Natural Variants of Photosystem II Subunit D1 Tune Photochemical Fitness to Solar Intensity

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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. ??, pp. 1–xxx, ???? ??, 2013 © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

Natural Variants of Photosystem II Subunit D1 Tune Photochemical Fitness to Solar Intensity*□ S

Received for publication, June 22, 2012, and in revised form, December 26, 2012 Published, JBC Papers in Press, December 27, 2012, DOI 10.1074/jbc.M112.394668

David J. Vinyard‡§¶1, Javier Gimpel!, Gennady M. Ananyev‡§, Mario A. Cornejo§, Susan S. Golden!, Stephen P. Mayfield!, and G. Charles Dismukes‡§2 From the ‡Department of Chemistry and Chemical Biology and §Waksman Institute of Microbiology, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854, the ¶Department of Chemistry, Princeton University, Princeton, New Jersey 08540, and the !San Diego Center for Algae Biotechnology, Division of Biological Sciences, University of California, San Diego, La Jolla, California 92093 AQ: A

Background: Cyanobacteria use multiple PSII-D1 isoforms to adapt to environmental conditions. Results: D1:2 achieves higher quantum efficiency of water oxidation and biomass accumulation rate at high light versus D1:1; the latter is more efficient at low light due to less charge recombination. Conclusion: A functional advantage for D1:1 is revealed for the first time. Significance: Improved photochemical efficiency at low light suggests an evolutionary advantage to retain D1:1. Photosystem II (PSII) is composed of six core polypeptides that make up the minimal unit capable of performing the primary photochemistry of light-driven charge separation and water oxidation in all oxygenic phototrophs. The D1 subunit of this complex contains most of the ligating amino acid residues for the Mn4CaO5 core of the water-oxidizing complex (WOC). Most cyanobacteria have 3–5 copies of the psbA gene coding for at least two isoforms of D1, whereas algae and plants have only one isoform. Synechococcus elongatus PCC 7942 contains two D1 isoforms; D1:1 is expressed under low light conditions, and D1:2 is up-regulated in high light or stress conditions. Using a heterologous psbA expression system in the green alga Chlamydomonas reinhardtii, we have measured growth rate, WOC cycle efficiency, and O2 yield as a function of D1:1, D1:2, or the native algal D1 isoform. D1:1-PSII cells outcompete D1:2-PSII cells and accumulate more biomass in light-limiting conditions. However, D1:2-PSII cells easily outcompete D1:1-PSII cells at high light intensities. The native C. reinhardtii-PSII WOC cycles less efficiently at all light intensities and produces less O2 than either cyanobacterial D1 isoform. D1:2-PSII makes more O2 per saturating flash than D1:1-PSII, but it exhibits lower WOC cycling efficiency at low light intensities due to a 40% faster charge recombination rate in the S3 state. These functional advantages of D1:1-PSII and D1:2-PSII at low and high light regimes, respectively, can be explained by differences in

* This work was supported in part by Division of Chemical Sciences, Geosci-

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ences, and Biosciences, Office of Basic Energy Sciences of the United States Department of Energy Grant DE-FG02-10ER16195 (to G. C. D.), Air Force Office of Scientific Research Grant FA9550-10-1-0052, and Department of Energy Grant DE-EE0003373, Consortium for Algal Biofuels Commercialization (CAB-COMM) (to S. P. M. and S. S. G.). □ S This article contains supplemental Figs. S1–S8, Tables S1 and S2, and an additional reference. 1 Supported by the Department of Defense, Army Research Office, National Defense Science and Engineering Graduate Fellowship 32CFR168a and National Science Foundation Graduate Research Fellowship DGE-0937373. 2 To whom correspondence should be addressed: Waksman Institute of Microbiology, Rutgers, The State University of New Jersey, 190 Frelinghuysen Rd., Piscataway, NJ 08854. Tel.: 732-445-6786; Fax: 732-445-5735; E-mail: [email protected].

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predicted redox potentials of PSII electron acceptors that control kinetic performance.

Photosystem II (PSII)3 is nature’s sole enzymatic solution to the challenging chemistry of water oxidation (1, 2). In PSII, solar energy is converted to chemical energy by splitting two molecules of water in the Mn4CaO5 (3) water-oxidizing complex (WOC) and reducing two plastoquinone (PQ) molecules to plastoquinols (H2PQ). The oxygen (O2) by-product from this reaction accounts for nearly all the O2 in the atmosphere (2). Regulation of solar energy conversion to these products in PSII is essential for cell survival and is controlled at multiple levels. The D1 protein of PSII is one of six core polypeptides that make up the minimal unit performing the primary photochemistry of light-driven charge separation and water oxidation in all oxygenic phototrophs. It provides most of the ligating amino acid residues for the WOC manganese core as well as binding pockets for the P680 chlorophyll-a (Chl-a) special pair, pheophytin (Pheo), and the secondary PQ acceptor. Because of its proximity to the WOC, it is frequently damaged by reactive oxygen species and is turned over faster than any other PSII protein subunit (4). The D1 protein is coded for by the gene psbA. Cyanobacteria contain 1–5 copies of psbA coding for 1–3 unique D1 isoforms per species. Cyanobacterial D1 isoforms are categorized into four groups as follows: D1m, D1!, D1:1, and D1:2 (4). D1m is most commonly associated with Synechocystis sp. PCC 6803 and is expressed under normal growth conditions (5, 6). D1! is up-regulated in low O2 (7) or microanaerobic (8) conditions. 3

The abbreviations used are: PSII, photosystem II; Chl, chlorophyll; ChlZ, sec" ondary electron chlorophyll donor to P680 ; cyt b559, cytochrome b559; DMBQ, 2,5-dimethyl-p-benzoquinone; E, Einstein; LED, light-emitting diode; P680, primary electron donor Chl special pair in PSII; Pheo, pheophytin; PQ, plastoquinone; QA, primary PQ acceptor in PSII; QY, quantum yield; TOF, turnover frequency; WOC, water-oxidizing complex; YD, PSII D2 polypeptide amino acid residue tyrosine 160; YO2, flash oxygen yield; YZ, PSII D1 polypeptide amino residue acid tyrosine 161; qPCR, quantitative PCR; STF, single turnover flash; FRR, fast repetition rate.

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Natural Variants of PSII Subunit D1 Tune Photochemical Fitness D1:1 and D1:2 were first identified and studied in Synechococcus elongatus PCC 7942 (hereafter Synechococcus 7942) (9) but have recently been examined in detail in Thermosynechococcus elongatus BP-1 (hereafter Thermosynechococcus) (10 –16). D1:1 is expressed during normal growth conditions at low or moderate light intensities (17). Upon exposure to high light (17–19), UV-B (20), chilling stress (21), or reductive stress (22), D1:2 is up-regulated and replaces D1:1 in PSII reaction centers. Algae and higher plants contain only one D1 isoform per species. It is coded in the chloroplast genome by two identical copies of the psbA gene in many green algae and typically one copy in higher plants (4). The algal/plant D1 isoform is structurally most similar to D1:2, given the presence of a Pheo hydrogen-bonding glutamate at position D1–130 (4). D1m, D1!, and D1:1 contain glutamine at position D1–130, which alters the midpoint potential of Pheo/Pheo$ by $33 mV in Synechocystis 6803 (23) and $17 mV in Thermosynechococcus (13, 24). In Synechococcus 7942 and Thermosynechococcus BP-1, PSII centers containing the D1:2 isoform have been shown to have higher O2 evolution rates (25), faster photoautotrophic growth " rates (26), more rapid tyrosine-Z (YZ) donation to P680 (24), and less sensitivity to photoinhibition (14, 20, 26 –29). However, “low light” D1:1 remains the dominant isoform in many cyanobacteria. Why have cyanobacteria maintained this seemingly inferior D1 isoform over billions of years of evolution? We hypothesized that under very low light intensities, D1:1 may have a functional advantage over D1:2. By expressing cyanobacterial D1 isoforms in a model green alga, we avoided background fluorescence interferences commonly encountered in experiments with cyanobacteria (30) and were thus able to quantitatively compare the D1:1, D1:2, and algal isoforms both in vivo and in vitro. Here, we show that when compared with the D1:2 and algal isoforms, D1:1 has higher WOC cycling efficiency at low light intensities, which is supported by the observation of a more stable S3 WOC intermediate. We attribute this improved efficiency to fewer competing PSII-cyclic electron $ transfers from native cofactors (including QA and cyt b559).

