ApcD is necessary for efficient energy transfer from phycobilisomes to photosystem I and helps to prevent photoinhibition in the cyanobacterium Synechococcus sp. PCC 7002

Biochimica et Biophysica Acta 1787 (2009) 1122–1128

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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a b i o

ApcD is necessary for efficient energy transfer from phycobilisomes to photosystem I and helps to prevent photoinhibition in the cyanobacterium Synechococcus sp. PCC 7002 Chunxia Dong a, Aihui Tang a, Jindong Zhao a,b,⁎, Conrad W. Mullineaux c, Gaozhong Shen d, Donald A. Bryant d,⁎ a

State Key Lab of Protein and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, China Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei 430072, China c School of Biological Sciences, Queen Mary University of London, London E1 4NS, UK d Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA b

a r t i c l e

i n f o

Article history: Received 15 February 2009 Received in revised form 14 April 2009 Accepted 15 April 2009 Available online 3 May 2009 Keywords: Cyanobacteria Phycobilisome Photosynthesis State transition Photosystem I Photosystem II Synechococcus sp. PCC 7002

a b s t r a c t Phycobilisomes (PBS) are the major light-harvesting, protein–pigment complexes in cyanobacteria and red algae. PBS absorb and transfer light energy to photosystem (PS) II as well as PS I, and the distribution of light energy from PBS to the two photosystems is regulated by light conditions through a mechanism known as state transitions. In this study the quantum efficiency of excitation energy transfer from PBS to PS I in the cyanobacterium Synechococcus sp. PCC 7002 was determined, and the results showed that energy transfer from PBS to PS I is extremely efficient. The results further demonstrated that energy transfer from PBS to PS I occurred directly and that efficient energy transfer was dependent upon the allophycocyanin-B alpha subunit, ApcD. In the absence of ApcD, cells were unable to perform state transitions and were trapped in state 1. Action spectra showed that light energy transfer from PBS to PS I was severely impaired in the absence of ApcD. An apcD mutant grew more slowly than the wild type in light preferentially absorbed by phycobiliproteins and was more sensitive to high light intensity. On the other hand, a mutant lacking ApcF, which is required for efficient energy transfer from PBS to PS II, showed greater resistance to high light treatment. Therefore, state transitions in cyanobacteria have two roles: (1) they regulate light energy distribution between the two photosystems; and (2) they help to protect cells from the effects of light energy excess at high light intensities. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Cyanobacteria are prokaryotes that perform oxygenic photosynthesis. Like eukaryotic algae and higher plants, oxygen-evolving cyanobacteria have two photosystems, photosystem (PS) I and II. The activities of these two photosystems must be carefully coordinated to maximize the efficiency of photosynthetic electron transfer. Phycobilisomes (PBS), which are attached to the stromal surfaces of the thylakoid membranes, are the major light-harvesting antenna complexes of cyanobacteria and red algal chloroplasts [1,2]. These water-soluble, supramolecular complexes are composed of phycobiliproteins and colorless linker proteins [1–4]. Although several

⁎ Corresponding authors. Jindong Zhao is to be contacted at College of Life Sciences, Peking University, Beijing, 100871 China. Tel.: +86 10 6275 6421; fax: +86 6275 1526. Donald A. Bryant, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA. Tel.: +1 814 865 1992; fax: +1 814 863 7024. E-mail addresses: [email protected] (J. Zhao), [email protected] (D.A. Bryant). 0005-2728/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbabio.2009.04.007

structural classes of PBS occur in cyanobacteria [4], the most common PBS structure is the hemidiscoidal form, which is composed of two substructural domains: the core and the peripheral rods. The core is mainly composed of allophycocyanin but additionally contains allophycocyanin-related proteins and two linker proteins, which together assemble into several cylindrical substructures [3,4]. The peripheral rods are composed of cylindrical assemblies of phycocyanin (PC), phycoerythrin or phycoerythrocyanin (if present), and several linker polypeptides; the peripheral rods are attached to and radiate from the surfaces of the core assembly. PBS are typically associated with PS II, and most of the light energy absorbed by phycobiliproteins is usually delivered to PS II reaction centers [5,6]. However, under unbalanced light conditions, when PS I receives too little light energy, light energy absorbed by PBS can also be delivered to PS I [7]. The processes by which light energy is redistributed to the two photosystems to produce an optimal rate of electron transfer are called “state transitions” [8,9]. Two important aspects of the state transition mechanism have been intensively investigated: (1) the mechanism by which the unbalanced excitation

