll absorption region. Samples were prepared to a concentration of 3.7 #g chlorophyll/ml and then incubated in the dark for 10 min to induce a high level of non-photochemical quenching (State II, low yield of PS II fluorescence). Replicate samples were incubated under blue illumination of 50 #mol m -2 s -1 to induce the minimal level of nonphotochemical quenching (State I, high yield of PS II fluorescence). All samples were then snap frozen in liquid nitrogen and kept in the dark until measurement. For the representative fluorescence spectra presented, the absorbance spectra of the two strains were nearly identical. The fluorescence spectra were deconvolved and the constituent peaks quantified using the PeakFit non-linear curve-fitting software from Jandel Scientific (Erkrath, Germany), following the peak parameters
used by Salehian and Bruce (1992). The spectra were not normalized and changes in peak heights between samples are indicative of absolute fluorescence yield changes.
Room temperature time resolvedfluorescence decay Fluorescence decay kinetics were analyzed with a single photon timing apparatus described in Bruce and Miners (1993). A Hamamatsu PLP-01 pulsed laser diode provided pulses of approximately 60 ps duration at 665 nm with a photon flux incident on the sample of 3.5 × 105 photons m -2 pulse -]. Fluorescence was detected at 90 ° to the excitation. Instrument response functions were measured by setting the emission monochromator to the excitation wavelength (665 nm) and collecting the signal from a suspension of latex
135 beads (1.09 # m diameter, Marivac Ltd., Halifax, Canada) in water. Fluorescence decay data was captured in 512 channels of 14.5 ps per channel, with collection continued until the peak channel contained 104 counts. 200 ml of cell culture (1-1.6 #g chl ml - l ) was cycled at 0.8 ml s -1 through a flow-through cuvette of 200 #1 volume, so that cells spent approximately 0.2 s in the measurement cuvette and 250 s in the reservoir. For measurements at the Fo level of fluorescence the cell culture reservoir was in darkness. For measurements at the FM fluorescence level, DCMU (10 #M final) was added to the reservoir, which was maintained under low light. A 3 ml bulb was inserted in the flow circuit immediately before the measurement cuvette and cells passing through this bulb were exposed to 2000 #mol photons m -2 s -1 for approximately 3.8 s, to close reaction centres, induce the State II to State I transition and generate maximal fluorescence. The ceils were exposed to approximately 0.5 s of dark time after this illumination before reaching the measuring laser beam. The Fo and FM fluorescence levels were verified by measuring room temperature fluorescence emission spectra using the same flow circuits described and the fluorescence spectroscope used for 77 K fluorescence spectra. For each experiment fluorescence decay curves were collected at 675, 680, 685, 690, 695 and 700 nm, and model curve fits were generated by a convolution of the instrument response function with a sum of exponential decay components. The decay curves from all six emission wavelengths were modelled simultaneously using a global fitting routine, to determine the spectral shape of each decay component. The fitting program used an optimized Levenberg/Marquat algorithm and was developed by Warren Zipfel (ICS, Ithaca, NY).
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Figure 2. Light response curves for Fluorescence and Quenching parameters determined from PAM fluorometryR2S2C3 (broken line) ('acclimated' DI:I only) or R2K1 (solid line) ('excessexcitation' Dl:2 only), n = 5, mean +/- S.E. A: Non-photochemical quenching (qr~); 1- ((F~,t - For)/(FM - Fo)); a measure of the dissipation of energyfromPS II not resulting in photochemicalelectron transport or fluorescence. B: Photochemical quenching (qp); (F~M- F)/F'M - Fo'); a measure of the proportion of PS II centres open to receive excitation and dissipate it via photochemicalelectron transport. C: F~/VM;a measure of the efficiencyof excitation energy capture by open PS I1centres.
