Proc. Natl. Acad. Sci. USA Vol. 90, pp. 11985-11989, December 1993 Biochemistry Two functionally distinct forms of the photosystem II reaction-center protein Dl in the cyanobacterium Synechococcus sp. PCC 7942 ( fluorescence//protein turnover) ADRIAN K. CLARKE*, VAUGHAN M. HuRRY, PETTER GUSTAFSSON, AND GUNNAR OQUIST Department of Physiology, University of Ume&, Ume& S-901 87, Sweden Communicated by Daniel J. Arnon, September 16, 1993

ABSTRACT The cyanobacterium Synechococcus sp. PCC form I ofthe Dl polypeptide (Dl:1), whereas the second form 7942 possesses a smallpsbA multigene family that codes for two (D1:2) is encoded by both the psbAII and psbAIII genes (4). distinct forms ofthe photosystem II reaction-center protein Dl The two forms of Dl differ in only 25 of the total 360 amino (D1:1 and D1:2). We showed previously that the normally acids, and 12 of these differences occur in the first 16 amino predominant Dl form (D1:1) was rapidly replaced with the acids and 7 within the putative transmembrane helices II and alternative D1:2 when cells adapted to a photon irradiance of III (4). As yet, no distinct functional difference between Dl:1 50 pmol mM2's'1 are shifted to 500 ,umol m-2 s-1 and that this and D1:2 has been reported. interchange was readily reversible once cells were allowed to The level of expression for the individual psbA genes in recover under the original growth conditions. By using thepsbA Synechococcus sp. PCC 7942 varies considerably with dif- inactivation mutants R2S2C3 and R2K1 (which synthesize only ferent photon irradiance. When grown at low irradiances, the D1:1 and D1:2, respectively), we showed that this interchange psbAImRNA makes up nearly 95% ofthe totalpsbA message between Dl forms was essential for limiting the degree of (4). The expression ofpsbAl, however, drops significantly at photoinhibition as well as enabling a rapid recovery of photo- higher irradiances, concomitant with an increase in the synthesis. In this report, we have extended these findings by combined expression of the psbAII and psbAIII genes (8). examining whether any intrinsic functional differences exist This shift in expression of the three psbA genes at high light between the two Dl forms that may afford increased resistance corresponds to changes in the proportion of Dl forms, with to photoinhibition. Initial studies on the rate ofDl degradation the relative amount of D1:1 in the thylakoid membranes at three photon irradiances (50, 200, and 500 #mol'm-2 s'1) decreasing along with a rise in D1:2 (9). Similar changes in the showed that the rates ofdegradation for both Dl forms increase expression of the three psbA genes in Synechococcus sp. with increasing photon flux density but that there was no PCC 7942 also occur when cells grown in low light are shifted signiicant difference between Dl:1 and D1:2. Analysis of to higher photon irradiance (10, 11). light-response curves for oxygen evolution for the mutants In a recent study, we further elucidated the molecular R2S2C3 and R2K1 revealed that cells with photosystem II response of Synechococcus sp. PCC 7942 to changes in reaction centers containing D1:2 have a higher apparent quan- photon irradiance by examining the proportion of D1:1 and tum yield (=25%) than cells possessing D1:1. Further studies D1:2 in the wild-type strain during a moderately high light using chlorophyll a fluorescence measurements confirmed that treatment (12). By producing specific antibodies that could R2K1 has a higher photochemical yield than R2S2C3; that is, effectively distinguish between the two forms of Dl, we a more efficient conversion of excitation energy from photon showed that the normally predominant Dl:1 polypeptide was absorption into photochemistry. We believe that the higher rapidly