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EXPERIMENTAL PROCEDURES Mutant Construction—Heterologous psbA genes from Synechococcus 7942 were expressed in the chloroplast genome of the model green alga Chlamydomonas reinhardtii (hereafter Chlamydomonas). Chlamydomonas contains two identical psbA copies in the inverted repeat region of its chloroplast genome. A strain generated from wild type 137c (CC-125 mt") in which both psbA copies had been inactivated and the native gene reintroduced at a distal single copy site in the chloroplast genome was available from a previous study (31). This strain, psbA-m-saa3"psbA-psbA, accumulates native D1 protein at wild type levels and does not express the mammalian protein introduced in the psbA-m-saa strain (31). Here, we refer to this strain as C. reinhardtii-PSII. Gene sequences for the Synechococcus 7942 D1:1 and D1:2 (9) were codon-optimized for Chlamydomonas chloroplast by modifying the endogenous psbA sequence only in the codons for which amino acid substitutions were required. Additionally, we replaced the cleaved C-terminal peptide with that of Chlamydomonas to avoid potential processing incompatibilities (supplemental Fig. S1). Gene syn-

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thesis was carried out by GeneArt (Germany). The synthetic genes were introduced into psbA-m-saa by particle bombardment into the same site as in C. reinhardtii-PSII (32). The coding sequences were under the control of the Chlamydomonas psbA promoter, 5!- and 3!-untranslated regions. Transformants were selected for resistance to kanamycin and confirmed by PCR through sequencing of the PCR products. Both strains were rendered homoplasmic for the transgene insertion by propagation in kanamycin and confirmed by PCR. The resulting strains are referred to in this work as D1:1-PSII and D1:2-PSII. Culturing Conditions, Growth Measurements, and Chlorophyll Determination—Chlamydomonas strains were grown in HS medium (33) supplemented with 5 mM NaHCO3. For fast repetition rate fluorometry experiments, cultures were maintained in a turbidostat (FMT-150, Photon Systems Instruments, Brno, Czech Republic) with continuous illumination at 100 !E m$2 s$1 at 25 °C. Optical density (OD) at 730 nm was maintained at 0.200 % 0.005. For growth rate measurements, 40-ml cultures were grown in HS medium and continuously bubbled with 2% CO2 in air. Growth was monitored as OD at 730 nm. Full growth curves were recorded and then fit to a four-component Gompertz function (34) to calculate doubling times. For biomass accumulation experiments, 9-liter cultures were grown in 12-liter glass carboys in HS medium and continuously bubbled with 2% CO2 in air. After the stationary phase was reached (monitored by OD730 nm), cells were harvested by centrifugation and dried at 90 °C overnight. Total dry weight was determined gravimetrically. Chl was extracted in methanol, and relative concentrations of Chl-a and Chl-b were determined spectrophotometrically using extinction coefficients reported by Porra et al. (35). qPCR and Western Blots—All starting material corresponded to mid-log phase cultures (&106 cells/ml) grown at 100 !E m$2 s$1 and 25 °C. The Concert Plant RNA Reagent (Invitrogen) was used for all RNA extractions, following the small scale protocol with 5 ml of culture. 320 ng of total RNA were reversetranscribed using the Verso cDNA synthesis kit (Thermo Scientific, Waltham, MA). 20-!l reactions were diluted 10-fold, and 2 !l were used for qPCR with the Absolute Fast kit for probes and without ROX (Thermo Scientific) according to the manufacturer’s instructions. qPCR assays were done in a CFX96 thermal cycler (Bio-Rad). Samples from each strain were run with five replicates. The rbcL gene was used as a reference (36). Expression levels were calculated with the CFX manager software (Bio-Rad) using the ''Ct method. Amplification efficiencies ranged from 90.7 to 93.2% and were also considered for the expression analysis. All qPCR primers and probes were purchased pre-mixed from Integrated DNA Technologies (Coralville, IA) and are shown in supplemental Table S1. Westerns blots were performed as described previously (37) with slight modifications. Total protein extracts were used instead of insoluble fractions. Total protein was quantified using the DC Protein Assay (Bio-Rad). The loading buffer also included 2 M urea, and denaturation was performed on ice for 30 min. The primary antibody corresponded to anti-D1 rabbit polyclonal raised against a conserved N-terminal peptide of the VOLUME 288 • NUMBER ?? • ???? ??, 2013

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protein (Agrisera, Sweden). Dylight 488-conjugated anti-rabbit was used as the secondary antibody (Thermo Scientific). Chl fluorescence from light-harvesting complex II monomers was used as a loading control (38). Fluorescence imaging was carried out with a Typhoon 8600 scanner (GE Healthcare). Image analysis and densitometry was performed with ImageJ (rsb.info.nih.gov). Competition Assays—An equal number of D1:1-PSII and D1:2-PSII cells (determined by hemocytometer) were inoculated in either TAP medium (dark control) or HS medium supplemented with 5 mM NaHCO3 in vented large surface area untreated tissue culture flasks. Mixed cultures in TAP medium were grown in complete darkness without shaking at 25 °C. Mixed cultures in HS medium were grown at either 3 or 290 !E m$2 s$1 with shaking (80 rpm) at 25 °C. For each time point, the total cell count of each flask was determined by a hemocytometer. Each resulting PCR used &1000 cells from the mixed culture for DNA template. Primers were designed that did not discriminate between the DNA sequences of D1:1-PSII or D1:2PSII (supplemental Fig. S1) as follows: forward primer, TTCTAACGCAATCGGTCT, and reverse primer, GAATAATGAACCACCGAAT, which results in a 379-bp gene fragment. By chance, the D1:2-PSII gene sequence contains a PvuII restriction site (CAG2CTG) that is not present in the D1:1PSII gene sequence (supplemental Fig. S1) and was used for identification. The 379-bp PCR fragment from D1:2-PSII DNA can be cleaved into 167- and 212-bp fragments by PvuII nuclease, although the 379-bp PCR fragment from D1:1-PSII DNA is unaffected (supplemental Fig. S2). Following 32 PCR cycles, two 10-!l aliquots were taken from each reaction mixture. 5 units of PvuII (New England Biolabs) were added to only 1 aliquot, and both were incubated at 37 °C for 40 min. Then both aliquots were run on a 1.2% agarose gel at 80 V for &45 min and visualized using ethidium bromide. The fluorescence intensity of the 379-bp fragment in the control aliquot (no PvuII) was proportional to the sum total of D1:1-PSII and D1:2PSII genes in the original mixed culture. The fluorescence intensity of the 379-bp fragment in the PvuII-treated fragment was proportional to the concentration of D1:1-PSII genes in the original mixed culture. These intensities were quantified using ImageJ and used to determine the fraction D1:1-PSII in the mixed culture at each specific time point. Knowledge of the total cell count and the fraction of D1:1-PSII enabled the determination of the number of D1:1-PSII cells and the number of D1:2-PSII cells. Isolation of Thylakoid Membrane Fragments—Thylakoid membrane fragments were isolated from Chlamydomonas strains using a modified procedure by Shim et al. and Gokhale and Sayre (39, 40), which is based in turn on the method for spinach by Berthold et al. (41). Briefly, &1-liter cultures were grown in HS medium supplemented with 5 mM NaHCO3 on a 12-h/12-h light/dark cycle at 100 !E m$2 s$1 and 25 °C. To synchronize growth phase, cells were harvested by centrifugation (3,500 ( g, 10 min) at 4 h into the third light cycle (42). Cells were resuspended in Buffer 1 (20 mM HEPES, pH 7.5, 350 mM sucrose, 2.0 mM MgCl2) at 1–2 mg of Chl/ml and then disrupted using a BeadBeater (BioSpec Products, Bartlesville, ???? ??, 2013 • VOLUME 288 • NUMBER ??