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of the two photosystems is sensed; and (2) the route through which the absorbed light energy is distributed to each photosystem. In both plants and cyanobacteria, the unbalanced light condition is sensed by the redox state of inter-photosystem electron carriers [10]. Therefore, both respiratory and cyclic electron transfer strongly influence the state transitions [11]. The distribution of absorbed light energy is differently regulated in plants and cyanobacteria. While PBS movement has been shown to be required for the state transition process in cyanobacteria [12,13], phosphorylation and movement of lightharvesting complex II is critical to light energy distribution in plants [14–16]. The mobility of PBS on the thylakoid surfaces of cyanobacteria has been studied by fluorescence recovery after photobleaching (FRAP). Under the conditions used for these measurements, PBS have been found to be moving constantly, and it has been suggested that the contacts of PBS with reaction centers of both photosystems are unstable and transient [12,13,17]. At present, little is known about the mechanism of PBS movement from one photosystem to another during state transitions. The transfer of the light energy absorbed by PBS to PS I requires ApcD in both Synechococcus sp. strain PCC 7002 (hereafter Synechococcus 7002) [18,19] and Synechocystis sp. strain PCC 6803 (hereafter Synechocystis 6803) [20]. Together with the core-membrane linker phycobiliprotein, ApcE, allophycocyanin-B (AP-B) is one of two terminal emitters of the PBS core [18, 21]. A Synechococcus 7002 mutant lacking ApcD, the AP-B alpha subunit, still assembles PBS [22,23], but is unable to perform state transitions normally [18]. PBS in the mutant cells are unable to distribute light energy to PS I but continue to deliver absorbed light energy principally to PS II even if the cells are incubated in the dark or illuminated with light 2, which is predominantly absorbed by PS II [18]. Another important gene involved in state transitions in cyanobacteria is rpaC [24], which encodes a small membrane protein that may be important in the interactions between PBS and PS II [25]. Although rpaC is required for state transitions and optimal growth of Synechocystis 6803 under weak illumination, it is not critical under higher light intensities. Changes in PS II activity under photoinhibitory light conditions were the same for the wild type and the rpaC mutant [15,24]. On the other hand, state transitions strongly influenced PS II activity and D1 (PsbA) protein turnover in Chlamydomonas reinhardtii [26]. It has been demonstrated that the photoinhibitory effect on PS II is dose-dependent in the cyanobacterium Synechococcus sp. PCC 6301 [27], and the absorption cross-section of PS II changed significantly between state 1 and state 2 [19]. It would thus be expected that state transitions could help in dissipating excessive energy absorbed by PBS, the major PS II lightharvesting complexes. In this study, we demonstrate that energy transfer from PBS to PS I occurs directly through AP-B, and we show that this process is particularly important for cell growth when the light is preferentially absorbed by PBS. Additionally, we show here that AP-B-mediated energy transfer to PS I and state transitions play important roles in protecting cells against photoinhibitory damage at high light intensities. 2. Materials and methods 2.1. Strains and chemicals The wild type (PR6000), the apcD mutant (PR6323), the apcF mutant (PR6325), the psbD1C mutant (PR6338) and apcD psbD1C mutant (PR6339) strains of Synechococcus sp. PCC 7002 were grown in medium A supplemented with 1 mg NaNO3 ml− 1 [28]. The growth temperature was 38 °C, and the light intensity was approximately 200 to 250 μmol photons m− 2 s− 1 provided from cool white fluorescent lamps. Blue light for growth was obtained according to Wang et al. [5]. All chemicals were purchased from Sigma (St. Louis, MO) unless otherwise noted. Cell densities were determined by measuring the optical density at 730 nm.