Results PAM room temperature fluorometry in cyanobacteria Pulse amplitude modulated (PAM) fluorometry is used with plants (Schreiber et al. 1986) and green algae (Ting and Owens 1992) for assessment of PS II photochemical efficiency (FvVF~!), the proportion of open PS II centres (qp), and the extent of non-photochemical dissipation of energy (qN), which competes with photochemistry and fluorescence to de-excite PS II. The technique has also proved useful with cyanobacteria although several key distinctions between green plants
and cyanobacteria necessitate adaptation of the method (Miller et al. 1991; Clarke et al. 1993b; Schreiber et al. 1995; Campbell and Oquist, unpub.). Figure 1A presents an annotated example of the protocol followed for the PAM measurements in this article. The modulated measuring beam used to determine dark-adapted Fo fluorescence must be weak (0.14 #mol photons m -2 s-l), as stronger measuring beams can alter PS II fluorescence yield. Furthermore, in marked contrast with plants, in dark-adapted cyanobacteria PS II variable fluorescence is generally very low (Figure IA), and qN is very high (Figure 2A), a condition
136 termed State II. Upon illumination fluorescence from PS II increases up to the growth light intensity (50 #mol photons m -2 s -1 in our case) where qN reaches a minimum, a condition termed State I. Therefore, a saturating flash given to dark-adapted cells stimulates maximal fluorescence in the dark adapted state, Ft,l(dark) (Figure 1A), which is generally considerably lower than the true maximal fluorescence yield, FM. Measurement of FM(dark) permits the calculation of qN in the dark and the extent of the dark to light State II to State I transition. For measurement of true FM, we add DCMU which eliminates photochemical quenching by closing all PS II reaction centres, and allows non-photochemical quenching mechanisms to collapse (Figure 1A). The two-phase rise in fluorescence after addition of DCMU distinguishes the fast rise related to PS II closure and loss of qp, and the slower rise related to relaxation of qN (Krause et al. 1982). Another special property of cyanobacteria is the phycobilisome antenna, which influences the Fo fluorescence (Figure 1B) and consequently affects the Fv/FM and F~,/F~ parameters. Under constant phycocyanin/chlorophyll, changes in Fv/FM and F~,/F~ are valid indicators of changing PS II efficiency, correlating well with alternate measures of PS II performance such as oxygen evolution (Clarke et al. 1993b, 1995; Campbell et al. 1995). Healthy cultures, as determined by oxygen evolution and growth rates, can however have Fv/FM values as low as 0.2, which in a plant would indicate severe photoinhibitory stress, but in the cyanobacterium can merely reflect a high phycocyanin content. The calculation of qp and qN are largely independent of the absolute level of Fo fluorescence and require only minimal mechanistic assumptions (van Kooten and Snel 1991), and so these parameters can be used to compare cultures with different phycocyanin/chlorophyll contents, as more robust measures of photosynthetic performance than the absolute values of Fv/FM and F~,/F~. The latter should be reserved for conditions of carefully standardized pigment content (Clarke et al. 1993b) or for linear measurements of a single culture over time (Clarke et al. 1995; Campbell et al. 1995). Quenching analysis ofR2S2C3 and R2K1 Figure 2 presents the results of a series of comparative measures of the fluorescence parameters of R2S2C3 and R2K1. In 5 independent replicates of R2S2C3, R2K1 and the wild-type strains, with a variety of PC/Chl values, the parameters of the wild-type con-
sistently fell between those of R2S2C3 and R2K1. For clarity of presentation the values from the wild-type have not been plotted. qN is significantly lower in R2K1 in the dark and up to around the growth light intensity (p < 0.05, n = 5) (Figure 2A). Each strain is, however, capable of a State II to State I transition during the shift from darkness to the growth light. As the growth light is exceeded the qN values tend to converge in the two strains, until at 4-5 times the growth light there is no significant difference between the mean values. Relaxation of qN is observed as the slow rise phase of the fluorescence yield after addition of DCMU (Figure 1A). qp is not significantly different in the two strains (Figure 2B), and remained high over the entire light range, up to 5 times the growth light regime. This indicates that electron transport efficiently removed electrons from PS II, even at light intensities strong enough to cause photoinhibition (220 and 272 #mol photons m -2 s-l; Clarke et al. 1995.). The F~,/F~t parameter is consistently lower in R2S2C3 at all light levels, indicating a lower efficiency of PS II photochemistry in this strain (Figure 2C). In Figure 2C, however, the magnitude of the distinction between the strains may be somewhat exaggerated because on average R2S2C3 accumulated somewhat more phycocyanin than R2K1. This higher phycocyanin content depresses Fv/F ~ M I for reasons independent of PS II efficiency. Nevertheless, when paired cultures of equal phycocyanin/chlorophyll ratios are compared, R2K1 still shows a higher F~,/F~t (Clarke et al. 1993b and data not presented). Active PS H content of the two strains In principle, a difference in the steady-state PS II content of the two mutant strains could account for some of the functional distinctions observed. Therefore, we determined PS II content using the well established oxygen flash yield method (Myers et al. 1980; Park et al. 1995). As shown in Table 1, no significant difference in PS II content was found between the strains. Furthermore, in individual determinations, measured PS II content did not correlate with the observed distinctions in PS II function. These measurements are corroborated by EPR measurements (B. Svensson and D. Campbell, unpub.) which did not detect any significant difference in the PS UPS II ratio of the two strains.
137
Table 1. PS II content of R2S2C3 and R2KI. Active PS II centres were measured by oxygen flash yield, as described in the 'Materials and methods'. PS I/PS II was calculated by assumingfixed chlorophyllcontentsof 52 chl/PS II and 118 chl/PS I. Averageddeterminationson 3 independentcultures, 4- S.E.
R2S2C3 (DI:I only) R2K1 (DI:2 only)
PS II/lO00chl
PS I/PS II
2.1 5:.25 2.3 5:.12
3.5/1 3.2/1
20000 o
15000 =o ~. 10000
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77 K fluorescence emission spectroscopy
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Figure 3. Absorbance spectra of the cultures used for 77 K fluorescence emission spectra. R2S2C3 ('acclimated' DI:I only) slightly higher at 630 nm than spectra of R2K1.