OK) with 0.5-mm zirconia beads. Thylakoid membranes were isolated by centrifugation (40,000 ( g, 20 min) to pellet thylakoids and whole cells, resuspended in Buffer 1, centrifuged (1200 ( g, 30 s) to remove cell debris and whole cells, and then centrifuged (40,000 ( g, 20 min) to pellet thylakoids. Purified thylakoids were resuspended in Buffer B (20 mM MES, pH 6.0, 15 mM NaCl, 5.0 mM MgCl2, 5.0 mM EDTA) at "2.86 mg Chl/ ml. 25% Triton X-100 (20 mM MES, pH 6.0, 15 mM NaCl, 5.0 mM MgCl2) was slowly added while stirring to a final Triton X-100/ Chl ratio of 20:1 and Chl concentration of 2.0 mg/ml. Following slow stirring for 25 min, membrane fragments were washed three times with Buffer B (40,000 ( g, 20 min) and flash-frozen in liquid N2 at &2 mg of Chl/ml in 25% glycerol, 50 mM MES, pH 6.0, 300 mM sucrose, 35 mM NaCl. All steps following cell harvesting were carried out at 4 °C under dim green light. All buffers contained 0.1 mM phenylmethanesulfonyl fluoride and 1.0 mM benzamidine as protease inhibitors. EPR Spectroscopy—EPR measurements on thylakoid membrane fragments were made on a Bruker Elexsys E580 at 9.45 GHz frequency. Specific measurement conditions are provided in supplemental Fig. S3. Spin quantification was performed using Fremy’s salt (K2NO(SO3)2, Sigma) standards, as described by Babcock et al. (43). Lithium Dodecyl Sulfate-PAGE—25 !g of total protein of thylakoid membrane fragments were denatured in 2% lithium dodecyl sulfate and 0.5% #-mercaptoethanol on ice for 10 min. Samples were loaded into wells of a 4 –16% polyacrylamide gel (Bio-Rad) and run at 4 °C at 1 watt constant power for &26 h. Bands were visualized using silver staining. O2 Evolution Rates—Steady-state O2 evolution rates were measured using a commercial Clark-type electrode (Hansatech) at 25 °C. Isolated membranes were diluted to 1–3.5 !g of Chl/ml in 40 mM MES, pH 6.0, 200 mM sucrose, 10 mM CaCl2, 10 mM MgCl2, and 10 mM NaCl. Freshly prepared 2 mM K3Fe(CN)6 and 0.1 mM 2,5-dimethyl-p-benzoquinone (DMBQ) or 2,5-dichloro-p-benzoquinone were added immediately before each experiment began. Continuous saturating illumination (for the Chl concentrations used) was provided by a red LED (680 !E m$2 s$1, $max # 627 nm). Flash O2—Flash O2 yields were measured amperometrically using a home-built Clark-type electrode (membrane-covered Pt-Ir electrode) as described previously (44). A red LED (6,200 !E m$2 s$1, $max # 627 nm) was used to provide single turnover flashes (STFs). Optimal STF duration for 0.5 !M PSII was determined to be 30 !s. Amperometric response to each STF was detected over a 1-s window and then integrated with baseline correction to determine the O2 yield per flash. Thylakoid membrane fragments were diluted to 0.5 !M PSII in 40 mM MES, pH 6.0, 200 mM sucrose, 10 mM CaCl2, 10 mM MgCl2, and 10 mM NaCl. Freshly prepared 2 mM K3Fe(CN)6 and 0.1 mM DMBQ were added immediately before each experiment began. Samples were dark-adapted for 5 min, pre-flashed with one 30-!s STF, dark-adapted for 5 min, and then subjected to 30 30-!s STFs applied at 0.1–1 Hz. Fast Repetition Rate (FRR) Fluorometry—FRR fluorometry measurements were performed on a home-built instrument as described previously (45). Samples of whole Chlamydomonas cells at 50 !g of Chl/ml were dark-adapted for 120 s and then JOURNAL OF BIOLOGICAL CHEMISTRY

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subjected to 50 STFs applied at 0.2–100 Hz. To prevent physiological adaptations to dark conditions during the &3-h experimental period, a 1-s flash from a blue LED (200 !E m$2 s$1) was applied prior to each 120-s dark adaptation period. Fourier Transform Analysis—The temporal frequencies comprising the oscillating traces from flash O2 and FRR fluorometry experiments were determined by Fourier transform as described previously (45, 46) with minor alterations. Prior to Fourier transformation, constant steady-state yields were subtracted from full traces to provide a base line of zero. Discrete Fourier transforms were calculated and fit to a cubic spline. The cycle$1 value corresponding to the maximal Fourier amplitude in the physical range of 0 – 0.5 cycle$1 was determined. The reciprocal of this value was defined as the period. The shortest theoretical period was 4. Kok Cycle Modeling—The rates of damping of flash O2 yield and flash Chl fluorescence ratio (Fv/Fm) were estimated by fitting to an extended version of the Kok model (47, 48), utilizing matrix analysis of the Markov process (49), extended to include S-state selective transitions. The transition probability matrix is shown in Scheme 1, where %, #, &, ', and ( represent misses, double hits, hits, backward transitions, and inactivations, respectively. Each matrix element aij represents the transition probability from Sj$1 to Si$1, where 1 ) i,j ) 5. The fifth S-state in this model is an inactive state S( accessed via inactivations of PSII centers during the experiment (50). Conservation of matter implies that the sum of each column must equal 1, and therefore the &?#n (hit) parameters are written as &0 # &1 # 1-%-#, &2 # 1-%-#-', and &3 # 1-%-#-'-(. Thus, the average hit probability &avg # 1-%-#-'/2-(/4. The yield of O2, YO2, produced by a given flash is proportional to the population passing through the S3 3 S0 transition (47). For this model, YO2(n) # (1-%-'-()S3(n $ 1) " #S2(n $ 1). We note that this model is similar to the extended Kok model proposed by Shinkarev (51) with the following alterations. First, the thermodynamically unreasonable backward transitions of S0 3 S3 and S1 3 S0 have been removed. We hypothesize that inactivations of the PSII reaction centers are most probable during the release of O2, which is known to generate reactive oxygen species (1). Thus, the inactive state, S(, is accessible solely through the precursor to the O2-evolving step, S3. Direct inactivation of S3 occurs with probability (. Double hit transitions passing through S3 access S( with probability #(. Limiting access to S( via S3 instead of allowing all S-states to directly feed into S( does not affect the calculated magnitude of (.4 Full oscillating traces from flash O2 or FRR fluorometry were normalized to achieve a steady-state value of 1. Fittings were 4

D. J. Vinyard, C. E. Zachary, G. M. Ananyev, and G. C. Dismukes, manuscript in preparation.

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FIGURE 1. Photoautotrophic growth curves of C. reinhardtii-PSII (●), D1:1-PSII ("), and D1:2-PSII (‚) at 15 !E m"2 s"1 in HS medium. Cultures were continually bubbled with 2% CO2 in air. Doubling times are inset. Data represent the means of four biological replicates with standard error.

numerically determined by minimizing an objective function proportional to the mean square deviation from the experimental curve using the BOBYQA algorithm (52) in the NLopt nonlinear optimization package (53). For flash O2 experiments, YO2 was followed as a function of flash number. For FRR fluorometry experiments, the population of S1 was followed. S3 Lifetime Measurements—Changes in the flash O2 oscillation pattern following two pre-flashes was used to monitor S3 lifetimes (54, 55). PSII samples as described above were darkadapted for 5 min, pre-flashed with one 30-!s STF, darkadapted for 5 min, pre-flashed twice with 30-!s STFs at 1 Hz, and then subjected to 30 30-!s STFs applied at 1 Hz. The resulting oscillation traces were fit to the extended Kok model as described above. The relative initial population of S3 (S3/(S0 " S1 " S2 " S3)) was plotted as a function of the delay time between the two pre-flashes and the 30 STF train. Data were fit to the two-component exponential decay function y # y0 " A1exp($x/*1) " A2exp($x/*2).

RESULTS Characterization of D1 Mutants—Chlamydomonas strains containing heterologous psbA genes from Synechococcus 7942 were photoautotrophic and grew at identical rates as C. reinhardtii-PSII at moderate light intensity (15 !E m$2 s$1, Fig. 1). At stationary phase, the OD730 nm of D1:1-PSII cultures was significantly higher than C. reinhardtii-PSII or D1:2-PSII, indicative of more cells or more absorbance per cell. Table 1 shows the ratio of Chl-a to Chl-b during exponential growth in photoautotrophic conditions. In whole cells, this ratio reflects the relative amount of reaction centers (Chl-a) to antenna (Chl-a"b). No significant difference in Chl-a/b was observed between D1:1-PSII and D1:2-PSII strains at any light intensity tested. However, both heterologous strains have a significantly higher Chl-a/b ratio than the C. reinhardtii-PSII strain. To better understand this phenotype, large scale cultures (9 liters) were inoculated in HS medium and bubbled with 2% CO2 in air. Upon reaching stationary phase (monitored by VOLUME 288 • NUMBER ?? • ???? ??, 2013

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Natural Variants of PSII Subunit D1 Tune Photochemical Fitness TABLE 1 Chl-a/b ratio of C. reinhardtii-PSII, D1:1-PSII, and D1:2-PSII intact cells in HS medium # 5 mM HCO3" Data represent means of four biological replicates with standard error.