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2.2. Measurement of oxygen evolution and fluorescence O2 evolution from cells of Synechococcus 7002 was measured with a Clark-type oxygen electrode at 37 °C. Sodium bicarbonate at a final concentration of 5 mM was added for measuring the rate of wholechain electron transport. Light was provided with a 300 W tungsten light source. Light intensities were adjusted as required by neutral filters. Fluorescence induction transients in the millisecond time range were measured as described by Zhao et al. [29]. The actinic light for these fluorescence induction experiments was provided with a 300 W tungsten light source and was passed through a heat filter, a cut-on filter, which allowed light of wavelengths longer than 500 nm to pass, and a cut-off filter, which allowed light with wavelengths shorter than 600 nm to pass. The photomultiplier was protected with a 680-nm (bandwidth: 10 nm) interference filter. The cells were either dark-adapted for 5 min or illuminated with blue light (100 μmol photons m− 2 s− 1) at room temperature before measurements. When specified, 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB) was added to a final concentration of 5 μM and 3(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) was added to a final concentration of 10 μM. Fluorescence emission spectra at 77 K were obtained as described by Schluchter et al. [30]. The excitation wavelength was either 440 nm, a wavelength primarily absorbed by chlorophyll (Chl), or 590 nm, a wavelength primarily absorbed by PC. The cells were dark-adapted as described above to place cells in state 2 or illuminated with blue light at 100 μmol photons m− 2 s− 1 in the presence of DCMU to place cells in state 1 prior to freezing in liquid nitrogen. 2.3. Measurement of absorption cross-section of PS I with light absorbed by phycobiliproteins P700 absorption changes of Synechococcus 7002 strains were measured with modulated light at 820 nm as described previously [7]. The cell suspension at a concentration of 25 μg Chl ml− 1 was excited by a single-turnover light-pulse from a dye laser tuned to 630 nm or 676 nm. The laser pulse traveled through the cell suspension for 3 mm before it reached the region illuminated by the measuring beam. The attenuation of the laser pulse by 3 mm of the cell suspension was measured directly at both excitation wavelengths so as to correct for self-shading by the sample. The laser pulse energy was measured using a pyrometer and converted into a relative number of photons per pulse by multiplying by the wavelength. The 820-nm absorbance change was measured at saturating pulse energy and subsequently at a series of subsaturating pulse energies. The relative absorption cross-section of PS I at each wavelength was calculated by plotting the 820 nm absorbance change against the relative number of photons in the pulse. In a plot of ln[(ΔAmax − ΔA) / ΔAmax] vs. flash intensity, the gradient at low flash intensity gives a measure of the average relative absorption cross-section of PS I [7]. The gradient of the plot was calculated by linear regression. Absorption spectra were measured for the same cell suspensions, and the contributions from Chl and PC were calculated using a formula derived from the absorption spectra of isolated thylakoid membranes and PBS of Synechococcus 7002, following the method used by Myers et al. [31]. Our formula was designed to separate the contributions of Chl and PC from the total cellular absorption at 630 nm and 676 nm. For Synechococcus 7002: Chl

A676 nm = 1:026 A676 nm − 0:101 A630 nm PC

A630 nm = 1:026 A630 nm − 0:258 A676 nm Chl where AN is the total cell absorbance at wavelength N, AN is the absorbance due to Chl at wavelength N, and ANPC is the absorbance

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Fig. 1. Fluorescence induction transients for Synechococcus 7002 wild type (panel A) and mutant strain PR6323 (ApcD−) (panel B). Fluorescence emission at 685 nm was recorded, and all experiments were conducted at room temperature. Cells were either pre-illuminated with blue light (440 nm, 100 μmol photons m− 2 s− 1) for 2 min to establish state 1 (curve a) or incubated in the dark for 5 min to establish state 2 (curve b) before the actinic light (500–600 nm, 820 μmol photons m− 2 s− 1) was switched on. Arrows indicate the Fo levels. The right-hand panels show fluorescence induction in the presence of electron transport inhibitors DCMU and DCMU + DBMIB for the wild type (panel C) and strain PR6323 (ApcD−) (panel D). Cells were incubated in the dark for 5 min before the actinic light was switched on.

due to PC at wavelength N. The relative absorption cross-section (C) of PS I at 630 nm and 676 nm were separated into Chl and PC components in the same way as the absorbance. The ratios Chl PC Chl CPC 630 nm/C676 nm and A630 nm/A676 nm were compared to give a measure of the relative contribution of PC to the absorption and PS I excitation spectra of the cells, which is an indication of the efficiency of energy transfer from PBS to PS I [7]. The quantum efficiency (Φ) of

energy transfer from PBS to PS I can be calculated by the following formula:     PC Chl PC Chl Φ = PI · C630 nm = C676 nm = A630 nm = A676 nm where PI is the proportion of photons absorbed by Chl which are transferred to PS I. The value of PI in Synechococcus 7002 is 0.85 [32].

