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ENVIRONMENTAL STRESS AND ADAPTATION TO STRESS

Iron Deficiency in Cyanobacteria Causes Monomerization of Photosystem I Trimers and Reduces the Capacity for State Transitions and the Effective Absorption Cross Section of Photosystem I in Vivo¹

Department of Biology and The Biotron, University of Western Ontario, London, Ontario, Canada N6A 5B7 (A.G.I., M. Krol, N.P.A.H.); Department of Plant Physiology, University of Umeå, Umea S–901 87, Sweden (A.G.I., D.S., E.S., S.S., G.O.); Department of Biological Sciences, Brock University, St. Catharines, Ontario, Canada L2S 3A1 (M. Koochek, S.V., D.B.); and Department of Biology, Chungnam National University, Daejon 305–764, Korea (Y.-I.P.)

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
The induction of the isiA (CP43') protein in iron-stressed cyanobacteria is accompanied by the formation of a ring of 18 CP43' proteins around the photosystem I (PSI) trimer and is thought to increase the absorption cross section of PSI within the CP43'-PSI supercomplex. In contrast to these in vitro studies, our in vivo measurements failed to demonstrate any increase of the PSI absorption cross section in two strains (Synechococcus sp. PCC 7942 and Synechocystis sp. PCC 6803) of iron-stressed cells. We report that iron-stressed cells exhibited a reduced capacity for state transitions and limited dark reduction of the plastoquinone pool, which accounts for the increase in PSII-related 685 nm chlorophyll fluorescence under iron deficiency. This was accompanied by lower abundance of the NADP-dehydrogenase complex and the PSI-associated subunit PsaL, as well as a reduced amount of phosphatidylglycerol. Nondenaturating polyacrylamide gel electrophoresis separation of the chlorophyll-protein complexes indicated that the monomeric form of PSI is favored over the trimeric form of PSI under iron stress. Thus, we demonstrate that the induction of CP43' does not increase the PSI functional absorption cross section of whole cells in vivo, but rather, induces monomerization of PSI trimers and reduces the capacity for state transitions. We discuss the role of CP43' as an effective energy quencher to photoprotect PSII and PSI under unfavorable environmental conditions in cyanobacteria in vivo.

Earlier studies reported major alterations in the composition of pigment-protein complexes in cyanobacteria under iron stress (Guikema and Sherman, 1983; Pakrasi et al., 1985a, 1985b) and suggested that CP43' could be associated not only with PSII but also with the PSI complex (Pakrasi et al., 1985a, 1985b). More recently, the induction of CP43' under iron stress was shown to cause the formation of an antenna ring of 18 molecules of CP43' around trimeric PSI in Synechococcus sp. PCC 7942 (Boekema et al., 2001) and Synechocystis PCC 6803 (Bibby et al., 2001a, 2001b). Our preliminary data also indicated that CP43' is preferentially associated with PSI but this association is not restricted to PSI trimers since CP43' is also detectable in PSI monomers (Huner et al., 2001). This was confirmed later in a study using PsaL-less Synechocystis PCC 6803 mutant grown under iron deficiency (Aspinwall et al., 2004).

The ring of CP43' around PSI trimers was suggested to increase the functional absorption cross section of PSI (Boekema et al., 2001) via efficient energetic coupling between the CP43' ring and the PSI reaction center core (Andrizhiyevskaya et al., 2002; Melkozernov et al., 2003). Later, more detailed structural analysis of PSI revealed the existence of various types of trimer and monomer PSI-IsiA supercomplexes, depending on the degree of the iron stress. This suggested a more dynamic and versatile role for the IsiA protein in light harvesting and/or photoprotection in response to iron stress (Yeremenko et al., 2004; Kouil et al., 2005).

Analyses of various cyanobacterial thylakoid membranes have indicated that PSI exists in trimeric and monomeric forms (Boekema et al., 1987; Shubin et al., 1993; Kruip et al., 1994; Komenda, 2000; Tucker and Sherman, 2000) and that the trimeric form of PSI predominates in vivo (Hladik and Sofrova, 1991; Karapetyan et al., 1999a, 1999b; Tucker and Sherman, 2000). It has been demonstrated that the PsaL subunit of PSI plays a key role in biogenesis and formation of stable PSI trimers (Chitnis et al., 1993; Chitnis and Chitnis, 1993) and the reconstitution of PSI trimers from isolated monomers is lipid dependent (Kruip et al., 1999). Indeed, the essential role of phosphatidylglycerol (PG) in the PsaL-dependent formation and stabilization of PSI trimers was recently established (Domonkos et al., 2004; Sato et al., 2004).