replaced with D1:2 following the shift to high light. photochemical efficiency of reaction centers containing D1:2 is Further, when cells were allowed to recover under the causally related to the preferential induction of D1:2 at high growth irradiance, the exchange between Dl forms was fully light and thus may be an integral component of the protection reversible so that D1:1 was again the major Dl species (12). mechanism within Synechococcus sp. PCC 7942 against pho- Corresponding light-shift experiments were also performed toinhibition. on the mutant strains R2K1 and R2S2C3 in which psbAl or psbAII/III, respectively, had been inactivated by insertion of The reaction center of photosystem II (PSII) consists of two antibiotic-resistance cassettes. These studies demonstrated structurally similar proteins, Dl and D2, as well as cy- that the induction of D1:2 synthesis during high light stress tochrome b559 and thepsbl gene product (for a recent review, was essential for limiting the extent ofphotoinhibition as well see ref. 1). The Dl and D2 polypeptides exist as a heterodimer as facilitating a rapid recovery ofphotosynthesis in Synecho- within the thylakoid membrane and bind the key components coccus sp. PCC 7942 (12). that mediate primary charge separation along with water Although we now know that Synechococcus sp. PCC 7942 oxidation. The Dl polypeptide found in is usually cells specifically interchange their Dl forms under variable coded for by a single psbA gene located on the chloroplast light conditions, it is still not known whether there are any genome. By contrast, all cyanobacteria studied so far possess differences in the physiological or biochemical properties of smallpsbA multigene families that code for two distinct forms D1:1 and D1:2 that might underlie this dynamic process. In of the Dl polypeptide (2-7). The genome of the unicellular this study, we have attempted to resolve this question by cyanobacterium Synechococcus sp. PCC 7942 contains three examining two potentially important properties of both Dl active psbA genes (psbAl, psbAII, and psbAIII), all ofwhich proteins in Synechococcus sp. PCC 7942. We already know are individually capable ofproviding sufficient Dl protein for that the synthesis of D1:1 and D1:2 is differentially induced normal photosynthetic activity (4). The psbAI gene codes for over a wide range oflight intensities. A possible difference in

The publication costs ofthis article were defrayed in part by page charge Abbreviations: PSII, photosystem II; DCMU, 3-(3,4-dichlorophe- payment. This article must therefore be hereby marked "advertisement" nyl)-1,1-dimethylurea. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 11985 Downloaded by guest on September 26, 2021 11986 Biochemistry: Clarke et al. Proc. Natl. Acad. Sci. USA 90 (1993) degradation rates, however, could also influence the degree Schott, Mainz, Germany): one to provide saturating flashes of high light inactivation of PSII reaction centers containing (FL 103) and a second to provide actinic illumination. A Walz either D1:1 or D1:2, and thus we have compared the rate of KS 101 suspension cuvette was used to contain the sample degradation of both forms of Dl at normal growth and high and maintain a constant environment during measurement. irradiances. Potential differences in photosynthetic perfor- The protocol followed is illustrated in Fig. 4. Prior to all mance between PSII with D1:1 and PSII with D1:2 in vivo fluorescence measurements, samples were concentrated to a were also examined, by using photochemical yield parame- chlorophyll content of 20 1g-ml1 and dark-adapted in the ters derived from both oxygen evolution and chlorophyll a Walz cuvette for 5 min. As shown in Fig. 4, minimum fluorescence. fluorescence (Fo) was determined by illuminating the dark- adapted cells with a low-intensity modulated light (ML) ('0.01 ,umol.m-2.s-1) from a light-emitting diode. Following MATERIALS AND METHODS determination ofFo, the actinic light (AL; in this example 250 Cell Strains and Growth Conditions. Synechococcus sp. ,molJm-2*s-1) was turned on and the sample was left until a PCC 7942 and the two psbA gene-inactivated mutants (strains stable fluorescence yield (F) was obtained. Fo', the minimum R2S2C3 and R2K1) described by Golden et al. (4) were grown fluorescence in the light-adapted state, was determined by in batch cultures in an inorganic medium (13). Cultures were briefly interrupting the actinic light with a far-red fiter (RG grown at 37°C under a continuous photon irradiance of 50 715; Schott), to ensure maximal oxidation of PSII electron gmol m-2 s'1 as measured with a Li-Cor quantum radiometer acceptors. Maximum fluorescence in the light-adapted state (Lambda Instruments, Lincoln, NB). Light sources were (FM') was then measured by adding a single 1-s pulse (Flash) Sylvania Hi-Light PAR 38 lamps (120 W, 240-250 V). Cul- of high-intensity white actinic light (15,000 ,umolm-2.s-1), tures were flushed with 5% CO2 in air to avoid changes in with the height of the resultant fluorescence peak represent- antennae size due to low inorganic carbon content in the ing FM'. Finally, maximal fluorescence (FM) was determined medium. Cells in the logarithmic phase of growth, as deter- by injecting 10 ,uM 3-(3,4-dichlorophenyl)-1,1-dimethylurea mined by cell density measurements (0D750, Shimadzu MPS (DCMU), the height of the resultant fluorescence peak was 2000 spectrophotometer), were used for all experiments. All taken to be FM, and the difference between the Fo level and comparative studies were carried out on cells with similar FM was taken to be the level ofmaximal variable fluorescence molar ratios of phycocyanin to chlorophyll a as calculated (FV). Photochemical (qp) and non-photochemical (qN) from A625/A679 ratios (14). quenching were calculated with the equations of van Kooten Analysis of Dl Degradation. Cells were harvested by cen- and Snel (17). The efficiency of excitation energy capture by trifugation at 4000 x g for 5 min and resuspended at a open PSII reaction centers (Fv'/FM') and the photon yield of chlorophyll concentration of 5 ,ug-mli1 in fresh growth me- PSII electron transport (OpsII) were calculated according to dium containing 10 mM NaHCO3. All treatments were car- Genty et al. (18). ried out in 50-ml transparent tubes in a water bath at 37°C. The light source was a halogen lamp (Power Star HQI-IS 400W; Osram, Berlin). The incident photon irradiance was RESULTS measured inside the tube by using a GaAsP diode (model Degradation of Dl:1 and D1:2 Proteins. The first analysis to G1125-02, Hamamatsu, Ichinocho, Japan) sensitive to white elucidate possible functional differences within PSII reaction light only and was recorded on a voltmeter (model 192; centers containing either D1:1 or D1:2 was to ascertain the Keithley). Voltages were calibrated against the quantum rate ofdegradation for both forms ofDl under various photon sensor described above and the desired photon irradiance irradiances. Degradation rates were measured by adding the obtained by the use of neutral density filters. Cells were antibiotic streptomycin 5 min before each of the light treat- bubbled with air to reduce the incidence of self-shading. Prior ments and then following the loss of Dl by using specific to each treatment, cells were incubated for 30 min at 50 antibodies capable ofdistinguishing the two forms ofDl (12). ,umol m-2s- with streptomycin (250 ,ugmli1) added during Synechococcus sp. PCC 7942 wild type (=95% D1:1) and the final 5 min to ensure the