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Figure 4. Fluorescence emission spectra at 77 K with excitation of the phycobilisome at 570 nm. R2S2C3 (broken line) ('acclimated' DI:I only); R2K1 (solid line) ('excess excitation' DI:2 only) A: Cells pre-adapted to State II. B: Cells pre-adapted to State I.
Table 2. Quantificationof the constituentpeaks after deconvolution of the fluorescenceemission spectra at 77 K, with excitationof the phycobilisomeat 570 nm. Peak areas are given as a percentage of the total area under each spectra; the percentage difference between the strains is also presented. Note large differencein 697/8 fluorescence(bold)
Peak (nm)
Fraction of total area R2S2C3 R2KI % Difference between strains
State I
657 679 686 697/8 719 752
27 7 12 16 32 6
27 7 ll 23 26 7
0 0 -8.5 44 -23 17
State lI
657 679 686 697/8 719 752
38 6 6 7 41 2
37 6 8 10 37 2
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Figure 3 presents the absorbance spectra of the cultures of R2S2C3 and R2K1 used for determination of 77 K fluorescence emission spectra (Figures 4 and 5). Note that the two cultures have virtually identical absorbance profiles, so distinctions in the 77 K emission spectra can be attributed to changes in fluorescence yield and not to differing pigment contents. Furthermore, in cyanobacteria, the chlorophyll contents of PS II and P S I are thought to be fixed (Myers et al. 1980), as is the phycobilisome/PS II stochiometry (Gantt 1994), and so cultures with identical absorbance spectra have very similar PS I/PS II ratios. Figure 4 presents representative fluorescence emission spectra from R2S2C3 and R2K1 at 77 K, with excitation of the phycobilisome antenna at 570 nm. The results of deconvolution and quantification of the constituent peaks of these spectra are presented in Table 2. Each strain was pretreated before freezing with either a dark incubation to induce State II (Figure 4A) or with
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Figure 5. Fluorescence emission spectra at 77 K with excitation of chlorophyll at 435 am. R2S2C3 (broken line) ('acclimated' D i d only); R2K] (solid line) ('excess excitation' D 1:2 only) A: Cells proadapted to State II by dark incubation immediately prior to freezing B: Cells pro-adapted to State I by incubation under 50/~mols photons m -2 s-1 of blue light immediately prior to freezing.
a blue light incubation for State I (Figure 4B). A peak at 657 nm arises from the phycobilisome, while allophycocyanin in the phycobilisome core fluoresces at 679 nm. A peak at 686 nm originates from PS II, with a second PS II peak at 697 nm in R2S2C3 and 698 nm in R2K1. One large peak at 719 nm arises primarily from PS I, although a shoulder (State II) or low peak (State I) at 752 nm is from PS II. Each of the strains is capable of a clear transition from State II, with low PS II fluorescence (Figure 4A), to State I with high PS II fluorescence (Figure 4B). This transition from State II to State I approximately doubles the sum of PS II fluorescence at 686, 697 and 752 nm (Figure 4 and Table 2), corroborating the change in qN measured at the growth temperature (Figure 2A). Two features notably distinguish the spectra obtained from the two strains. In R2K1 the PS II peak at 698 nm is about 40% larger than the equivalent peak in R2S2C3. This difference is consistent in both
State I and State II. (Figure 4 and Table 2) and supports the measurements at growth temperature which show higher PS II fluorescence yield and lower nonphotochemical dissipation of energy in R2K1 (Figure 2A). Conversely, the 719 nm P S I peak is 10-20% smaller in R2K1 than in R2S2C3 (Figure 4a, b), consistent with a decreased transfer of excitation energy from PS II to P S I in R2K1 compared with R2S2C3. There are only small differences between the strains in the other PS II peaks at 686 and 752 nm. In each strain the 657 nm phycobilisome peak is comparable and larger in State II than in State I, although slightly blue-shifted in R2K1. The net result of these differences is that although each strain is capable of a state transition, R2K1 appears shifted towards the State I condition of higher PS II fluorescence yield, while R2S2C3 tends towards the State II condition of lower PS II fluorescence. These same specific 77 K spectral distinctions have been found in the cultures grown for room temperature quenching analysis and PS II/chl measurements, with 5% CO2 in air at 37 °C and 50 #mol photons m -2 s - t (data not presented). Figure 5 presents the 77 K fluorescence emission spectra of the same samples of R2S2C3 and R2K1 under excitation of chlorophyll at 435 nm, from ceils in State II (Figure 5A) and State I (Figure 5B). In this case, the fluorescence emissions from the phycobilisome are absent, and the PS I fluorescence at 719 nm is enhanced since the bulk of the cellular chlorophyll is associated with PS I. Once again, the 697/8 nm PS II peak is about 40% larger in R2K1 than in R2S2C3, while the other PS II peaks are more comparable. The PS I peak is also smaller in R2K1 than in R2S2C3, as found with excitation of the phycobilisome. Since the two strains display the same spectral distinctions under both phycobilisome and chlorophyll excitation, the coupling of the phycobilisome to the PS II reaction centre is not involved in this difference, suggesting the functional distinction conferred by DI:I and D1:2 lies within the reaction centre. These 77 K data provide precise support for the in vivo PS II fluorescence analysis. The 697/8 nm PS II peak is thought to arise from a specific population of low energy chlorophylls associated with CP47 (van Dorssen et al. 1987). In R2K1 this emission is larger than in R2S2C3, even though the other PS II emissions are quite comparable and the Phycobilisome/Photosystem II/Photosystem I ratios were similar in the two strains. Even at 77 K in the absence of electron transport, a process quenches this fluorescence more in R2S2C3 than in R2K1.