Fig. 2. Growth rate measurements for Synechococcus 7002 wild type cells (circles) and strain PR6323 (squares) at 38 °C under different light conditions. Optical densities at 550 nm of the cultures are plotted on a semi-logarithmic scale as a function of time. Panels A and B show growth of the wild type (circles) and PR6323 (squares) under white light, which was provided with cool white fluorescence bulbs, at 250 μmol photons m− 2 s− 1 and 100 μmol photons m− 2 s− 1, respectively. Panels C and D show growth of the wild type (circles) and PR6323 (squares) under green light at 112 μmol photons m− 2 s− 1 (panel C) and 64 μmol photons m− 2 s− 1 (panel D). The green light was produced by passing cool white fluorescence light through a dark green cellulose acetate filter. The numbers in panels indicate the calculated doubling times. The data presented are the average of three individual measurements for each condition and strain.

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and placed cells in state 1, or cells were incubated in the dark to place cells in state 2. A higher Fm was obtained for the wild type when the cells were pre-adapted to state 1 than when the cells that were adapted to state 2 (Fig. 1A). On the other hand, the Fm level of PR6323 was the same for both pretreatment conditions (Fig. 1B). DCMU and DBMIB are inhibitors that inhibit electron transfer at the QB reduction site of PS II and the Qo site of cytochrome (Cyt) b6f complex, respectively. When fluorescence induction was performed with wild type cells in the presence of DCMU, a state 2 to state 1 transition was observed, and this transition was prevented by further addition of DBMIB (Fig. 1C). Because PR6323 cells were in state 1 in the presence of DCMU, DBMIB, or both inhibitors based on their initial fluorescence level, no state transition was observed in PR6323 under the same conditions (Fig. 1D). To determine whether the inability to perform state transitions had an effect on growth, the wild-type Synechococcus 7002 strain PR6000 and mutant strain PR6323 lacking ApcD/AP-B were grown under various light conditions (Fig. 2). Mutant strain PR6323 grew only slightly more slowly than the wild type under a saturating white light intensity (250 μmol photons m− 2 s− 1; Fig. 2A) or under a lower light intensity (100 μmol photons m− 2 s− 1; Fig. 2B). This result indicated that the absence of ApcD/AP-B did not significantly impair lightharvesting and energy transfer when cells were grown under balanced light conditions. However, when the cells were grown in green light, which is preferentially absorbed by PBS, the growth rate of PR6323 was significantly slower than that of the wild type. Under a moderateintensity green light (112 μmol m− 2 s− 1; Fig. 2C), the doubling times of the wild type and strain PR6323 were 12.0 h and 16.1 h, respectively.

Fig. 3. Comparison of oxygen evolution rate (μmol oxygen (mg Chl)− 1 h− 1) as a function of light intensity at three illumination conditions for Synechococcus 7002 wild type (circles) and strain PR6323 (ApcD−) (squares). Panel A, 500–750 nm light; panel B, 580 nm light; panel C, 680 nm light.

2.4. Other methods PBS were isolated from Synechococcus 7002 according to Bryant et al. [33]. The isolation of thylakoid membranes from Synechococcus 7002 and the determination of P700:Chl and PS II:Chl ratios were performed as previously described [32]. The cell density was measured with a Cary-14 spectrophotometer modified for computerized acquisition of data by On-Line Systems, Inc (Bogart, GA). 3. Results 3.1. ApcD is required for state transitions and optimal growth under light preferentially absorbed by PBS When cyanobacteria are excited with green light (500–600 nm, 1000 μmol photons m− 2 s− 1) at room temperature, the fluorescence emission mostly occurs from PS II at 680 nm, and the variable fluorescence indicates how much light is transferred from PBS to PS II. Fig. 1 shows fluorescence induction transients under various conditions in the wild type and mutant strain PR6323, in which the apcD gene is insertionally inactivated [22, 23]. The cells were pretreated by illumination with blue light, which is predominantly absorbed by PS I

Fig. 4. Comparison of the absorption spectra of wild-type and mutant cells of Synechococcus 7002 with action spectra for P700 photooxidation. Absorption spectra of the cultures of the wild type (PR6000) and strain PR6323 (ApcD−) were recorded and are shown as solid lines. The actinic light wavelength for P700 photooxidation was adjusted with a tuneable dye laser and the actinic absorption spectra are shown as dashed lines. All experiments were performed at room temperature.