cessation of new Dl synthesis. R2K1 (100% D1:2) were used to determine the degradation Light treatments were carried out for 2 hr at a photon rates of D1:1 and D1:2, respectively. The mutant R2S2C3, irradiance of 50, 200, and 500 ,umol m-2 s-1. Samples for which synthesizes only D1:1, was not used in these experi- protein isolation were taken at the start of each treatment and ments due to the presence of the streptomycin-resistance every 30 min thereafter. Cell proteins were isolated as gene within the cassette used to inactivate the psbAII gene described (12). (4). Protein samples containing equivalent amounts of chloro- The rate of degradation for both D1:1 and D1:2 increases phyll (1.5 Mg) were separated in lithium dodecyl sulfate/15% with higher irradiance (Fig. 1). Densitometric quantification polyacrylamide gels (15). Proteins were transferred electro- of Dl breakdown showed that after the first 30 min, the level phoretically to nitrocellulose and immunoblot analysis was ofboth Dl:1 and D1:2 decreased from 58% under the growth performed (16). Density scanning of immunoblots was car- irradiance (50 umolJm-2.s-1) to 45-55% and 28-37% at 200 ried out with a DU-8 spectrophotometer (Beckman) accord- and 500 ,umolJm-2 s-1, respectively (Fig. 2). By comparison, ing to the manufacturer's recommendations. there was little difference between the degradation rates of Oxygen Evolution. Photosynthetic oxygen evolution was D1:1 and D1:2, especially at the two higher photon irradi- measured with a Clark-type oxygen electrode (Hansatech ances. Thus, the degradation rate ofboth Dl forms increased Instruments, Norfolk, U.K.) (12). Measurements were car- with increasing light intensity but the two forms have similar ried out within a range of photon irradiances from 0 to 175 degradation rates at irradiances up to 500 ,mol mM2 s-1. ,umolm-2s-1. Prior to each measurement, 10 ,ul of 1 M Oxygen-Evolution Capacity of R2K1 and R2S2C3. Given NaHCO3 was added to each sample (2 ml) to ensure satu- that the two forms of Dl in Synechococcus sp. PCC 7942 do rating CO2 conditions. Photosynthetic rates were calculated not degrade from the PSII reaction center at significantly as ,umol of 02 evolved per mg of chlorophyll per hr and are different rates, a more physiological study was carried out to presented as the mean ± SD (n = 3). test for potential functional differences. Light-response Chlorophyll Fluorescence. Steady-state chlorophyll a fluo- curves of oxygen evolution were measured for the two rescence was measured at 37°C with a modulated fluorometer mutants R2S2C3 and R2K1 to compare both the photosyn- (PAM chlorophyll fluorometer; Walz, Effeltrich, Germany) thetic efficiency and capacity of cells with PSII containing with the PAM 103 accessory and two Schott lamps (KL 1500; either D1:1 or D1:2 (Fig. 3). The strain R2K1, with PSII Downloaded by guest on September 26, 2021 Biochemistry: Clarke et al. Proc. Natl. Acad. Sci. USA 90 (1993) 11987

A 50 pmol m-' s-' 100

80

60 DI:2 moo* *moo *.::: -- 40 0 30 60 90 120 B 200 pmol m-2 s-' 20 o 0 100 D1:2 c) 08 80 0 30 60 90 120 60

'4-4 C 500 pimol m-2 s-' 0 40 bb 20 D1:1 cvm D1:2 0 100 0 30 60 90 120 Time (min) 80

FIG. 1. Degradation of D1:1 and D1:2, shown by immunodetec- 60 tion of residual D1:1 and D1:2 in wild-type and R2K1 cells, respec- tively, during a 2-hr treatment in the presence of streptomycin at a photon irradiance of 50 (A), 200 (B), or 500 (C) p&molm- s-1. 