C. reinhardtii-PSII D1:1-PSII D1:2-PSII

T2

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T3

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25 !E m"2 s"1

80 !E m"2 s"1

160 !E m"2 s"1

3.13 % 0.08 3.94 % 0.09 3.90 % 0.12

3.43 % 0.12 3.87 % 0.12 3.97 % 0.11

3.05 % 0.09 3.70 % 0.08 3.92 % 0.18

OD730 nm), the biomass yield was measured gravimetrically. As shown in Table 2, D1:1-PSII accumulates 11% more biomass than C. reinhardtii-PSII (p # 0.0269) and 12% more biomass than D1:2-PSII (p # 0.0474) in the light-limiting conditions of this experiment. To test for a growth advantage between the D1:1-PSII and D1:2-PSII cells at various light intensities, an equal number of cells from each strain was inoculated into a mixed culture. The relative genotype of the mixed culture was monitored over time as described under “Experimental Procedures.” Standardization of the method established a linear correlation (R2 # 0.9777, see supplemental Fig. S2). In cells grown heterotrophically in darkness (TAP medium), no discernable difference between strains was observed (Fig. 2A). Mixed cultures grown photoautotrophically (HS medium " 5 mM HCO3$) at 3 !E m$2 s$1 contained &30% more D1:1-PSII cells than D1:2-PSII cells after 10 days (Fig. 2B). By contrast, at high light fluxes (290 !E m$2 s$1), D1:2-PSII cells were in 500% excess after 8 days compared with D1:1-PSII cells (Fig. 2C). RT-PCR results of psbA transcript levels are listed in Table 3. When normalized to total Chl concentration and the C. reinhardtii-PSII strain, D1:1-PSII and D1:2 strains accumulate &30 – 45% less psbA transcript. These data are in agreement with D1 protein quantification (Table 3 and supplemental Fig. S3) in which an approximate 40% decrease in D1 accumulation was observed for D1:1-PSII and D1:2-PSII compared with C. reinhardtii-PSII. Characterization of Thylakoid Membrane Fragments—PSII concentration in thylakoid membrane fragments prepared from C. reinhardtii-PSII, D1:1-PSII, and D1:2-PSII was estimated through spin quantification of tyrosine YD# detected using EPR. Light minus dark difference spectra were obtained that allow separation of P700" and the fast decaying tyrosine YZ# from the slow decaying YD# (43). Spectra of samples that were illuminated for 30 s at room temperature and then darkadapted for 30 min at 0 °C (supplemental Fig. S4) were integrated (supplemental Fig. S5), and the results are listed in Table 3. YD# decays very slowly in the dark at pH ) 7.2 with biphasic kinetics. At 21 °C, Vass and Styring (56) measured these kinetics in PSII-enriched spinach grana membranes as t1⁄2f # 41 min (21%) and t1⁄2s # 510 min (79%). The faster component represents the oxidation of S0 by YD# and has an activation energy of 30 kJ/mol (55). Using these values, we can estimate that at 0 °C, less than 7% of YD# has been reduced during the 30-min dark adaptation. D1:1-PSII and D1:2-PSII thylakoid membrane fragments have 2-fold higher Chl content per YD# than those from C. reinhardtii-PSII. We emphasize that these preparations also contain significant amounts of PSI as evidenced by the lightadapted EPR spectra (supplemental Fig. S4) and lithium dode???? ??, 2013 • VOLUME 288 • NUMBER ??

TABLE 2 Photoautotrophic biomass accumulation at stationary phase of 9-liter cultures at 60 !E m"2 s"1 in HS medium bubbled with 2% CO2 in air Dry weight C. reinhardtii-PSII D1:1-PSII D1:2-PSII

g/liter

0.237 % 0.003 0.263 % 0.007 0.235 % 0.007

FIGURE 2. Competition assays of D1:1-PSII (") and D1:2-PSII ($) cells at 0 !E m"2 s"1 in TAP medium (A), 3 !E m"2 s"1 in HS medium # 5 mM HCO3" (B), and 290 !E m"2 s"1 in HS medium # 5 mM HCO3" (C). Data represent the means of four (C) or five (A and B) biological replicates with standard error. Experimental design is detailed in the text.

cyl sulfate-polyacrylamide gel (supplemental Fig. S6). This feature arises naturally due to the lack of well ordered grana in the Chlamydomonas chloroplast. JOURNAL OF BIOLOGICAL CHEMISTRY

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Natural Variants of PSII Subunit D1 Tune Photochemical Fitness TABLE 3 Quantification of psbA transcript and D1 protein in whole cells and YD in thylakoid membrane fragments

ZSI ZSI

Transcript and protein data are normalized to total Chl-a"b and values of the C. reinhardtii-PSII strain and represent means of five (qPCR) or three (Western blot) biological replicates with standard error. Thylakoid membrane fragments were prepared from cells growth at 100 !E m$2 s$1 (detailed under “Experimental Procedures”). For EPR quantification of YD in thylakoid membrane fragments, dark-adapted spectra (supplemental Fig. S3) were integrated twice to calculate the area under the absorbance spectra. Spin quantification was performed using Fremy’s salt (supplemental Fig. S4). O2 evolution was measured at 25 °C under saturating light intensity in the presence of 0.1 mM DMBQ and 2 mM ferricyanide. Whole cells

psbA mRNA

D1 protein

Thylakoid membrane fragments [Chl]

Chl-a/b

mg/ml

C. reinhardtii-PSII D1:1-PSII D1:2-PSII

ZSI

F3 ZSI F4

F5

ZSI F6

1.00 % 0.07 0.71 % 0.06 0.55 % 0.03

1.00 % 0.14 0.63 % 0.03 0.59 % 0.01

2.01 2.47 2.57

Chl/YD

!M

2.46 2.75 2.71

Knowledge of the Chl/YD ratios and the O2 evolution rates normalized to Chl (Table 3) allowed for the determination of PSII turnover frequencies (TOFs). With DMBQ, D1:2-PSII had a slightly faster TOF (31.1 s$1) followed by D1:1-PSII (26.9 s$1) and then C. reinhardtii-PSII (21.9 s$1). The use of an alternative electron acceptor (2,5-dichloro-p-benzoquinone) did not affect this trend, and the values closely agree with the DMBQ results (supplemental Table S2). These TOFs are similar to rates reported for Chlamydomonas PSII-enriched thylakoid membrane fragments (45 s$1) (39) and core particles (27 s$1) (57). In Vitro Flash O2 Yield—Flash O2 yield from thylakoid membrane fragments was measured for 30 STFs at repetition frequencies from 0.1 to 1 Hz. Excluding the dark adaptation period, these STF frequencies correspond to constant illumination light intensities of 0.02– 0.2 !E m$2 s$1. A representative oscillation trace at 0.5 Hz is shown in Fig. 3A. Full data are available in supplemental Fig. S7. The O2 yield from 30 STFs, YO2, was averaged for each trace as shown in Fig. 4. When normalized to YD-PSII concentration D1:2-PSII produces the most O2 per flash followed by D1:1-PSII. C. reinhardtii-PSII has significantly lower YO2 over the STF frequencies tested. In Vitro WOC Cycling Efficiency—The rate of damping of oscillations in YO2 was monitored by Fourier transform (model independent, Fig. 5A) or fitting to our extended Kok model (Fig. 5B). In general, both period$1 and average Kok cycle hits increase with increasing STF frequencies. Both methods indicate that oscillations are of higher quality (slower rate of damping) in D1:1-PSII followed by D1:2-PSII. C. reinhardtii-PSII has significantly lower period$1 and Kok model hits (&avg). Variable Chl-a Fluorescence Yield—FRR fluorometry analysis allows in vivo measurements of whole cells over a wider range of frequencies than O2 detection using a Clark-type electrode (45). The variable Chl-a fluorescence yield (Fv/Fm) for whole cells was measured for 50 STFs at repetition frequencies from 0.2 to 100 Hz. Excluding the dark adaptation period, these STF frequencies correspond to constant illumination light intensities of 0.16 – 80 !E m$2 s$1. A representative oscillation trace at 0.5 Hz is shown in Fig. 3B. Full data are available in supplemental Fig. S8. The average Fv/Fm value from the first 50 STFs, denoted *Fv/Fm+, is shown in Fig. 6. *Fv/Fm+ decreases with increasing STF frequency for all strains. Whole cells containing C. reinhardtii-PSII have the highest average Fv/Fm values followed by D1:2-PSII and then D1:1-PSII.

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[YD#] 6.80 4.10 4.32

331 674 666

O2 evolution rate

PSII turnover frequency

!mol O2 (mg Chl)$1 h$1

mol O2 (mol PSII)$1 s$1

267 161 188

21.9 26.9 31.1

In Vivo WOC Cycling Efficiency—The rate of damping of oscillations in Fv/Fm (supplemental Fig. S8) was monitored by Fourier transform (model-independent, Fig. 7A) or fitting to our extended Kok model (model-dependent, Fig. 7B). In agreement with WOC cycling efficiency in vitro measured by YO2, in vivo FRR fluorometry experiments indicate a general increase in both period$1 and Kok model hits with increasing STF frequencies. C. reinhardtii-PSII whole cells have significantly faster damping of Fv/Fm oscillations at most STF frequencies tested. At low STF frequencies ()1 Hz), D1:1-PSII whole cells have the highest quality Fv/Fm oscillations followed by D1:2PSII then C. reinhardtii-PSII. S3 State Lifetime—The lifetime of the S3 intermediate of the Kok cycle was monitored in thylakoid membrane fragments from the O2 yield. As shown in Fig. 8, S3 decays significantly more slowly in D1:1-PSII compared with D1:2-PSII and C. reinhardtii-PSII.