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Fig. 5. Semi-logarithmic plots of P700 photooxidation as a function of flash intensity at wavelengths 630 nm (closed circles and dashed lines) or 676 nm (open circles and solid lines). The cells of wild type strain PR6000 (panel A), PR6323 (ApcD−; panel B), PR6338 (PsbD1− PsbC−; panel C) and PR6339 (ApcD− PsbD1− PsbC−; panel D) of Synechococcus 7002 were dark-adapted before the actinic flash was turned on. Light wavelengths were adjusted with a tuneable dye laser. Line fits were performed by linear regression. The data obtained with 630 nm light in panel A (closed circles) were biphasic and line fit of the data generated two dashed lines (see text for details). All experiments were performed at room temperature.

Under a lower green light intensity (64 μmol m− 2 s− 1; Fig. 2D), the doubling times of the wild type and mutant PR6323 were 38.0 h and 51.4 h, respectively. Thus, the doubling time of the apcD mutant was approximately 35% longer than that of the wild type under either green light intensity. Oxygen evolution rates for the wild type and the ApcD/AP-B-less mutant strain PR6323 were measured as a function of light intensity under different light wavelengths, and the results are shown in Fig. 3. When the cells were illuminated with white light (500–750 nm) absorbed by PBS and Chl, the light saturation behavior for oxygen evolution of the two strains was identical (Fig. 3A). Similarly, when cells were illuminated with red light (680 nm; Fig. 3C) or blue light (440 nm; data not shown), wavelengths that are predominantly absorbed by Chl, the apcD mutant strain also had the same saturation behavior for oxygen evolution as the wild type. However, when cells were illuminated with 580-nm light (Fig. 3B), which is absorbed predominantly by PBS, oxygen evolution of strain PR6323 increased more slowly in response to increasing light intensity than the wild type. These results were consistent with the growth experiments described above and showed that the apcD mutant strain PR6323 was unable to utilize light wavelengths principally absorbed by PBS as efficiently as the wild type.

photooxidation of P700 induced by single-turnover flashes was measured at two different wavelengths: 630 nm, which is absorbed predominantly by PC in PBS, and 676 nm, which is absorbed predominantly by Chl. Fig. 5 shows the semi-logarithmic plots of the differential P700 bleaching signal versus flash intensity from different strains after dark adaptation to state 2. When the actinic flash wavelength was 676 nm, which is preferentially absorbed by Chl, P700 oxidation for the wild type (Fig. 5A) varied in a monophasic manner as a function of flash intensity. However, when the flash wavelength was 630 nm, which is preferentially absorbed by PC, the P700 oxidation signal varied in a biphasic manner as a function of the flash intensity. This phenomenon has also been observed in other cyanobacteria [7], and it indicates that a portion of PS I-associated P700 more readily receives PBS-absorbed light than the remainder [7, 34]. Panels B through D of Fig. 5 show the semi-logarithmic plots of the differential P700 oxidation signals versus flash intensity from the strains PR6323 (ApcD−), PR6338 (PsbD1− PsbC−), and PR6339 (ApcD− PsbD1− PsbC−), respectively. Strains PR6338 and PR6339 had the psbD1 and psbC genes deleted and therefore contained no active PS II. The average absorption cross-section (C) of PS I in these strains was estimated from the initial slopes of the plots, which were fitted through linear regression. The quantum efficiency of light energy transfer from PBS to PS I (Φ) was then calculated according to the equation given in the Materials and methods section, and the results are shown in Table 1. When cells were pre-adapted to state 2, the quantum efficiency of light energy transfer from PBS to PS I in the wild type was 0.89. When ApcD was eliminated genetically, the quantum efficiency of energy transfer from PBS to PS I was greatly reduced to 0.31. The effect of apcD inactivation on the quantum efficiency in a PS II-less strain (psbD1 psbC mutant) was also examined. Strain PR6338, which has no detectable PS II and thus can only grow photoheterotrophically (data not shown), could still efficiently transfer PBSabsorbed light energy to PS I (Φ = 0.63). However, a large decrease in the quantum efficiency of energy transfer from PBS to PS I (Φ = 0.27) was again observed when the apcD gene was inactivated in a PS II-less background (strain PR6339: apcD psbD1 psbC). This result showed that light energy transfer to PBS to PS I does not occur through PS II. 3.3. State transition alleviates photoinhibition under excessive light Compared to most cyanobacteria, Synechococcus 7002 is remarkably resistant to photoinhibition [35]. Because PBS are the major lightharvesting complexes for PS II in cyanobacteria, and because the absorption cross-section of PS II changes during state transitions, we compared the ability of the wild type and strain PR6323 (ApcD−) to