40 Streptomycin (250 gmlm1) was added 5 min before each light treatment. Protein extracts were taken every 30 min and separated 20 by lithium dodecyl sulfate/PAGE, with samples loaded on the basis of equal chlorophyll (1.5 ug). Each Dl form was detected by 0 immunoblotting with specific polyclonal antibodies. Results are 0 30 60 90 120 representative ofthree separate experiments foreach light treatment. Time (min) reaction centers containing only D1:2, exhibited higher rates FIG. 2. Rate ofDl:l (o) and Dl:2 (o) degradation in wild-type and of oxygen evolution at a given irradiance than R2S2C3 cells, R2K1 cells, respectively, during a 2-hr treatment in the presence of which possess PSII reaction centers with Dl:1. Furthermore, streptomycin at a photon irradiance of 50 (A), 200 (B), or 500 (C) the apparent quantum yield for oxygen evolution, calculated pmol*mM2.s-1. The relative amounts of D1:1 and D1:2 were deter- from the initial slope of the light-response curve, was 25% mined by density scanning ofimmunoblots. Where indicated, values higher for R2K1 than for R2S2C3 cells. represent means ± SD (n = 3). Chlorophyll a Fluorescence of R2K1 and R2S2C3. The apparent difference in quantum yield of photosynthesis be- yield (F) and to loss in fluorescence peak yield (FM'). tween the two types ofPSII in R2K1 and R2S2C3 was further However, we found that ifa single flash ofvery high intensity examined by chlorophyll a fluorescence. However, despite (15,000 ,umol m-2*s-') was applied after steady-state fluores- the widespread use ofchlorophyll a fluorescence to elucidate cence was achieved (F), then the peak height (FM') resulting the functioning of PSII in plants, its application to cyano- from the flash was comparable to the linear portion of the requires several modifications in the methodology. fluorescence rise following addition of DCMU (Fig. 4), Synechococcus sp. PCC 7942 undergoes a rapid transition consistent with both methods giving similar estimates of qp. from state 2 in the dark to state 1 upon illumination (19). The difference in photochemical yield of PSII between the Consequently, the fluorescence yield from a high-intensity two psbA inactivation strains R2K1 and R2S2C3, as mea- flash in the dark-adapted state (normally denoted FM) is lower sured by oxygen evolution (Fig. 3), was also observed in the than the fluorescence from a similar flash in the yield light FV/FM ratios measured by chlorophyll a fluorescence. The (data not shown), reflecting the greater energy-capturing FvIFM ratio, which is an estimate of the photochemical capacity of PSII in the light-adapted state, state 1. Synecho- of PSII when all reaction centers are was coccus sp. PCC 7942, therefore, does not exhibit conven- efficiency open, tional fluorescence patterns, and fluorescence parameters 0.700 ± 0.027 for R2K1 and 0.642 ± 0.011 for R2S2C3. These cannot be measured by the light-doubling technique of values were consistent with the differences in Fv/FM previ- Schreiber et al. (20). To overcome this limitation, we have ously found by using 77 K chlorophyll fluorescence (22). The measured the maximal fluorescence yield in the presence of possibility that the lower photochemical yield observed in the DCMU and actinic light, after first determining the steady- strain R2S2C3 was due to the growth irradiance (50 state fluorescence values, and we refer to this value as FM ,umolmM2 s-1) being mildly photoinhibitory was also tested (see Fig. 4). Miller et al. (21) reported a similar fluorescence by shifting R2S2C3 cells to 30 ,mol.m-2*s' for 30 min. No technique for Synechococcus UTEX 625. We tested a range change in the Fv/FM ratio was observed after this shift, of cell densities, flash times, flash frequencies, and flash indicating that photoinhibition at 50 ,umolmM-2's1 was not the intensities and found that repeated high-intensity flashes cause of the lower photochemical efficiency of PSII in resulted in continual increases in background fluorescence R2S2C3. Downloaded by guest on September 26, 2021 11988 Biochemistry: Clarke et al. Proc. Natl. Acad. Sci. USA 90 (1993) Fm-.