DISCUSSION Varying the Relative Amounts of the D1 Isoforms in Response to Light Intensity Provides a Growth Advantage—D1:1-PSII is uniquely able to achieve higher biomass accumulation at low light intensities. 1) D1:1-PSII cells grown photoautotrophically at low light intensity (15 !E m$2 s$1) achieved higher OD730 nm (cell density) at stationary phase than either C. reinhardtii-PSII or D1:2-PSII (Fig. 1). 2) This observation was corroborated in large scale mono-cultures under light-limiting conditions as follows: D1:1-PSII accumulated 11–12% more biomass (dry weight) than either C. reinhardtii-PSII or D1:2-PSII (Table 2). 3) In shaded mixed cultures at 3 !E m$2 s$1, D1:1-PSII cells outcompeted D1:2-PSII cells by 30% after 10 days (Fig. 2B). However, D1:2-PSII was clearly far better adapted to grow at very high light intensities. At 290 !E m$2 s$1, D1:2-PSII cells outcompeted D1:1-PSII cells by 500% after 8 days (Fig. 2C). Similar growth competition experiments using Synechococcus 7942 mutants that express only D1:1 or D1:2 indicated that D1:2 is especially important during a transition period after cells are exposed to higher light intensities (58). Chlamydomonas Cells Adapt Their Chl/PSII Ratio When Expressing Heterologous psbA Genes—Chlamydomonas expressing cyanobacterial D1:1 and D1:2 produced 37% less PSII centers ([YD#]) and 37– 41% less D1 protein (Western blots) than the recombinant C. reinhardtii control (Table 3). This drop in PSII reaction centers was accompanied by a 2-fold VOLUME 288 • NUMBER ?? • ???? ??, 2013

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FIGURE 3. Comparison of flash O2 yield of thylakoid membrane fragments (using DMBQ as an artificial electron acceptor and denoted in vitro in the main text) (A) and in vivo variable Chl-a fluorescence, Fv/Fm, of intact cells (B) at 0.2 Hz for C. reinhardtii-PSII (●), D1:1-PSII ("), and D1:2-PSII (‚). Data sets were normalized to steady-state values and offset to directly compare the damping of oscillations. Fourier transforms are inset.

FIGURE 4. Flash O2 yields of thylakoid membrane fragments (in vitro, as in Fig. 3) prepared from C. reinhardtii-PSII (●), D1:1-PSII ("), and D1:2-PSII (‚) when normalized to PSII-YD. Data represent average flash O2 yield over the initial 30 STFs for three replicates with standard error. Chl concentrations corresponding to 0.5 !M PSII were C. reinhardtii (114 !g/ml), D1:1 (214 !g/ml), and D1:2 (197 !g/ml).

increase in the ratio of Chl:PSII and higher Chl-a/b ratios (Table 3). The higher Chl:PSII ratio likely results from the lower steady-state pool of psbA transcripts in these strains compared with the C. reinhardtii-PSII strain (Table 3). Given that all three constructs utilize the same native psbA promoter and thylakoid-targeting sequence from Chlamydomonas, we hypothesize that the heterologous transcripts are either less stable in vivo or that cells adapt to the presence of the photochemically more efficient hybrid PSII enzyme by making less psbA product. However, the modified codons in the synthetic genes might also play a role in decreasing translation efficiency (59, 60). The higher Chl-a/b ratios seen for D1:1-PSII and D1:2-PSII compared with C. reinhardtii-PSII (Tables 1 and 3) may result from either an increased competition between Chl-a and Chl-b for binding to LHC antenna proteins or the smaller total number of PSII centers (which bind only Chl-a). Alternatively, the PSI content in D1:1-PSII and D1:2-PSII thylakoid membrane frag???? ??, 2013 • VOLUME 288 • NUMBER ??

ments may significantly differ compared with C. reinhardtiiPSII. Our present data do not allow distinction between these options. Normalization of O2 Rates to PSII Concentration Reveals That D1:2-PSII Has a Higher Intrinsic TOF—Given the measured differences in D1 accumulation in whole cells, accumulation of tyrosine-D (YD) radical in thylakoid membranes and Chl content, O2 measurements were normalized to the absolute concentration of PSII centers instead of total Chl. The trend in D1 protein content in whole cells quantitatively tracks with the YD radical content in thylakoid membranes, which further corroborates the normalization to PSII. The TOF in thylakoid membranes is proportional to the intrinsic efficiency of PSII and is independent of downstream electron acceptors due to use of an exogenous quinone electron acceptor. D1:1-PSII Has Higher WOC Cycling Efficiency at Low STF Frequencies—The inverse period and &avg, representing intrinsic WOC cycling efficiency, are substantially higher for D1:1PSII than for D1:2-PSII at low STF frequencies, and both mutants are much higher than C. reinhardtii-PSII at all measured frequencies (Figs. 5 and 7). These data reveal the significant operational advantage of the D1:1 isoform over D1:2 at low light intensities that was not previously recognized. One way to interpret this observation is that cytochrome (cyt) b559 is more readily photo-reduced in D1:2-PSII (and C. reinhardtii-PSII) than D1:1-PSII as originally hypothesized by Sander et al. (26). Spectroscopically, contributions from such side pathway reactions were found to have a lower quantum efficiency in isolated Thermosynechococcus PSII core complexes containing the D1:1 isoform (11). In addition to YZ donation and cyclic electron " flow around PSII through cyt b559, the hole in P680 may be filled " $ by the nonradiative recombination of {P680QA } (61, 62). The thermodynamics and kinetics of this process are discussed below. Fv/Fm Does Not Predict YO2 and WOC Cycling Efficiency—In vitro, YO2 increases with increasing STF frequency (0.1–1 Hz) (Fig. 4A), whereas in vivo, the average *Fv/Fm+ decreases with increasing STF frequency (0.2–100 Hz) (Fig. 6). In both cases, JOURNAL OF BIOLOGICAL CHEMISTRY

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FIGURE 5. A, period$1 of oscillations of flash O2 yield from thylakoid membrane fragments (in vitro, as in Fig. 3) as determined by the peak of the Fourier transform. B, average hit parameter (&avg) of oscillations calculated from fitting to an extended Kok model. C. reinhardtii-PSII (●), D1:1-PSII ("), and D1:2-PSII (‚). Full oscillations are presented in supplemental Fig. S5. Data represent means of three replicates with standard error.

FIGURE 6. Average variable Chl-a fluorescence yield (*Fv/Fm+) summed over 50 STFs of intact cells of C. reinhardtii-PSII (●), D1:1-PSII ("), and D1:2-PSII ($). The slopes of the three traces obtained by fitting to a straight line are $0.0651, $0.0342, and $0.0471, respectively. Data represent means of three biological replications with standard error.

the quality of transient oscillations improves with increasing STF frequency (Figs. 5 and 7). This longer coherence of oscillations is quantified both by the (model-independent) period$1 and the (model-dependent) Kok hit parameter (&avg). Both reflect the cycling efficiency of the WOC. Fv/Fm is proportional to the quotient of quantum yield (QY) of PSII charge separation divided by the total Chl emission yield (2). Accordingly, it does not depend solely on PSII and is not the intrinsic QY of PSII photochemistry. However, YO2 reports on the net conversion as follows: 2H2O 3 O2 " 4H" " 4e$, and thus measures the intrinsic photochemical QY of the WOC. We therefore draw attention to TOF, YO2, period$1, and &avg as the more relevant measures of PSII function. Discrepancies between Fv/Fm and YO2 have been observed previously at low light intensities (analogous to low STF frequencies) and were attributed to cyclic electron flow around PSII through cyt b559 (63). Following an actinic pulse, cyt b559 is reduced in chloroplasts with a t1⁄2 of &100 ms (64). In vitro experiments in spinach have indicated that cyt b559 can then " reduce oxidized ChlZ", which fills the hole in P680 with a t1⁄2 of &500 ms (65, 66). These relatively slow kinetics are faster than the dark time between STFs in the YO2 experiments in this " work. If such a pathway is active, P680 would be competitively reduced by YZ (which in turn oxidizes the WOC releasing O2)

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and cyt b559/ChlZ. Thus, Fv/Fm does not scale directly with YO2 (WOC photochemical QY), period$1, and &avg (WOC cycling efficiency) and should be interpreted accordingly. At all STF frequencies, Fv/Fm is higher in C. reinhardtii-PSII followed by D1:2-PSII and least in D1:1-PSII (Fig. 6). Because of the large increase in Chl content (and hence F0 fluorescence) in the mutants, this sequence is not surprising but also not particularly revealing. However, the change in Fv/Fm values over the 500-fold STF frequency range, i.e. slope, decreases at different rates and should reflect the Fv contribution (photochemical charge separation within PSII). The average slope obtained by least squares fitting to a straight line reveals the following trend: C. reinhardtii-PSII ($0.065) , D1:2-PSII ($0.047) , D1.1PSII ($0.034). We suggest the larger slope for C. reinhardtiiPSII reflects greater regulation of PSII charge separation within the native strain than the mutants. Although the cyanobacterial D1 isoforms outperform the algal D1 isoform at all flash frequencies as revealed both by WOC cycling efficiency (Fig. 5) and by WOC photochemical QY (Fig. 6), stronger regulation of PSII charge separation in the native D1 isoform may be an advantage for overall survival fitness (i.e. photoprotection). Structure-Function Analyses of D1 Isoforms and Evolutionary Implications—The midpoint potential (Em) of Pheo/Pheo$ is regulated by the presence of a glutamine (D1:1-PSII) or glutamate (D1:2-PSII and C. reinhardtii-PSII) at D1 position 130. Recently, Em values for Pheo/Pheo$ in Thermosynechococcus BP-1 were determined at $522 mV for D1:1 (24) and $505 mV for D1:2 (13). This difference was found to be more subtle than previous experiments in Synechocystis 6803 in which substitution of Gln-130 with Glu-130 resulted in a $33 mV difference in the Em of Pheo/Pheo$ (23). The less negative Em of Pheo/ Pheo$ in D1:2 supports enhanced charge separation efficiency by increasing the free energy gap between P680* and Pheo (23, 24, 61, 62). Given the results from these previous studies, it is reasonable to expect Em(Pheo/Pheo$) to be more negative (higher energy) in D1:1-PSII (containing Gln-130) compared with D1:2-PSII and C. reinhardtii-PSII (both containing Glu-130). The Em of $ QA/QA also varies with the D1 isoform. In Thermosynechococ$ cus, Em (QA/QA ) was found to be $140 mV in D1:1 (15) and $ $103 mV in D1:2 (14). Variations in the Em of QA/QA control " $ the kinetics of {P680QA } recombination as discussed by Vass and Cser (62) as follows. Because the free energy gap between $ " QA and P680 is very large (,1 V), the kinetics of this tunneling VOLUME 288 • NUMBER ?? • ???? ??, 2013