139 Table 3. Lifetimes and wavelength maxima for components of timeresolved room temperature fluorescence emission from R2S2C3 and R2KI. Note the large difference in 323--463 ps components (bold) Lifetime (ps)
Maximum (nm)
Assignment
A: Four component global analysis of fluorescence emission at Fo R2S2C3 14 690 PSI X2=1.08 109 685 PS II 685 PS II 323 R2KI X2=l.II
1600 14 102
417 2000 B: Four component R2S2C3 8 X2=1.2 119 340 1300 R2KI X2=1.19
12 117 463 1200
685 PS II 690 PSI 685 PS II 685 PS II 685 PS II global analysis of fluorescence emission at FM 690 PSI 685 PS II 685 PS II 685 PS II 690 PSI 685 PS II 685 PS II 685 PS II
Picosecond time-resolved fluorescence lifetime analysis To further elucidate the energy transfer properties of the two strains we measured the decay of fluorescence emission after excitation by a flash from a diode laser at 665 nm. The fluorescence decay profile was captured at six emission wavelengths (675,680, 685, 690, 695 and 700 nm), to reveal the spectral distribution of the emission. A circulating system and flow-through cuvette were used to permit light or dark pre-adaptation of the cells. Each strain was measured when darkadapted, with reaction centres open (Fo level) and also with reaction centres closed (FM level) by exposure to DCMU and saturating white light immediately before measurement. For each sample fluorescence decay profiles were deconvoluted using a global analysis of the emissions at all six wavelengths. The software iteratively seeks an optimal fit to the set of decay profiles from the six detection wavelengths. The lifetime of each component is fixed over all detection wavelengths but the amplitude of each component is permitted to vary between wavelengths (Mullineaux and Holzwarth 1991). This global analysis reveals the spectral properties of each component, assisting in their assignment to specific pigments
in the cell. Furthermore, the global analysis increases the confidence of the lifetime estimations since data from six separate emission wavelengths are pooled for the determination of component lifetimes. The maximum error associated with all decay lifetimes is 10% except for the fastest decay component (ca. 10 to 15 ps) which carries a maximum error of 20ps. Components originating from PS II have a maximum at 685 nm and change lifetime and or amplitude during the transition from open to closed reaction centres. In contrast, components from P S I are more stable over the change from Fo to FM, and are red-shifted relative to PS II (Bittersmann and Vermass 1991; Mullineaux and Holzwarth 1991). A summary of these global analyses are presented in Table 3 and Figure 6 for strains R2S2C3 and R2K1, at the Fo and FM fluorescence levels. In all cases the data were best fit with a sum of four exponential decays. Five component fits did not have significantly lower chi-squared values and most often collapsed into four components. Panel A of Figure 6 compares the decay associated spectra of the fastest lifetime component at both Fo and FM in R2S2C3 and R2K1. The spectra and lifetimes (14 ps) were essentially identical at Fo for R2S2C3 and R2K1. Upon trap closure the lifetimes at FM decreased somewhat (to 8 ps for R2S2C3 and 12 ps for R2K1) and the amplitudes increased. However, the relative contribution of this component to total decay (the product of lifetime times amplitude) was essentially constant between Fo and FM and the peak amplitude was in all cases at 690 nm. The short lifetime, similar relative contribution to total fluorescence decay at Fo and FM and long wavelength maxima indicate that this component is PSI associated. There was no significant difference between R2S2C3 and R2K1 in this fast PS I associated decay component. The lifetime of this PS I decay component is faster than previously reported (20 to 40 ps) in intact cyanobacteria (Bittersman and Vermaas 1991; Mullineaux and Holzwarth 199 I) but these fast lifetimes are at the limit of resolution of our apparatus and carry a relatively large uncertainty (20 ps). Panel B of Figure 6 compares the spectra of the second decay component at Fo and FM for R2S2C3 and R2K1. In both mutants the amplitude of this decay component was maximum in open reaction centres (at Fo) and decreased upon trap closure. The lifetimes were very similar in both mutants at Fo and FM (109 and 119 ps for R2S2C3; 102 and 117 ps for R2K1). However, the amplitude at Fo in R2K1 was larger than in R2S2C3 and the decrease in amplitude upon
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Figure 6. Spectra of time-resolved fluorescence decay components after excitation by a 665 nm laser pulse. Open symbols, emissions from cells with open PS II reaction centres, at Fo level fluorescence. Closed symbols, emissions from cells with dosed PS II reaction centres, at FMlevel fluorescence Circles (O), R2S2C3 samples (DI:I only); Squares ([~), R2K1 samples (Dl:2 only) A. Component 1; lifetime 8-14 ps B. Component 2; lifetime 102--119 ps C. Component 3; lifetime 323-463 ps D. Component 4; lifetime 1200-2000 ps