.------Fm

120

-40 bo - - - - F Fo-II - -I .- --FoI 0 20 40 60 80 100 120 140 160 180 Fo----J t t t Flash aCMU Photon Irradiance (ILrmol m S ) A FIG. 3. Light-response curve of photosynthesis for the psbA AL inactivation mutants R2K1 and R2S2C3. Oxygen-evolution activity (Jumol of 02 per mg of chlorophyll per hr) of R2K1 (e) and R2S2C3 (o) was measured at irradiances from 0 to 175 ,umolm-2-s-1. Cells of 60s both strains were grown under identical temperature and light conditions. Cells in logarithmic phase of growth with similar molar ML ratios of phycocyanin to chlorophyll a were used for each experi- ment. Prior to each measurement, 10 A4 of 1 M NaHCO3 was added FIG. 4. Illustration of characteristic chlorophyll a fluorescence to each sample (2 ml, 10 Ag of chlorophyll) to ensure saturating CO2 emissions for psbA inactivation mutants R2K1 and R2S2C3. The conditions. The apparent quantum yield of photosynthesis for both cells were concentrated to 20 pg of chlorophyll per ml and dark- R2K1 and R2S2C3 was determined from the initial slope of each adapted for5 min before the experiment was started by turning on the curve by using the linear regression derived from the first four modulated light (ML). After a stable Fo level was obtained, the oxygen-evolution activities above the compensation point (dotted actinic light (AL; 250 ,umol.m-2.s-1 in this example) was turned on line). Where indicated, values represent means ± SD (n = 3). and fluorescence was monitored until a stable light level (F) was established. The actinic beam was then interrupted by inserting a The effect of increasing light intensity on photochemical far-red filter (RG 715; Schott) into the light path (FR) to fully oxidize quenching (qp), nonphotochemical quenching (qN), efficiency PSII electron acceptors and measure minimal fluorescence in the of energy capture by open PSII reaction centers light (Fo'). When a stable fluorescence yield was reestablished (F), (Fv'/FM'), a single 1-s flash of high intensity light (Flash; 15,000 .mol m-2.s-1) and yield of PSII electron transport (4Psu) of the two psbA was applied to measure maximal fluorescence in the light (F;O. inactivation strains R2K1 and R2S2C3 is shown in Fig. 5. Maximal fluorescence yield (FM) was then measured by poisoning Both mutants showed an identical decline in qp when the the cells with 10 pM DCMU. isradiance increased above 25 yumoljm2S-1, indicating an equal rate of reaction-center closure. However, in both DISCUSSION mutants an increase in Fv'/FM' (i.e., the trapping efficiency The results show that the rate of degradation for both forms ofopen reaction centers) was observed up to an irradiance of of in PCC 7942 increases with 50 ,umoljm-2*s-1. This initial increase in FV'/FM' is almost Dl Synechococcus sp. higher certainly associated with the dark/light transition from state 0. 6 0.66 2 to state 1. This state transition would also account for the relatively high level ofqN forthe dark-adapted cells. It is clear 0.4 0.4 that in both mutants the full transition to state 1 is not achieved until the cells are exposed to an i-radiance equiv- 0.2 0.2 alent to that to which they have been acclimated. Once the

full transition to state 1 is achieved, both qp and Fv'/FM' 0.0 . begin to decline in a manner similar to that observed in leaves 1.0 1.0 of higher plants. The consequence of these two responses is that the estimated yield ofPSII electron transport (FPsn = qp x Fv'/Fm') for both mutants increases at low irradiance as 0.6z0.6 the cells move from state 2 to state 1 and then declines as the light intensity increases above the growth isradiance. How- 0.2 0.2 ever, despite the similarity in the fluorescence patterns of ,,,,,,,,,,,,,,,,, both mutants, R2K1 exhibits higher FV'/FM' at all light 0 100 200 300 400 500 0 100 200 300 400 500 intensities measured, with the result that R2K1 shows a Photon Irradiance (,mol m-2s-1) higher yield for electron transport over PSII (¢psu). Con- FIG. 5. Effect of increasing photon irradiance on chlorophyll comitant changes in qN occur during both the transition from fluorescence parameters for thepsbA inactivation