Earlier studies have implicated the monomer-trimer equilibrium of PSI in regulation of state transitions (Kruip et al., 1994; Schluchter et al., 1996) and protection against photodamage (Karapetyan et al., 1999a, 1999b). It is generally accepted that state transitions in cyanobacteria strongly depend on and are triggered by changes in the redox state of the plastoquinone (PQ) pool (Mullineaux and Allen, 1990). It is also established that dark-adapted cyanobacterial cells are usually in state II, which is characterized by low fluorescence yield of PSII (Mullineaux and Allen, 1990; Falk et al., 1995). In contrast, iron-stressed cells are locked in state I, associated with characteristic increase of PSII fluorescence at 685 nm and a decrease of PSI fluorescence peak (Öquist, 1974a, 1974b; Falk et al., 1995; Ivanov et al., 2000).

The important findings in recent years regarding iron stress-induced derepression of the IsiA gene clearly underline the dynamic rearrangements of the cyanobacterial antenna and its possible implications on the composition of PSI and PSII Chl-protein complexes in response to iron stress. Despite the continued interest in the induction of isiA during iron stress in cyanobacteria, the functional role of CP43' in vivo remains equivocal. In this study we address the trimer-monomer equilibrium of PSI in regulating the energy distribution between PSII and PSI and the specific role of CP43' in balancing the energy flow under iron-deficient conditions in vivo.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Fluorescence Measurements

Modulated Chl fluorescence measurements revealed low PSII fluorescence yield (Fig. 1A ) in control dark-adapted Synechococcus cells characteristic for state II in cyanobacteria (Mullineaux and Allen, 1986, 1990). Upon illumination with actinic white light (AL), the quenching of PSII was released and maximum PSII fluorescence in the light-adapted state (F_m') increased relative to maximum PSII fluorescence in the dark-adapted state (F_m), characteristic for cells that have undergone a transition to state I (Fig. 1A). In contrast, iron-stressed cells exhibited minimal changes in F_m' relative to F_m upon illumination with AL (Fig. 1C), suggesting that the cells are locked in state I (Falk et al., 1995).

View larger version (13K): [in this window] [in a new window]   Figure 1. Typical modulated Chl fluorescence traces of control (A and B) and iron-stressed (C and D) Synechococcus cells. Nontreated cells, A and C; HgCl2 (40 µM)-treated cells, B and D. ML, Measuring modulated light (650 nm, 0.12 µmol m–2 s–1); SP, saturated light pulse (0.8 s, 2,800 µmol m–2 s–1); AL, actinic light (50 µmol m–2 s–1).

View larger version (19K): [in this window] [in a new window]   Figure 2. A, Fluorescence emission spectra at 77 K of iron-sufficient (solid line) and iron-depleted (250x, dashed line; 500x, dotted line) Synechocystis cells. B, 77 K fluorescence emission spectra of control (iron sufficient) Synechococcus cells (solid line) and after a 48 h shift from control BG-11 medium to iron-deficient conditions (dotted line). C, Time course of the PSII fluorescence at 685 nm () and the position of PSI peak in Synechococcus cells () after transfer to iron-deficient medium. All fluorescence spectra are averages from four to eight corrected scans. Chl fluorescence was excited at 436 nm. Mean values ± SE were calculated from four to eight independent measurements. D, Representative immunoblots of CP43' polypeptide during the shift of Synechococcus cells from iron-sufficient to iron-deficient medium.

The 77 K fluorescence measurements also demonstrated that HgCl₂-treated control Synechococcus cells shifted the fluorescence emission spectrum from state II to state I (Fig. 3A ) and the observed emission spectrum resembled that of iron-stressed cells (Figs. 2B and 3B). In contrast, HgCl₂ had no significant effects on the 77 K spectra of iron-stressed cells (Figs. 1D and 3B). The concentration of HgCl₂ used in this study (40 µM) did not affect the donor side of PSII and electrons generated in the presence of HgCl₂ were able to reduce P700⁺ (Ivanov et al., 2000).

View larger version (12K): [in this window] [in a new window]   Figure 3. Effect of HgCl2 (40 µM) on the 77 K fluorescence emission spectra of dark-adapted control (A) and iron-stressed (B) Synechococcus cells after a shift from control BG-11 medium to iron-deficient conditions. The presented fluorescence spectra are averages from five to six corrected scans.