trap closure much greater in R2K1. Although smaller than in R2K1, in R2S2C3 the decrease in amplitude was still significant at five of the six detection wavelengths. A component of similar lifetime (40 to 130 ps) and spectral shape (685 nm peak) which disappears upon complete trap closure has been observed in intact cyanobacteria (Bittersman and Vermaas 1991; Mullineax and Holzwarth 1991), thylakoids and isolated PS II particles (Roelofs et al. 1992; Shatz et al. 1988; Vass et al. 1993) and is associated with energy transfer to and trapping by open reaction centres. This component decreased but did not disappear at FM in our study, suggesting incomplete trap closure. This makes the assignment of decay components at FMmore difficult as we expect overlapping components due to the presence of both open and closed reaction centres. However, the larger relative decrease in this component from Fo to FM in R2K1 suggests more effective trap closure, supporting a higher photochemical efficiency in R2K1 (Clarke et al. 1993b; this paper). The third decay component is presented in panel C of Figure 6. The amplitude of this component was
larger at FM than at Fo in both mutants and had a peak emission at 685 nm in all cases. The dependence on trap closure and peak wavelength associate this component with PS II. Trap closure increased the lifetime of this component in both mutants, but the lifetimes were significantly shorter in R2S2C3 than in R2K1 (323 vs. 417 ps at Fo and 340 vs. 463 ps at FM), the only significant difference in component lifetimes between the mutants. The increase in amplitude of this component upon trap closure was larger in R2KI than in R2S2C3. Components with similar lifetimes at Fo have been associated with charge stabilization (electron transfer from Phe- to QA) in thylakoids and isolated PS II particles (see review by Dau 1994). At FM, lifetimes in this range have been proposed to reflect the slower trapping kinetics of closed PS II centres (Dau 1994). It is likely that these two components are mixed in our FM data due to incomplete trap closure. Nevertheless the larger amplitude change of this component upon trap closure in R2K1 than R2S2C3 is again consistent with the higher photochemical efficiency of R2K1. The faster decay of this component in R2S2C3 is not eorrelat-
141 ed with more efficient electron transport from Phe- to QA (higher photochemical efficiency) and it therefore probably reflects a competitive non-photochemical deexcitation pathway, consistent with the higher qN in R2S2C3. The fourth component is presented in panel D of Figure 6. Increased amplitude upon trap closure, especially in R2K1, and the wavelength maxima at 685 nm associate this component with closed PS II centres. The lifetimes were similar between the mutants but were significantly longer at Fo than at FM. At FM this component has been proposed to reflect the decay of the primary radical pair (P680 +, Phe-) in the presence of QA- (Dau 1994). As with the other PS II associated components the smaller yield change from Fo to FM in R2S2C3 than in R2K1 is consistent with less trap closure and a lower photochemical efficiency in R2S2C3.
Discussion
The chlorophyll fluorescence quenching coefficient qN measures the non-photochemical dissipation of excitation energy from PS II, in competition with photochemistry and fluorescence. In our experiments a large change in qN occurs during the State Transition, a pronounced feature of the photosynthetic system of cyanobacteria. In the dark-adapted State II qN is very high and PS II fluorescence is low when detected by 77 K fluorescence emission spectroscopy or at the growth temperature by PAM fluorometry. Upon illumination qN decreases until minimal qN and maximal PS I! fluorescence are achieved at about the growth light intensity, when the cells are in State I. qN again develops (Krause et al. 1982) as the light surpasses the growth level. State transitions may help balance the excitation distribution between PS II and PSI under low and variable light (Vincent 1979, 1990) or during metabolic changes (Dominy and Williams 1987; Miller et al. 1991; Mullineaux and Allen 1986; Romero et al. 1992; Turpin and Bruce 1990). Photochemical quenching of fluorescence measures the proportion of PS II centres in which QA is oxidized and ready to receive an electron from P680 upon excitation. In plants and green algae qp drops steadily as light surpasses the growth level and excitation pressure on PS II exceeds energy dissipation via electron transport or by the qN mechanisms. Incipient photoinhibition can be predicted by declining qp in both plants (Oquist et al. 1992; Huner et al. 1993) and
green algae (Maxwell et al. 1994). In marked contrast,