mutants R2K1 and state 2 to state 1 and as the irradiance increases above the R2S2C3. R2K1 (e) and R2S2C3 (o) cells were concentrated to 20 pg growth irradiance (Fig. 5). qN is high for both mutants when of chlorophyll per ml and dark-adapted for 5 min. Chlorophyll a they are in state 2 and it declines rapidly in response to the measurements were carried out as described in Fig. 4, with the following parameters measured: Fv'/FM', efficiency of excitation transition to state 1, reaching a minimum at 50 ,umoljmM2-s-1. energy capture by open PSII reaction centers; 4psn, photochemical At is-adiances above 50 ,umol-m-2.s-1, qN steadily increases yield of PSII electron transport; qp, photochemical quenching; qN, for both mutants, with R2S2C3 maintaining higher levels than nonphotochemical quenching. Where indicated, values represent R2K1. means ± SD (n = 4). Downloaded by guest on September 26, 2021 Biochemistry: Clarke et al. Proc. Natl. Acad. Sci. USA 90 (1993) 11989 photon irradiance but that there is no significant difference in possibility for forming oxidizing radicals, thus protecting the the degradation rates between D1:1 and D1:2, especially reaction center from photoinhibition. Since the major amino under high irradiance (200 and 500 jmol m-2*s-1). A differ- acid differences between the two Dl forms are located in the ential rate of degradation, therefore, is not a distinctive N terminus, to which the phycobilisome is presumably at- feature ofthe two Dl forms and cannot explain why wild-type tached, and in the putative membrane-spanning helices II and cells completely replace D1:1 with D1:2 during high light III, of which helix III contains the electron donor (Tyrz) to stress. This finding was perhaps not unexpected, considering the P680 chlorophyll, we believe that more detailed studies on the putative regions ofthe chloroplast Dl protein believed to both donor side modifications and antennae attachment may be primary cleavage sites. Three such cleavage sites have help explain the differential photochemical properties of the been proposed. The first is situated on the stromal side ofthe two types of PSII reaction centers. Dl polypeptide between the transmembrane helices IV and V (23, 24), whereas the other two are located on the lumenal We thank Prof. Susan S. Golden (Department of Biology, Texas side of the thylakoid membrane between either helices I and A&M University) for her continued support in providing the various II (24) or helices III and IV (25). In all three of these psbA inactivation mutants of Synechococcus sp. PCC 7942. We interhelical regions, the corresponding amino acids found in thank Mr. Arto Soitamo and Dr. Eva-Mari Aro (Department of the two forms of Dl in Synechococcus sp. PCC 7942 are Biology, University of Turku, Turku, Finland) for critical discus- identical to each other (4). Although this does not exclude the sions. This research was supported by the Swedish Natural Science possibility of an additional cleavage site unique to the Syn- Research Council and the Nordic Council for Research in Agricul- echococcus sp. PCC 7942 D1:1 and/or D1:2 polypeptides, the ture. high level of conservation between the cyanobacterial and chloroplast Dl protein (26) suggests that this is remote. 1. Barber, J. & Andersson, B. (1992) Trends Biochem. Sci. 17, 61-66. Despite the lack of any difference in the degradation rates 2. Curtis, S. E. & Haselkorn, R. (1984) Plant Mol. Biol. 3, of the two Dl forms, the photosynthetic measurements 249-258. carried out in this study suggest that a significant functional 3. Mulligan, B., Schultes, N., Chen, L. & Bogorad, L. (1984) difference does exist between PSII reaction centers contain- Proc. Natl. Acad. Sci. USA 81, 2693-2697. ing either D1:1 or D1:2. In strains that synthesize only one 4. Golden, S. S., Brusslan, J. & Haselhorn, R. (1986) EMBO J. 5, form of the Dl protein, the apparent quantum yield of 2789-2798. photosynthesis, as measured by oxygen evolution, was 5. Jansson, C., Debus, R. J., Osiewacz, H. D., Gurevitz, M. & =25% higher in the strain with PSII reaction centers con- McIntosh, L. (1987) Plant Physiol. 