Table 1 Estimated quantum efficiency of energy transfer from PBS to PS I in the wild type and three mutant strains of Synechococcus 7002. Strain

C630/C676 a

1.06 ± 0.12 0.49 ± 0.07 0.67 ± 0.06 0.41 ± 0.06

Chl CPC 630/C676

A630/A676

Chl APC 630/A676

Quantum efficiency

0.91 ± 0.13 0.25 ± 0.07 0.45 ± 0.06 0.16 ± 0.07

0.90 0.88 0.90 0.80

0.87 0.69 0.71 0.60

0.89 ± 0.13 0.31 ± 0.09 0.63 ± 0.08 0.27 ± 0.10

3.2. Action spectrum and quantum efficiency of energy transfer from PBS to PS I for Synechococcus 7002

PR6000 PR6323 PR6338 PR6339

To measure the efficiency of energy transfer from PBS to PS I in the wild type and the apcD mutant, we first compared the absorption spectra of the whole cells with the action spectra of P700 oxidation. Whole-cell absorption spectra and action spectra for P700 oxidation in the strains PR6000 and PR6323 are shown in Fig. 4. The absorption spectra of the wild type and the ApcD/AP-B-less mutant strain PR6323 were similar, because PBS are assembled normally in the absence of ApcD [22]. However, the efficiency of energy transfer from PBS to PS I was greatly reduced in the apcD mutant strain PR6323 in comparison to wild type strain PR6000. To measure the efficiency of energy transfer from PBS to PS I, the light intensity-dependent

The relative absorption cross-sections for PS I were calculated from the slopes of the semi-logarithmic plots of P700 photooxidation as a function of flash intensity. Absorption cross-sections were measured at two wavelengths: 630 nm and 676 nm. The relative absorption cross-sections at 630 nm (C630) and 676 nm (C676) were Chl deconvoluted into the phycocyanin contribution (CPC 630) and the Chl contribution (C676) using the simultaneous equations described in the Materials and methods. The absorbance (A) of cells at 630 nm and 676 nm were deconvoluted in the same way. Dividing the deconvoluted absorption cross-section ratio by the deconvoluted absorption ratio gives a measure of the efficiency of energy transfer from PBS as compared to that from Chl. The ratio was multiplied by the proportion of Chl associated with PS I. The proportion is 0.85 in PR6000 (wild type) and PR6323 (ApcD−), and assuming that all Chl is associated with PS I in the absence of PS II, is 1.0 in PR6338 (PsbD1− PsbC−) and PR6339 (PsbD1− PsbC− ApcD−). a Each entry is the average of five individual measurements.

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Fig. 6. Photoinhibition of the Synechococcus 7002 wild type and strains PR6323 (ApcD−) and PR6325 (ApcF−) by white light at 2500 μmol photons m− 2 s− 1 (panel A) and at 3200 μmol photons m− 2 s− 1 (panel B). Cells of the wild type (circles), PR6323 (squares) and PR6325 (triangles) at a concentration of 5 μg Chl ml− 1 were exposed to the photoinhibitory light at 35 °C for up to 1 h. The cultures were bubbled with air plus 1% (v/v) CO2 during the illumination period. At the times indicated, aliquots of the cultures were withdrawn, and the oxygen evolution rates of the cells were determined. The data are presented as percentages of the initial rates before the photoinhibitory treatment. The initial oxygen evolution activities of the wild type (PR6000), PR6323 and PR6325 strains were similar: 365, 358 and 315 μmol oxygen (mg Chl)− 1 h− 1, respectively.

tolerate high light stress (Fig. 6). When cultures were bubbled with air plus 1% (v/v) CO2 at 35 °C, the oxygen evolution rate of the wild type and strain PR6323 decreased exponentially as a function of exposure time under high intensity white light (2500 μmol photons m− 2 s− 1) (Fig. 6A). However, oxygen evolution was lost about 30% faster in strain PR6323 (ApcD−) than in the wild type. Overall, oxygen evolution decreased by ∼ 60% in mutant PR6323 in 40 min, while oxygen evolution in the wild type only decreased by about ∼40% inhibition under the same conditions. Strain PR6325, which carries an inactivated apcF gene and exhibits less efficient energy transfer from PBS to PS II [23], was much more resistant to the high light treatment than the wild type (Fig. 6A). The oxygen evolution of strain PR6325 only decreased by about 20% after 40 min. However, when the light intensity was increased to 3500 μmol photons m− 2 s− 1, all three strains showed similar rates and extents of photoinhibition of O2 evolution activity (Fig. 6B). These results demonstrated that photoinhibition of PS II was dosage-dependent and suggested (1) that the absorption cross-section of PS II was critical to the level of damage to PS II; and (2) that state transitions and ApcD/AP-B play an important role in protecting cells from photoinhibition. 4. Discussion 4.1. Physiological significance of state transitions Most cyanobacteria and red algae use PBS as their major lightharvesting antenna systems. Because light is the energy source for growth, it is one of the most important environmental factors for these organisms, and both light quality (i.e., wavelength) and light intensity are critical factors that determine the optimal growth conditions for a given organism. Therefore, it is not surprising that