Synechococcus sp. PCC 7942 and other cyanobacteria maintain a high qp at light intensities 5 times higher than the growth light regime (Clarke et al. 1993b), well into the photoinhibitory range of light (Clarke et al. 1995). The qp parameter is nevertheless applicable to cyanobacteria, as demonstrated by treatment with DCMU which inhibits electron transport by blocking re-oxidation of the QA site, and results in a rapid drop in qp as the reaction centres close. Since qp remains high under excess light, electron transport away from PS II does not appear to become immediately limiting, probably a consequence of the complex and flexible patterns of electron flow in cyanobacteria (Peschek 1987; Shyam et al. 1993; Campbell and Oquist, unpub.), and the generally high ratio of PS I to PS II (Fujita and Murakami 1987). Our results show that the amino acid differences between D 1:1 and D 1:2 cause a decrease in steady state fluorescence in cells with D I:I (R2S2C3), resulting from increased non-photochemical quenching. This is accompanied by decreased emission at 77 K from lowenergy Chl a molecules associated with CP47 (van Dorssen et al. 1987), faster decay of the picosecond fluorescence component indicative of charge stabilization (electron transfer from Phe- to QA) and increased PSI fluorescence emission yield at 77 K. These functional differences between cells containing DI: 1 and D 1:2 occur in cells with very similar pigment and PS II contents, are not dependent on phycobilisome to PS II coupling nor on differences in the oxidation state of PS II. In addition, changes in the amplitudes of picosecond decay components associated with PS II trap closure were smaller in cells with DI: 1, supporting the previous report that DI:I confers lower photochemical efficiency (Fv/FM) ' t than does Dl:2 (Clarke et al. 1993b). The shorter lifetime of the charge-stabilization fluorescence decay component in cells with D1:1 compared to cells with Dl:2 most likely reflects a nonphotochemical quenching pathway which competes with charge stabilization (electron transfer to QA). The specific quenching of 695 nm fluorescence emission at 77 K suggests involvement of CP47 in this mechanism. This quenching of PS II fluorescence in cells with D 1:1 coincides with an increase in the absolute yield of fluorescence from P S I at 77 K, suggesting that DI:I is associated with a 'super' state II condition with increased excitation of PSI at the expense of PS II. The presence of DI:I in low light grown cells is therefore functionally correlated with down regulation
142 of overall PS II activity in favour of PS I. This could result from either higher non-photochemical quenching in each individual reaction centre, or from a higher proportion of the reaction centres in State II under a given condition. Further work is necessary to determine whether the room temperature fluorescence lifetime and 77 K fluorescence yield changes reflect an actual increase in energy transfer to PS I. The counter-intuitive finding that the constitutive DI:I confers relative down-regulation of PS II, in comparison to centres with Dl:2, leads us to consider the biological pressure which maintains DI:I in Synechococcussp. PCC 7942. Under moderate, steady light cells with only DI:I grow somewhat faster than cells with only Dl:2 (Krupa et al. 1991). The natural light regime is not steady and DI:I might be of even greater selective advantage under low or fluctuating light regimes, where variable PS II down-regulation and an extreme State II may confer overall metabolic benefits (Oquist et al. 1995). In wild-type cells at optimal temperatures Dl:2 content remains maximal for only a few hours after a shift from low to high light (Clarke et al. 1995), possibly corresponding to high light periods resulting from mixing or noon sun, with the cells expressing Dl:2 shifted toward State 1. The Dl:2 centres possess somewhat increased intrinsic resistance to ongoing excess light (Krupa et al. 1991), possibly through more rapid dissipation of excess excitation pressure by feeding electrons to the (non-saturated) electron transport chain (Campbell and 0quist, unpub.). This rapid modulation of Photosystem II activity allows the cells to minimize photoinhibition (Clarke et al. 1993b; reviewed in t3quist et al. 1995) without altering their phycobilisome antenna content, which responds more slowly to longer term influences such as CO2 supply (Reuter and Mtiller 1993; Clarke et al. 1995). Our current work aims to define the exact amino acid differences involved in the functional distinctions between DI:I and D1:2, and the biological utility of this novel system of light acclimation in cyanobacteria.
Acknowledgements Drs Adrian K. Clarke and Vaughan M. Hurry have generously assisted in the design, discussion and interpretation of the experiments. An anonymous reviewer provided valuable criticisms. We are grateful to Prof. Susan S. Golden (Department of Biology, Texas A &
M University) for her gifts of the psbA inactivation mutants of Synechococcussp. PCC 7942. This work was supported by grants from the Swedish Natural Science Research Council to G.O. and P.G. and by grants to D.B. from the Natural Sciences and Engineering Research Council of Canada.