85, 1021-1025. taining D1:2. This result is supported by chlorophyll a 6. Gingrich, J. C., Buzby, J. S., Stirewalt, V. L. & Bryant, D. A. fluorescence studies. The open PSII reaction centers in the (1988) Photosynth. Res. 16, 83-99. strain R2K1 were -25% more efficient in capturing excitation 7. Bouyoub, A., Vernotte, C. & Astier, C. (1993) Plant Mol. Biol. energy, as shown by the Fv'/FM' ratios, than open PSII 21, 249-258. 8. Schaefer, M. R. & Golden, S. S. (1989) J. Bacteriol. 171, reaction centers in R2S2C3. Since at the same time there was 3973-3981. little difference in the oxidation state ofthe quinone acceptor 9. Schaefer, M. R. & Golden, S. S. (1989) J. Biol. Chem. 264, QA and thus the proportion of open to closed PSII reaction 7412-7417. centers (Fig. 5), the higher yield of PSII electron transport 10. Bustos, S. A., Schaefer, M. R. & Golden, S. S. (1990) J. ((bps) in R2K1 compared with R2S2C3 was fully accounted Bacteriol. 172, 1998-2004. for by the higher trapping efficiency of PSII in R2K1. This 11. Kulkarni, R. D., Schaefer, M. R4 & Golden, S. S. (1992) J. suggests that PSII reaction centers containing D1:2 exhibit Bacteriol. 174, 3775-3781. higher oxygen-evolution rates than those containing D1:1, as 12. Clarke, A. K., Soitamo, A., Gustafsson, P. & Oquist, G. (1993) a consequence of harnessing a greater proportion of excita- Proc. Natl. Acad. Sci. USA 90, 9973-9977. tion energy, while at the same time maintaining the same 13. Krupa, Z., Oquist, G. & Gustafsson, P. (1990) Plant Physiol. proportion of PSII reaction centers in an open configuration, 93, 1-6. by 14. Myers, J., Graham, J. R. & Wang, R. T. (1978) J. Phycol. 14, presumably having a greater capacity for quenching 513-518. excitation energy through photosynthetic electron transport. 15. Clarke, A. K. & Critchley, C. (1990) PlantPhysiol. 94,567-576. Thus, R2K1 is not only more photochemically efficient under 16. Clarke, A. K. & Critchley, C. (1992) Plant Physiol. 100, conditions of limiting light but is able to maintain this 2081-2089. advantage at light intensities of up to 5 times the growth 17. van Kooten, 0. & Snel, J. F. H. (1990) Photosynth. Res. 25, irradiance through a persistently higher efficiency for energy 147-150. capture and for energy dissipation through photosynthetic 18. Genty, B., Briantais, J. M. & Baker, N. R. (1989) Biochim. electron transport. Biophys. Acta 990, 87-92. The fact that the variable photochemical yield of PSII 19. Mullineaux, C. W. & Allen, J. F. (1990) Photosynth. Res. 23, reaction centers in Synechococcus sp. PCC 7942 is 297-311. achieved 20. Schreiber, U., Schliwa, W. & Bilger, U. (1986) Photosynth. by varying at most only 25 amino acids within the 360 that Res. 10, 51-62. comprise the Dl protein suggests that some if not all of these 21. Miller, A. G., Espie, G. S. & Canvin, D. T. (1991) Can. J. Bot. differences between D1:1 and D1:2 are significant. The 69, 1151-1160. trapping efficiency of PSII (Fv'/FM') depends on the prob- 22. Krupa, Z., Oquist, G. & Gustafsson, P. (1991) Physiol. Plant. abilities of energy trapping, back-transfer from the reaction 82, 1-8. center to the antennae, and energy exchange between PSII 23. Greenberg, B. M., Gaba, V., Mattoo, A. K. & Edelman, M. units (27). It is not at present pQssible to decide on the (1987) EMBO J. 6, 2865-2869. mechanistic reason behind PSII reaction centers with D1:1 24. De Las Rivas, J., Andersson, B. & Barber, J. (1992) FEBSLett. 301, 246-252. showing a lower photochemical yield than those containing 25. Barbato, R., Frizzo, A., Friso, G., Rigona, F. & Giacometti, G. D1:2. Further, it is difficult to relate the increased trapping (1992) FEBS Lett. 304, 136-140. efficiency of PSII reaction centers with D1:2 to the prefer- 26. Svensson, B., Vass, I., Cedergren, E. & Styring, S. (1990) ential induction of D1:2 during high light stress. It is possible EMBO J. 9, 2051-2059. that the increased capacity of PSII with D1:2 to dissipate 27. Havaux, M., Strasser, R. J. & Greppin, H. (1991) Photosynth. excitation energy through photochemistry may reduce the Res. 27, 41-55. Downloaded by guest on September 26, 2021