Immunoblot analysis of proteins from iron-stressed Synechococcus cells revealed the appearance of the isiA gene product, the CP43' Chl-binding polypeptide typical for iron-stressed cells (Pakrasi et al., 1985a; Falk et al., 1995; Ivanov et al., 2000; Sandström et al., 2002) within the first 24 h and no further changes occurred after prolonged iron starvation (Fig. 2D). This was accompanied with a decrease in the level of CP43 polypeptide relative to control cells (data not shown). In contrast, NAD(P)H dehydrogenase protein (Ndh-H) decreased gradually following the shift to iron-deficient condition and its minimal value of 17% was reached after 96 h (Fig. 4, A and B ). Iron deficiency also induced a concomitant decrease in the relative abundance of the PSI-associated, PsaL polypeptide (Fig. 4, A and B).

View larger version (31K): [in this window] [in a new window]   Figure 4. A, Representative western blots of polypeptides from Synechococcus cells probed with antibodies raised against PsaL and NDH-H proteins during the shift of control (iron sufficient) cells to iron-deficient medium. B, Densitometric analysis of PsaL and NDH-H polypeptides. The numerical data for the relative protein abundance were normalized to the maximal values in control (iron sufficient) cells. Mean values ± SE were calculated from three independent experiments.

Nondenaturating PAGE of thylakoid membranes from control (Fig. 5A ) and iron-stressed (Fig. 5D) Synechococcus cells resolved five distinct bands corresponding to the major Chl-protein complexes of PSI and PSII (Komenda, 2000; Tucker and Sherman, 2000). Densitometric scans of the gels exhibited considerable differences between the control and iron-stressed cells, the most distinctive difference being the appearance of a new peak (band 5) in iron-stressed cells (Fig. 5E). Concomitantly, the relative abundance of the PSI trimer (band 1) decreased by 34% in iron-stressed cells compared to the control.

View larger version (32K): [in this window] [in a new window]   Figure 5. Nondenaturating PAGE profiles (A and D) and densitograms (B and E) of the Chl-protein complexes (1–5) in thylakoid membranes of control (A and B) and iron-stressed (D and E) Synechococcus cells. Band 1 is designated to the PSI trimer (PsaA/PsaB) core complex; band 2 is designated to the PSII (D1 + CP43) reaction center complex; band 3 is a mixture of PSII and PSI; band 4 is designated to the PSII (D1 + CP43) complex; and band 5 is PsaA/PsaB + CP43'. The traces in B and E represent averages from three to five independent experiments. C and F, Immunodetection of PsaB, D1:1, CP43, and CP43' polypeptides in Chl-protein complexes separated by nondenaturating PAGE in control and iron-deficient Synechococcus cells, respectively.

Quantitative analysis of the densitometric scans revealed a very small proportion of PSI monomers (band 5) in control cells resulting in a high PSI_TRIMER/PSI_MONOMER ratio (14.60 ± 2.29, n = 3). In contrast, iron-stressed cells exhibited a greater proportion of PSI monomers, and as a consequence, a reduced PSI_TRIMER/PSI_MONOMER ratio (0.61 ± 0.04, n = 5).

Lipid Composition

The lipid composition of control (iron sufficient) Synechococcus sp. PCC 7942 cells was similar (Table II ) to that published earlier (Deshnium et al., 1997). Lipid analysis of iron-stressed Synechococcus cells revealed an almost 2-fold reduction in PG compared to control cells. Digalactosyldiacylglycerol and sulfoquinovosyldiacylglycerol increased 1.7- and 1.4-fold, respectively, while the amount of monogalactosyldiacylglycerol did not change (Table II). In addition, the lipid/Chl ratio was slightly higher in cells subjected to iron stress.

The apparent rise in F_o' after a light-to-dark transition was used as a measure of the dark reduction of the PQ pool by cytosolic reductants (Mano et al., 1995). A transient increase of F_o' level over a period of 20 sec was detected in iron-sufficient cells after turning off the AL (Fig. 6A ). Addition of far-red background light, which preferentially excites PSI and thus drives the oxidation of the PQ pool, completely eliminated the postilluminated increase in F_o' (Fig. 6B). The presence of HgCl₂ also completely inhibited the postillumination increase of F_o (data not shown). In contrast, iron-stressed cells did not exhibit a rise in the F_o' level after a light-to-dark transition (Fig. 6C).