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Natural Variants of PSII Subunit D1 Tune Photochemical Fitness

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FIGURE 7. A, Period$1 of oscillations of Fv/Fm in intact cells as determined by the peak of the Fourier transform. B, average hit parameter (&avg) of oscillations calculated from fitting to an extended Kok model. C. reinhardtii-PSII (●), D1:1-PSII ("), and D1:2-PSII ('). Full oscillations are presented in supplemental Fig. S6. Data represent means of three biological replicates with standard error.

FIGURE 8. S3 state population measured as the decay kinetics in thylakoid membrane fragments prepared from C. reinhardtii-PSII (●), D1:1-PSII ("), and D1:2-PSII ($). Lines represent fits to a two-component exponential decay. Lifetimes are inset. Data represent means of three replicates with standard error. Asterisks indicate statistical significance (p ) 0.05) between D1:1-PSII and D1:2-PSII values.

reaction are described as existing in an inverted Marcus region (62). Thus, a smaller 'Em is associated with faster recombina$ tion. It therefore follows that D1:2 (lower Em(QA/QA )) has " $ faster {P680QA } recombination kinetics than D1:1 (24, 26, 62, 67). " $ The nonradiative direct {P680 QA } recombination contributes to photoprotection at high light intensities. Increasing the rate of this recombination decreases the probability of forming tri" plet 3{P680 Pheo$}, which generates singlet O2 molecules that are known to damage the photosynthetic apparatus (62). It has thus been concluded by Vass and Cser (62), Sugiura et al. (24), and Rutherford et al. (61) that D1:2 is specifically tuned for enhanced photoprotection at high light intensities, whereas D1:1 is more prone to photoinhibition, which has been repeatedly demonstrated in the literature (14, 20, 26 –29). Why then do cyanobacteria such as Synechococcus 7942, Thermosynechococcus, and others predominantly express D1:1? Our data reveal a significant advantage in terms of WOC ???? ??, 2013 • VOLUME 288 • NUMBER ??

cycling efficiency at low light intensities for PSII centers containing D1:1 over D1:2 and an algal D1 isoform at low light intensities. In both in vitro flash O2 (Fig. 5) and in vivo FRR fluorometry (Fig. 7) studies, D1:1-PSII has more efficient WOC cycling at low STF frequencies. The difference is striking at 0.2 Hz (Fig. 7) where Fourier transform analysis estimates D1:1PSII has a flash cycle period of 4.68 % 0.04, whereas D1:2-PSII and C. reinhardtii-PSII have significantly poorer periods (farther from idealized four) of 5.03 % 0.10 and 4.95 % 0.06, respectively. A similar conclusion is reached from Kok modeling of the average hit probability, &avg to be 0.590 % 0.010 in D1:1-PSII versus 0.499 % 0.016 in D1:2-PSII and 0.510 % 0.020 in C. reinhardtii-PSII. The observation of improved oscillations in YO2 at low STF in D1:1-PSII is consistent with whole cells measurements of Synechocystis 6803 mutants by Tichy et al. (29) that expressed Synechococcus 7942 D1 isoforms. At one flash frequency (1 Hz), the strain expressing D1:1 has peak YO2 on flashes three and eight, and the strain expressing D1:2 has peak YO2 on flashes four and nine. Fitting to the classic Kok model (47) gave miss parameters (%) of 0.241 and 0.271 for D1:1 and D1:2, respectively (29). The enhanced quality of oscillations in D1:1-PSII at low STF frequencies is the result of a more stable S3 Kok cycle intermediate. We find that the fast component of S3 decay kinetics in vitro is 40% faster in D1:2-PSII and C. reinhardtii-PSII compared with D1:1-PSII (Fig. 8). These experiments, which were performed at 24 °C, produce half-times consistent with previous reports in spinach PSII membranes. Using a similar YO2 method, Messinger et al. (55) calculated the kinetics of S3 decay at 20 °C to be tf1⁄2 # 7 s (28%) and ts1⁄2 # 94 s (72%), where tf1⁄2 is the fast half-time and ts1⁄2 is the slow half-time. More recently, these kinetics were directly measured at 20 °C via EPR by Chen et al. (68) and found to be tf1⁄2 # 10 % 6 s (11%) and ts1⁄2 # 124 % 6 s (89%). In the time scales studied here, S3 is reduced by the acceptor side of PSII and not YD (68). Thus, the result of faster S3 decay in D1:2-PSII versus D1:1-PSII is consistent with the " $ faster {P680 QA } recombination kinetics of D1:2-PSII. The biphasic nature of the decay has been interpreted as representing distinct hydrogen bonding environments around YZ, which " modulate the kinetics of P680 reduction (68). A PSII-WOC that features a more stable S3 intermediate is uniquely equipped to survive at very low light intensities. For example, cyanobacteria living in biomats, shaded terrestrial JOURNAL OF BIOLOGICAL CHEMISTRY

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Natural Variants of PSII Subunit D1 Tune Photochemical Fitness

FIGURE 9. Structure-function relationships of PSII centers containing varying D1 isoforms. 'GCS refers to the change in free energy of charge separation * " " " (Em(P680 /P680 ) $ Em(Pheo/Pheo$)). 'GR refers to the change in free energy of direct [P680 QA$] recombination (Em(QA/QA$) $ Em(P680/P680 )). Midpoint potentials are based on measurements in T. elongatus (12–14, 16).

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environments, or in the lower mixed layer of the oceanic water column must subside on very low incident light intensities (2, 69, 70). If the rate of PSII-WOC cycling becomes limited by the incident photon flux (and subsequent charge separation), the presence of the D1:1 isoform uniquely enables the WOC to extend the lifetime of higher and less stable S-states. Structurally, this is accomplished by minimizing the recombination " $ kinetics of {P680 QA }. This effect is observed directly in the lightlimiting biomass accumulation and competition experiments described in this work; subtle changes in acceptor side midpoint potentials do have a real impact on cell growth and competition in vivo. We summarize the structure-function relationship of D1 isoforms in the context of our model in Fig. 9. In D1:1-PSII, glutamine is present at position D1–130, causing the reduction potential (Em) of Pheo/Pheo$ to be more negative (Fig. 9, left panel) and resulting in less charge separation and a lower O2 $ QY. The Em of QA/QA is proportionally more negative, which " $ decreases the {P680QA } recombination rate. At low light intensities, this is an advantage. S3 is stabilized, and WOC cycling efficiency improves. However, at high light intensities, inhibi" $ tion of the nonradiative {P680 QA } recombination results in photoinhibition as flux is directed to alternative pathways (e.g. " 3 {Pheo$P680 }) that generate singlet O2. In D1:2-PSII and C. reinhardtii-PSII, glutamate is present at position D1–130, causing the Em of Pheo/Pheo$ to be less negative and resulting in a higher probability of charge separation $ and a higher O2 QY. The Em of QA/QA is proportionally less " $ negative, which increases the {P680QA } recombination rate. At low light intensities, this is a disadvantage. S3 is destabilized, and WOC cycling efficiency decreases. However, at high light " $ intensities, the enhancement of the nonradiative {P680 QA } recombination results in photoprotection as flux is directly " away from pathways (e.g. 3{Pheo$P680 }) that generate singlet O2. Whereas this model physically satisfies the observation of an enhanced WOC cycling efficiency phenotype in D1:1-PSII, it does not fully explain the differences between Fv/Fm, YO2, period$1, and &avg. We postulate that in addition to thermodynamic differences of Pheo and QA, cyclic electron flow around PSII involving cyt b559 may also contribute more in D1:2-PSII than D1:1-PSII, as described above. The functional role of PSII