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PBS-containing organisms have evolved several mechanisms for adaptation to changes in light conditions. State transitions are a mechanism that produces a short-term adaptation to an unbalanced light regime that favors excitation of either PS I or PS II [8– 11, 16]. It has also been shown that light intensity influences the distribution of absorbed light energy [36]. There are several gene products that are critical to energy transfer and distribution from PBS to photosystems. In Synechococcus 7002, ApcF, which has sequence similarity to allophycocyanin β subunits, plays an important role in energy transfer from PBS to PS II [23], although in Synechocystis 6803 ApcF also seems to be important in energy transfer from PBS to PS I [20]. AP-B, one of the terminal emitters in PBS, is required for energy transfer from PBS to PS I in both PBScontaining organisms [18, 19]. The importance of the ability to transfer light energy from PBS to PS I is demonstrated in the growth measurements presented in this study (Fig. 2). A 35% difference in the growth rates between the wild type and strain PR6323 (ApcD−) is highly significant, and it shows that the inability to transfer PBS-absorbed light energy to PS I causes a large growth disadvantage when light conditions favor PS II excitation. The growth rate phenotype of PR6323 is similar to that of the rpaC mutant strain of Synechocystis 6803, which grows approximately 25% more slowly than the wild type when cells are grown under light principally absorbed by PBS [24]. RpaC is not a component of the PBS, it bears no chromophore for light energy transfer, and it is found in organisms such as Prochlorococcus sp. that do not have PBS. Recent evidence suggests that RpaC plays a role in complex formation between PBS and PS II [25]. The similar growth-rate phenotypes of PR6323 and of an rpaC mutant under light absorbed by PBS suggest that mutations in genes involved in state transitions might produce similar mechanistic defects. As revealed by the fluorescence induction studies presented in this study, a significant amount of excitation energy is transferred from PBS to PS I under state 2 conditions (Fig. 1, Table 1). By using modulated fluorescence measurements, Campbell et al. [36] showed that exposure to high light induces a higher non-photochemical quenching of fluorescence (NPQ), and changes in the PS II absorption cross-section in state transitions are observed in wild type Synechococcus 7002 but not in its apcD mutant [19]. NPQ in cyanobacteria is largely due to state transitions [37] and blue-lightinduced quenching, which is dependent on a soluble orange carotenoid protein [38]. An apcD mutant of another cyanobacterium, Nostoc sp. PCC 7120, has normal blue light induced NPQ [39], and the orange carotenoid protein, which is encoded by open reading frame SynPCC7002_A2810 in Synechococcus 7002, is not expected to be altered in PR6323. Thus, we conclude that the inability to alter the absorption cross-section of PS II by state transitions in the apcD mutant under strong illumination results in more severe photoinhibition in the mutant than in wild type. The following evidence supports this conclusion: (1) PS II is the major site of photoinhibition, and damage to PS II is dependent on light dosage in cyanobacteria [27]; (2) when apcF is inactivated, energy transfer from PBS to PS II is less efficient, and the mutant shows a higher tolerance to high light stress (Fig. 6); because the apcF mutant did not show a significant increase in fluorescence, the absorbed energy could be dissipated through quenching; and (3) PS II activity and PsbA (D1) protein turnover are influenced by state transitions in C. reinhardtii [26]. Therefore, state transitions are not only important in regulating energy distribution under an unbalanced light regime, but they are also important in alleviating photoinhibitory damage under excessive light conditions. The phenotype of strain PR6323 (ApcD−) under strong light is different from the phenotype of the rpaC mutant of Synechocystis 6803, which showed little ability to prevent photoinhibition under strong illumination conditions [15]. Thus, although both ApcD and RpaC are involved in state transitions, these observations imply that their functions are different.