References Aro EM, Virgin I and Andersson B (1993) Photoinhibition of Photosystem II. Inactivation, protein damage and turnover. Biochim Biophys Acta 1143:113-134 Barber J and Andersson B (1992) Too much of a good thing: Light can be bad for photosynthesis. Trends Biocbem Sci 17:61--66 Bittersmann E and Vermass W (1991) Fluorescence lifetime studies of cyanobacterial Photosystem II mutants. Biochim Biophys Acta 1098:105-116 Brimble S and Bruce D (1989) Pigment orientation and excitation energy transfer in Porphyridium cruentum and Synechoccocus sp. PCC 6301 cross-linked in light state 1 and light state 2 with ghitaraldehyde. Biochim Biophys Acta 973:315-323 Bruce D and Miners J (1993) Use of a pulsed laser diode to measure picosecond fluorescence lifetimes. Photochem Photobio158: 464-468 Bustos SA, Schaefer MR and Golden SS (1990) Different and rapid responses of four cyanobacterial psbA transcripts to changes in light intensity. J Bacteriol 172:1998-2004 Campbell D Zhou G Gustaffson P Oquist G and Clarke AK (1995) Electron transport regulates exchange of two forms of Photosystem II D1 protein in the cyanobacterium Synechococcus. EMBO J 14 (22): 5457-5466 Clarke AK, Soitamo A, Gustafsson G and t)quist G (1993a) Rapid interchange between two distinct forms of cyanobacterial PS II reaction-center protein D1 in response to photoinhibition. Proc Nati Acad Sci USA 90:9973-9977 Clarke AK, Hurry VM, Gustafsson P and t)quist G (1993b) Two functionally distinct forms of the PS II reaction-center protein D1 in the cyanobacterium Synechococcus sp. PCC 7942. Proe Natl Acad Sci USA 90:11985-11989 Clarke AK, Campbell D, Gustafsson P, and Oquist G (1995) Dynamic responses of Photosystem II and phycobilisomes to changing light in the cyanobacterium Synechococcus sp. PCC 7942. Planta 197:553-562 Dan H (1994) Molecular mechanisms and quantitative models of variable Photosystem II fluorescence. Photochem Photobiol 60: 1-23 Dominy PJ and Williams WP (1987) The role of respiratory electron flow in the control of excitation energy distribution in blue-green algae. Biochim Biophys Acta 892:264-274 Fujita Y and Murakami A (1987) Regulation of electron transport composition in cyanobacterial photosynthetic system: Stoichiometry among Photosystem I and II complexes and their lightharvesting antennae and cytochrome b ~ complex. Plant Cell Physiol 28:1547-1553 G antt E (1994) Supramolecular membrane organization. In: Bryant D (ed) The Molecular Biology of Cyanobacteria, pp 119-138. Kluwer Academic Publishers, Dordrecht Genty B, Briantais JM and Baker NR (1989) The relationship between the quantum yield of photosynthetic electron transport
143 and quenching of chlorophyll fluorescence. Biochim Biophys Acta 990:87-92 Golden SS (1995) Light-responsive gene expression in cyanobacteria. J Bacteriol 177:1651-1654 Golden SS, Brusslan J and Haselkorn R (1986) Expression of a family ofpsbA genes encoding a Photusystem II polypeptide in the cyanobacterium Anacystis nidulans R2. EMBO J 5: 27892798 Golden SS, Cho D-Sand McNalty MS (1989) Two functional psbD genes in the cyanobacterium Synechococcus sp. strain PCC 7942. J Bacteriol 171:4707-4713 Huner NPA, 0quist,G, Hurry VM, Krol M, Falk S and Griffith M (1993) Photosynthesis, photoinhibition and low temperature acclimation in cold tolerant plants. Photosynth Res 37:19-39 Kranse GH, Vernotte C and Briantais J-M (1982) Photoinduced quenching of chlorophyll fluorescence in intact chloroplasts and algae. Resolution into two components. Biochim Biophys Acta 679:116-124 Krupa Z, Oquist G and Gustafsson P (1990) Photoinhlbition and recovery of photosynthesis in psbA gene-inactivated strains of cyanobacterium Anacystis nidulans. Plant Physiol 93:1-6 Krupa Z, 0quist G and Gustafsson P (1991) Photoinhibition of photosynthesis and growth responses at different light levels in psbA gene mutants of the cyanobacterium Synechococcus. Physiol Plant 82:1-8 Kulkarni RD and Golden SS (1994) Adaptation to high light intensity in Synechococcus sp. strain PCC 7942: Regulation of three psbA genes and two forms of the D1 protein. J Bacteriol 176:959-965 Long SP, Humphries S and Falkowski PG (1994) Photoinhibition of photosynthesis nature. Annu Rev Plant Physiol Plant Mol Biol 45:633-62 Maxwell DP, Falk S, Trick CG and Huner NPA (1994) Growth at low temperature mimics high-light acclimation in ChloreUavulgaris. Plant Physiol 105:535-543 Miller AG, Espie GS and Canvin DT (1991) The effects of inorganic carbon and oxygen on fluorescence in the cyanobactedum Synechococcus UTEX 625. Can J Bot 69:1151-1160 Mullineaux CW and Allen .IF (1986) The state 2 transition in the cyanobacterium Synechococcus6301 can be driven by respiratory electron flow into the ptastoquinone pool. FEBS Lett 205: 155160 Mullineaux CW and Holzwarth AR (1991) Kinetics of excitation energy transfer in the cyanobacterial phycobilisome-Photosystem II complex. Biochim Biophys Acta 1098:68-78 Myers J, Graham JR and Wang RT (1980) Light harvesting in Anacystis nidulans studied in pigment mutants. Plant Physiol 66: 1144-1149