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cyclic electron flow, if such exists, could serve to dissipate light energy, or to pump protons into the lumen to support the pH gradient/electromotive force. No evidence to support the latter has emerged to date. As previously mentioned, the changes in Em values for Pheo/ $ Pheo$ and QA/QA measured in Thermosynechococcus between D1:1 and D1:2 are more subtle than when the single point mutant Q130E was prepared in Synechocystis 6803 (23). Given that &25 out of 360 amino acids vary between D1:1 and D1:2 in both Synechococcus 7942 and Thermosynechococcus, we concur with the previous hypothesis (24, 71) that other amino acids besides D1–130 partially compensate for changes in the Pheo hydrogen bonding environment. Current work is underway to uncover these contributions using our versatile Chlamydomonas model system. In conclusion, through the heterologous expression of Synechococcus 7942 D1:1 and D1:2 in the Chlamydomonas chloroplast, we have directly compared the efficiency of the cyanobacteria D1 isoforms and the algal D1 isoform. At low light intensities (low STF frequencies), D1:1-PSII has significantly better WOC cycling than D1:2-PSII and C. reinhardtii-PSII due to a more stable S3 intermediate. This difference in WOC cycling efficiency is explained structurally through alterations $ of the Em values of Pheo/Pheo$ and QA/QA that functionally " $ control the efficiency of charge separation and {P680 QA } recombination. The phenotypes exhibited by D1:1-PSII are consistent with improved fitness for cyanobacteria living at very low light fluxes in natural environments. Acknowledgments—We thank Dr. Clyde Cady for assistance with EPR measurements and Dr. Chase Zachary for Kok modeling support. REFERENCES 1. Muh, F., and Zouni, A. (2011) Light-induced water oxidation in photosystem II. Front. Biosci. 16, 3072–3132 2. Falkowski, P. G., and Raven, J. A. (2007) Aquatic Photosynthesis, 2nd Ed., pp. 81–117, Princeton University Press, Princeton, NJ 3. Umena, Y., Kawakami, K., Shen, J.-R., and Kamiya, N. (2011) Crystal structure of oxygen-evolving photosystem II at a resolution of 1.9 Å. Nature 473, 55– 60 4. Mulo, P., Sicora, C., and Aro, E.-M. (2009) Cyanobacterial psbA gene family. Optimization of oxygenic photosynthesis. Cell. Mol. Life Sci. 66, 3697–3710

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Natural Variants of PSII Subunit D1 Tune Photochemical Fitness 5. Bouyoub, A., Vernotte, C., and Astier, C. (1993) Functional analysis of the two homologous psbA gene copies in Synechocystis PCC 6714 and PCC 6803. Plant Mol. Biol. 21, 249 –258 6. Mohamed, A., Eriksson, J., Osiewacz, H. D., and Jansson, C. (1993) Differential expression of the psbA genes in the cyanobacterium Synechocystis 6803. Mol. Gen. Genet. 238, 161–168 7. Summerfield, T. C., Toepel, J., and Sherman, L. A. (2008) Low-oxygen induction of normally cryptic psbA genes in cyanobacteria. Biochemistry 47, 12939 –12941 8. Sicora, C. I., Ho, F. M., Salminen, T., Styring, S., and Aro, E.-M. (2009) Transcription of a “silent” cyanobacterial psbA gene is induced by microaerobic conditions. Biochim. Biophys. Acta 1787, 105–112 9. Golden, S. S., Brusslan, J., and Haselkorn, R. (1986) Expression of a family of psbA genes encoding a photosystem II polypeptide in the cyanobacterium Anacystis nidulans R2. EMBO J. 5, 2789 –2798 10. Boussac, A., Sugiura, M., and Rappaport, F. (2011) Probing the quinonebinding site of photosystem II from Thermosynechococcus elongatus containing either PsbA1 or PsbA3 as the D1 protein through the binding characteristics of herbicides. Biochim. Biophys. Acta 1807, 119 –129 11. Hughes, J. L., Cox, N., Rutherford, A. W., Krausz, E., Lai, T.-L., Boussac, A., and Sugiura, M. (2010) D1 protein variants in photosystem II from Thermosynechococcus elongatus studied by low temperature optical spectroscopy. Biochim. Biophys. Acta 1797, 11–19 12. Kato, Y., Shibamoto, T., Yamamoto, S., Watanabe, T., Ishida, N., Sugiura, M., Rappaport, F., and Boussac, A. (2012) Influence of the PsbA1/PsbA3, Ca2"/Sr2" and Cl$/Br$ exchanges on the redox potential of the primary quinone QA in photosystem II from Thermosynechococcus elongatus as revealed by spectroelectrochemistry. Biochim. Biophys. Acta 1817, 1998 –2004 13. Kato, Y., Sugiura, M., Oda, A., and Watanabe, T. (2009) Spectroelectrochemical determination of the redox potential of pheophytin a, the primary electron acceptor in photosystem II. Proc. Natl. Acad. Sci. U.S.A. 106, 17365–17370 14. Ogami, S., Boussac, A., and Sugiura, M. (2012) Deactivation processes in PsbA1-photosystem II and PsbA3-photosystem II under photoinhibitory conditions in the cyanobacterium Thermosynechococcus elongatus. Biochim. Biophys. Acta 1817, 1322–1330 15. Shibamoto, T., Kato, Y., Sugiura, M., and Watanabe, T. (2009) Redox potential of the primary plastoquinone electron acceptor QA in photosystem II from Thermosynechococcus elongatus determined by spectroelectrochemistry. Biochemistry 48, 10682–10684 16. Sugiura, M., Iwai, E., Hayashi, H., and Boussac, A. (2010) Differences in the interactions between the subunits of photosystem II-dependent on D1 protein variants in the thermophilic cyanobacterium Thermosynechococcus elongatus. J. Biol. Chem. 285, 30008 –30018 17. Schaefer, M. R., and Golden, S. S. (1989) Light availability influences the ratio of two forms of D1 in cyanobacterial thylakoids. J. Biol. Chem. 264, 7412–7417 18. Schaefer, M. R., and Golden, S. S. (1989) Differential expression of members of a cyanobacterial psbA gene family in response to light. J. Bacteriol. 171, 3973–3981 19. Bustos, S. A., Schaefer, M. R., and Golden, S. S. (1990) Different and rapid responses of four cyanobacterial psbA transcripts to changes in light intensity. J. Bacteriol. 172, 1998 –2004 20. Campbell, D., Eriksson, M.-J., Oquist, G., Gustafsson, P., and Clarke, A. K. (1998) The cyanobacterium Synechococcus resists UV-B by exchanging photosystem II reaction-center D1 proteins. Proc. Natl. Acad. Sci. U.S.A. 95, 364 –369 21. Campbell, D., Zhou, G., Gustafsson, P., Oquist, G., and Clarke, A. K. (1995) Electron transport regulates exchange of two forms of photosystem II D1 protein in the cyanobacterium Synechococcus. EMBO J. 14, 5457–5466 22. Sippola, K., and Aro, E.-M. (1999) Thiol redox state regulates expression of psbA genes in Synechococcus sp. PCC 7942. Plant Mol. Biol. 41, 425– 433 23. Merry, S. A., Nixon, P. J., Barter, L. M., Schilstra, M., Porter, G., Barber, J., Durrant, J. R., and Klug, D. R. (1998) Modulation of quantum yield of primary radical pair formation in photosystem II by site-directed mutagenesis affecting radical cations and anions. Biochemistry 37, 17439 –17447

