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 (chlorophyll fluorescence/photosynthesis/protein turnover) ADRIAN K. CLARKE*, VAUGHAN M. HuRRY, PETTER GUSTAFSSON, AND GUNNAR OQUIST Department of Plant 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 plants 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).