0quist G, Chow WS and Anderson JM (1992) Photoinhibition of photosynthesis represents a mechanism for the long-term regulation of Photosystem II. Planta 186:450--460 Oquist G, Campbell D, Clarke AK and Gustafsson P (1995) The cyanobacterium Synechococcus modulates Photosystem II function in response to excitation stress through D1 exchange. Photosynth Res 46:151-158 Park Y-I, Chow WS, and Anderson JM (1995) Light inactivation of functional Photosystem II in leaves of peas grown in moderate light depends on photon exposure. Planta 196:401-411 Peschek GA (1987) Respiratory electron transport. In: Fay P and Van Baalen C (eds) The Cyanobacteria, pp 119-161. Elsevier, Amsterdam Reuter W and MUller C (1993) Adaptation of the photosynthetic apparatus of cyanobacteria to light and CO2. J Photochem Photobiol B: Bio121:3-27
Rippka R, Deruelles J, Waterbury JB, Herdman M and Stanier RY (1979) Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111: 1-61 Roelofs TA, Lee C-H and Holzwarth AR (1992) Global target analysis of picosecond chlorophyll fluorescence kinetics from pea chloroplasts. Biophys J 61:1147- 1163 Romero JM, Lara C and Sivak MN (1992) Effect of carbon and nitrogen assimilation on chlorophyll fluorescence emission by the cyanobactedum Anacystis nidulans. Physiol Plant 85: 433438 Salehian O and Bruce D (1992) Distribution of excitation energy in photosynthesis: quantification of fluorescence yields form intact cyanobacteria. J Luminescence 51: 91-98 Samuelsson G, Lonneborg A, Gustafsson P and Oquist G (1987) The susceptibility of photosynthesis to photoinhibition and the capacity of recovery in high and low light grown cyanobacteria Anacystis nidulans. Plant Physiol 83:438-441 Schaefer MR and Golden SS (1989) Differential expression of members of a cyanobacterial psbA gene family in response to light. J Bacteriol 171: 3973-3981 Schaefer MR and Golden SS (1989b) Light availability influences the ratio of two forms of D1 in cyanobacterial thylakoids. J Biol Chem 264:7412-7417 Shatz G, Brock H and Holzwarth AR (1988) A kinetic and energetic model for the primary processes in Photosystem II. Biophys J 54: 397-405 Schreiber U, Schliwa U and Bilger W 0986) Continuous recording of photochemical and non-photochemical chlorophyll fluorescence quenching with a new type of modulation fluorometer. Photosynth Res 10:51-62 Schreiber U, Endo T, Mi H and Asada K (1995) Quenching analysis of chlorophyll fluorescence by the saturation pulse method: Particular aspects relating to the study of eukaryotic algae and cyanobacteda. Plant Cell Physiol 36(5): 873-882 Shyam R, Raghavendra AS and Sane PV (1993) Role of dark respiration in photoinhibition of photosynthesis and its reactivation in the cyanobacterium Anacystis nidulans. Physiol Plant 88: 446452 Tandeau de Marsac N and Houmard J (1993) Adaptation of cyanobacteria to environmental stimuli: New steps towards molecular mechanisms. FEMS Microbioi Rev 104:119-190 Ting CS and Owens TG (1992) Limitations of the pulse-modulated technique for measuring the fluoresence characteristics of algae. Plant Physiol 100:367-373 Tsinoremas NF, Schaefer MR and Golden SS (1994) Blue and red light reversibly control psbA expression in the cyanobacterium Synechococcus sp. Strain PCC 7942. J Biol Chem 269(23): 16143-16147 Turpin DH and Bruce D (1990) Regulation of photosynthetic light harvesting by nitrogen assimilation in the green alga Selenastrum minutum. FEBS Lett 263:99-103 van Dorssen RJ, Breton J, Plijter JJ, Satoh K, van Gorkom HJ and Amesz J (1987) Spectroscopic properties of the reaction center and of the 47 kDa chlorophyll protein of Photosystem II. B iochlm Biophys Acta 893:267-274 van Kooten O and Snel JFH (1990) The use of chlorophyll fluorescence nomenclature in plant stress physiology. Photosynth Res 25:147-150 Vass I, Gatzen G and Holzwarth AR (1993) Picosecond timeresolved fluorescence studies on photoinhibition and double reduction of QA in Photosystem II. Biochim Biophys Acta 1183: 388-396
144 Vincent WF (1979) Mechanisms of rapid photosynthetic adaptation in natural phytoplankton communities I: Redistribution of excitation energy between Photosystems I and II. J Phycol 15:429--434 Vincent WF (1990) The dynamic coupling between photosynthesis and light in the phytoplankton environment. Verh Intemat Ver Limno124:25--37
Wtinschmann G and Brand JJ (1992) Rapid turnover of a component required for photosynthesis explains temperature dependence and kinetics of photoinhibition in a cyanobacterium, Synechococcus 6301. Planta 186:426--433 Walker D (1987) The Use of the Oxygen Electrode and Fluorescence Probes in Simple Measurements of Photosynthesis. Hansatech Instruments Limited, Kings Lynn, UK