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24. Sugiura, M., Kato, Y., Takahashi, R., Suzuki, H., Watanabe, T., Noguchi, T., Rappaport, F., and Boussac, A. (2010) Energetics in Photosystem II from Thermosynechococcus elongatus with a D1 protein encoded by either the psbA1 or psbA3 gene. Biochim. Biophys. Acta 1797, 1491–1499 25. Clarke, A. K., Hurry, V. M., Gustafsson, P., and Oquist, G. (1993) Two functionally distinct forms of the photosystem II reaction-center protein D1 in the cyanobacterium Synechococcus sp. PCC 7942. Proc. Natl. Acad. Sci. U.S.A. 90, 11985–11989 26. Sander, J., Nowaczyk, M., Buchta, J., Dau, H., Vass, I., Deák, Z., Dorogi, M., Iwai, M., and Rögner, M. (2010) Functional characterization and quantification of the alternative psbA copies in Thermosynechococcus elongatus and their role in photoprotection. J. Biol. Chem. 285, 29851–29856 27. Clarke, A. K., Soitamo, A., Gustafsson, P., and Oquist, G. (1993) Rapid interchange between two distinct forms of cyanobacterial photosystem II reaction-center protein D1 in response to photoinhibition. Proc. Natl. Acad. Sci. U.S.A. 90, 9973–9977 28. Kulkarni, R. D., and Golden, S. S. (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 29. Tichý, M., Lupínková, L., Sicora, C., Vass, I., Kuviková, S., Prásil, O., and Komenda, J. (2003) Synechocystis 6803 mutants expressing distinct forms of the photosystem II D1 protein from Synechococcus 7942. Relationship between the psbA coding region and sensitivity to visible and UV-B radiation. Biochim. Biophys. Acta 1605, 55– 66 30. Schreiber, U. (2004) in Chlorophyll a Fluorescence: A Signature of Photosynthesis (Papageorgiou, G. C., and Govindjee, eds) pp. 279 –319, Springer, Dordrecht, The Netherlands 31. Manuell, A. L., Beligni, M. V., Elder, J. H., Siefker, D. T., Tran, M., Weber, A., McDonald, T. L., and Mayfield, S. P. (2007) Robust expression of a bioactive mammalian protein in Chlamydomonas chloroplast. Plant Biotechnol. J. 5, 402– 412 32. Rasala, B. A., Muto, M., Sullivan, J., and Mayfield, S. P. (2011) Improved heterologous protein expression in the chloroplast of Chlamydomonas reinhardtii through promoter and 5!-untranslated region optimization. Plant Biotechnol. J. 9, 674 – 683 33. Sueoka, N. (1960) Mitotic replication of deoxyribonucleic acid in Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. U.S.A. 46, 83–91 34. Zwietering, M. H., Jongenburger, I., Rombouts, F. M., and van ’t Riet, K. (1990) Modeling of the bacterial growth curve. Appl. Environ. Microbiol. 56, 1875–1881 35. Porra, R. J., Thompson, W. A., and Kriedemann, P. E. (1989) Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim. Biophys. Acta 975, 384 –394 36. Chen, R., Gyokusen, M., Nakazawa, Y., and Gyokusen, K. (2010) Selection of housekeeping genes for transgene expression analysis in Eucommia ulmoides Oliver using real time RT-PCR. J. Bot. 2010 37. Mayfield, S. P., Cohen, A., Danon, A., and Yohn, C. B. (1994) Translation of the psbA mRNA of Chlamydomonas reinhardtii requires a structured RNA element contained within the 5!-untranslated region. J. Cell Biol. 127, 1537–1545 38. Schuster, G., Timberg, R., and Ohad, I. (1988) Turnover of thylakoid photosystem II proteins during photoinhibition of Chlamydomonas reinhardtii. Eur. J. Biochem. 177, 403– 410 39. Shim, H., Cao, J., Govindjee, and Debrunner, P. G. (1990) Purification of highly active oxygen-evolving photosystem II from Chlamydomonas reinhardtii. Photosynth. Res. 26, 223–228 40. Gokhale, Z., and Sayre, R. T. (2009) in The Chlamydomonas Sourcebook (Stern, D. B., ed) pp. 573– 602, Academic Press, Amsterdam 41. Berthold, D. A., Babcock, G. T., and Yocum, C. F. (1981) A highly resolved, oxygen-evolving photosystem II preparation from spinach thylakoid membranes. FEBS Lett. 134, 231–234 42. Mason, C. B., Bricker, T. M., and Moroney, J. V. (2006) A rapid method for chloroplast isolation from the green alga Chlamydomonas reinhardtii. Nat. Protoc. 1, 2227–2230 43. Babcock, G. T., Ghanotakis, D. F., Ke, B., and Diner, B. A. (1983) Electron donation to photosystem II in reaction center preparations. Biochim. Bio-

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ARTNO: M112.394668

Natural Variants of PSII Subunit D1 Tune Photochemical Fitness phys. Acta 723, 276 –286 44. Ananyev, G. M., and Dismukes, G. C. (1996) Assembly of the tetra-Mn site of photosynthetic water oxidation by photoactivation. Mn stoichiometry and detection of a new intermediate. Biochemistry 35, 4102– 4109 45. Ananyev, G., and Dismukes, G. C. (2005) How fast can photosystem II split water? Kinetic performance at high and low frequencies. Photosynth. Res. 84, 355–365 46. Kolling, D. R., Brown, T. S., Ananyev, G., and Dismukes, G. C. (2009) Photosynthetic oxygen evolution is not reversed at high oxygen pressures. Mechanistic consequences for the water-oxidizing complex. Biochemistry 48, 1381–1389 47. Kok, B., Forbush, B., and McGloin, M. (1970) Cooperation of charges in photosynthetic O2 evolution-I. A linear four-step mechanism. Photochem. Photobiol. 11, 457– 475 48. Forbush, B., Kok, B., and McGloin, M. P. (1971) Cooperation of charges in photosynthetic O2 evolution-II. Damping of flash yield oscillation, deactivation. Photochem. Photobiol. 14, 307–321 49. Lavorel, J. (1976) Matrix analysis of the oxygen evolving system of photosynthesis. J. Theor. Biol. 57, 171–185 50. Delrieu, M. J., and Rosengard, F. (1987) Fundamental differences between period-4 oscillations of the oxygen and fluorescence yield induced by flash excitation in inside-out thylakoids. Biochim. Biophys. Acta 892, 163–171 51. Shinkarev, V. P. (2005) Flash-induced oxygen evolution in photosynthesis: simple solution for the extended S-state model that includes misses, double-hits, inactivation, and backward-transitions. Biophys. J. 88, 412– 421 52. Powell, M. J. D. (2009) The BOBYQA Algorithm for Bound Constrained Optimization Without Derivatives. Technical Report NA2009/06, Cambridge, UK 53. Johnson, S. G. (2011) The NLopt Nonlinear Optimization Package, Version 2.2.4, Massachusetts Institute of Technology, Cambridge, MA 54. Joliot, P., and Kok, B. (1975) in Bioenergetics of Photosynthesis (Govindjee, ed) pp. 387– 412, Academic Press, New York 55. Messinger, J., Schröder, W. P., and Renger, G. (1993) Structure-function relations in photosystem II. Effects of temperature and chaotropic agents on the period four oscillation of flash-induced oxygen evolution. Biochemistry 32, 7658 –7668 56. Vass, I., and Styring, S. (1991) pH-dependent charge equilibria between tyrosine-D and the S states in photosystem II. Estimation of relative midpoint redox potentials. Biochemistry 30, 830 – 839 57. Sugiura, M., and Inoue, Y. (1999) Highly purified thermo-stable oxygenevolving photosystem II core complex from the thermophilic cyanobacterium Synechococcus elongatus having His-tagged CP43. Plant Cell Physiol. 40, 1219 –1231

12 JOURNAL OF BIOLOGICAL CHEMISTRY

58. Kulkarni, R., and Golden, S. (1995) Form II of D1 is important during transition from standard to high light intensity in Synechococcus sp. strain PCC 7942. Photosynth. Res. 46, 435– 443 59. Morton, B. R. (1996) Selection on the codon bias of Chlamydomonas reinhardtii chloroplast genes and the plant psbA gene. J. Mol. Evol. 43, 28 –31 60. Mayfield, S. P., Manuell, A. L., Chen, S., Wu, J., Tran, M., Siefker, D., Muto, M., and Marin-Navarro, J. (2007) Chlamydomonas reinhardtii chloroplasts as protein factories. Curr. Opin. Biotechnol. 18, 126 –133 61. Rutherford, A. W., Osyczka, A., and Rappaport, F. (2012) Back-reactions, short-circuits, leaks and other energy wasteful reactions in biological electron transfer. Redox tuning to survive life in O2. FEBS Lett. 586, 603– 616 62. Vass, I., and Cser, K. (2009) Janus-faced charge recombinations in photosystem II photoinhibition. Trends Plant Sci. 14, 200 –205 63. Prasil, O., Kolber, Z., Berry, J. A., and Falkowski, P. G. (1996) Cyclic electron flow around photosystem II in vivo. Photosynth. Res. 48, 395– 410 64. Whitmarsh, J., and Cramer, W. A. (1978) A pathway for the reduction of cytochrome b-559 by photosystem II in chloroplasts. Biochim. Biophys. Acta 501, 83–93 65. Buser, C. A., Diner, B. A., and Brudvig, G. W. (1992) Photooxidation of cytochrome b559 in oxygen-evolving photosystem II. Biochemistry 31, 11449 –11459 66. Shinopoulos, K. E., and Brudvig, G. W. (2012) Cytochrome b559 and cyclic electron transfer within photosystem II. Biochim. Biophys. Acta 1817, 66 –75 67. Sane, P. V., Ivanov, A. G., Sveshnikov, D., Huner, N. P., and Oquist, G. (2002) A transient exchange of the photosystem II reaction center protein D1:1 with D1:2 during low temperature stress of Synechococcus sp. PCC 7942 in the light lowers the redox potential of QB. J. Biol. Chem. 277, 32739 –32745 68. Chen, G., Han, G., Göransson, E., Mamedov, F., and Styring, S. (2012) Stability of the S3 and S2 state intermediates in photosystem II directly probed by EPR spectroscopy. Biochemistry 51, 138 –148 69. Dyer, B. D. (2003) A Field Guide to Bacteria, pp. 232–263, Cornell University Press, Ithaca, NY 70. Stambler, N., and Dubinsky, Z. (2007) in Algae and Cyanobacteria in Extreme Environments (Seckbach, J., ed) pp. 81–97, Springer, Dordrecht, The Netherlands 71. Loll, B., Broser, M., Kós, P. B., Kern, J., Biesiadka, J., Vass, I., Saenger, W., and Zouni, A. (2008) Modeling of variant copies of subunit D1 in the structure of photosystem II from Thermosynechococcus elongatus. Biol. Chem. 389, 609 – 617

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