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4.2. Quantum efficiency and route of energy transfer from PBS to PS I Measurements of the quantum efficiency of energy transfer from PBS to PS I in the PS II-less mutant strain PR6338 demonstrated that PBS can transfer energy to PS I directly through an ApcD/AP-Bdependent route with no significant contribution from PS IIdependent Chl spillover (Figs. 4, 5 and Table 1). This conclusion differs from that of Shimada et al. [40] studied energy transfer from PBS to PS I in a psbC mutant of Synechocystis 6803 at low temperature. The results in Table 1 also show that Synechococcus 7002 has very high quantum efficiency in energy transfer from PBS to PS I. It has the highest efficiency (0.89) of light energy transfer from PBS to PS I among all cyanobacteria measured so far. Synechococcus sp. PCC 6301 has an efficiency of 0.60 [7] while Synechocystis 6803 has an efficiency of 0.40 [34]. Because this quantum efficiency seems to correlate well with the relative growth rates of these three organisms, we speculate that the quantum efficiency of energy transfer from PBS to PS I could be an important factor that influences the growth rates of the cyanobacteria. Although a strain lacking both AP-B and PS II could still transfer some energy to PS I (Table 1), the results showed that energy transfer to PS I occurs much less efficiently when ApcD/AP-B was missing (Table 1, Figs. 4 and 5). One possibility is that some PCabsorbed light energy could be transferred to PS I directly by alternative phycobiliprotein complexes containing PC as suggested by Kondo et al. [41]. These two populations of PS I complexes would likely have different optical cross-sections, and the biphasic lines in the cross-section measurements shown in Fig. 5 are in agreement with this suggestion. Another possibility is that ApcE (Lcm), the second terminal emitter in PBS cores, transfers some energy to PS I when ApcD/AP-B is missing. Finally, energy transfer might occur directly from allophycocyanin to PS I but with a lower efficiency than when ApcD/AP-B is present. Further studies will be required to understand the detailed pathway of energy transfer from PBS to PS I. Acknowledgments The research in this report was supported by grants from The National Natural Science Foundation of China (no. 30230040) and the Ministry of Science and Technology of China (2009CB11850) to J.Z.; by a grant from the Wellcome Trust and the Biotechnology and Biological Sciences Research Council to C.W.M.; and by grant MCB-0519743 from the U.S. National Science Foundation to D.A.B. References [1] A.N. Glazer, The light guide, J. Biol. Chem. 264 (1989) 1–4. [2] R. MacColl, Cyanobacterial phycobilisomes, J. Struct. Biol. 124 (1998) 311–334. [3] R. MacColl, Allophycocyanin and energy transfer, Biochim. Biophys. Acta 1657 (2004) 73–81. [4] W.A. Sidler, Phycobilisomes, in: D.A. Bryant (Ed.), The Molecular Biology of Cyanobacteria, Kluwer Academic Publishers, Dordrecht, 1994, pp. 139–216. [5] R.T. Wang, C.L.R. Stevens, J. Myers, Action spectra for photoreactions I and II of photosynthesis in the blue-green alga Anacystis nidulans, Photochem. Photobiol. 25 (1977) 103–108. [6] T. Katoh, E. Gantt, Photosynthetic vesicles with bound phycobilisomes from Anabaena variabilis, Biochim. Biophys. Acta 546 (1979) 383–393. [7] C.W. Mullineaux, Excitation energy transfer from phycobilisomes to photosystem I in a cyanobacterium, Biochim. Biophys. Acta 1100 (1992) 285–292. [8] P. Bonaventura, J. Myers, Fluorescence and oxygen evolution from Chlorella pyrenodoisa, Biochim. Biophys. Acta 189 (1969) 366–383. [9] N. Murata, Control of excitation transfer in photosynthesis I. Light-induced change of chlorophyll a fluorescence in Porphyridium cruentum, Biochim. Biophys. Acta 172 (1969) 242–251. [10] J.F. Allen, State transition—a question of balance, Science 299 (2003) 1530–1532. [11] C. Huang, X. Yuan, J. Zhao, D.A. Bryant, Kinetic analyses of state transitions of the cyanobacterium Synechococcus sp. PCC 7002 and its mutant strains impaired in electron transport, Biochim. Biophys. Acta 1607 (2003) 121–130. [12] C.W. Mullineaux, M.J. Tobin, G.R. Jones, Mobility of photosynthetic complexes in the thylakoid membranes, Nature 390 (1997) 421–424.

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