View larger version (14K): [in this window] [in a new window]   Figure 6. Postillumination transients from steady-state fluorescence to Fo' after the AL (50 µmol photons m–2 s–1, 8 min) was turned off in Synechococcus cells grown under control (+Fe; A and B) and iron-deficient (C and D) conditions. The intensity of far-red (FR) light applied after turning off the AL was 10 W m–2 (B and D).

The relative antenna size of PSI in whole cells of Synechococcus sp. PCC 7942 was determined in 3-(3,4-dichlorophenyl)-1,1-dimethylurea (10 µM) pretreated samples by measuring the level of P700 photooxidation as a function of single-turnover flash intensity following the absorbance change at 820 nm (A_820–860; Mauzerall and Greenbaum, 1989; Samson and Bruce, 1995; Lunde et al., 2000). The light-saturation curves of P700 photooxidation in control (Fig. 7A ) and iron-stressed cells (Fig. 7B) were comparable and fit a single-hit Poisson distribution (Mauzerall and Greenbaum, 1989; Samson and Bruce, 1995). However, quantitative analysis revealed that the effective absorption cross sections () of PSI in vivo determined from the flash-saturation curves of P700 photooxidation was 32% lower in cells grown under iron-deficient conditions compared to controls (Table I). Similar results were observed in iron-stressed and iron-replete Synechocystis sp. PCC 6803. Iron-stressed cells showed no significant change in PSI absorbance cross section when grown with 250x lower iron (data not shown) and showed a 36% decrease in PSI cross section when grown under 500x lower iron conditions (Table I).

View larger version (12K): [in this window] [in a new window]   Figure 7. Flash-saturation curves of the relative extent of PSI absorbance changes at 820 nm (A820–860 nm) in control (A) and iron-stressed (B) whole cells of Synechococcus sp PCC 7942. The experimental data points were acquired by flashing the samples with single-turnover flashes of varying intensity and measuring the resulting P700 photooxidation. Mean values ± SE were calculated from six to nine measurements in three independent experiments. The solid curves represent the fit of the experimental data.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Since the photosynthetic and respiratory electron transfer chains of cyanobacteria share common redox components (Scherer, 1990), it is generally accepted that state transitions in cyanobacteria are controlled by the redox state of the PQ pool (Mullineaux and Allen, 1986, 1990). The lack of postillumination increase of F_o' fluorescence (Fig. 6C), which was used as a measure for the dark reduction of the PQ pool (Asada et al., 1993; Mano et al., 1995), indicates that the PQ pool remains mostly oxidized in dark-adapted, iron-stressed cells compared to control Synechococcus (Fig. 6A). These results are consistent with earlier reports indicating that the state II transition in cyanobacteria can be induced by the respiratory electron flow into the PQ pool (Mullineaux and Allen, 1986). Inhibition of NAD(P)H dehydrogenase by HgCl₂ (Mi et al., 1992; Haldimann and Tsimilli-Michael, 2002) resulted in a transition from state II to state I in dark-adapted control cells (Figs. 1B and 3A). This effect was limited in iron-stressed cells corroborating the immunoblot analyses that indicated a lower abundance of Ndh-H in cells grown under iron-stress conditions (Fig. 4). These results are in agreement with the limited capacity for state I-state II transitions reported in the ndhF^– strain of Synechococcus sp. PCC 7002, providing direct evidence for the role of NDH-dependent electron flow in controlling state transitions in cyanobacteria (Huang et al., 2003). Since the NAD(P)H dehydrogenase is one of the most iron-abundant complexes within the cyanobacterial thylakoid membranes, its down-regulation would result in minimal contribution of stromal reductants to the intersystem electron flow in iron-stressed cells. This would minimize dark reduction of the PQ pool and maintain iron-stressed cells in state I. Indeed, an earlier report has demonstrated that the mutant M55 of Synechocystis sp. PCC 6803 lacking the gene encoding subunit B of the NAD(P)H-dehydrogenase complex (Ogawa, 1992) is locked in state I (Schreiber et al., 1995).

In addition, monomer-trimer equilibrium of PSI has been also suggested to play a role in regulation of state transitions in cyanobacteria (Kruip et al., 1994; Schluchter et al., 1996). The observed lower relative abundance of PsaL (Fig. 4) and the reduced amount of PG (Table II), which are thought to be the two key factors controlling the oligomerization state of PSI in cyanobacteria (Chitnis et al., 1993; Chitnis and Chitnis, 1993; Domonkos et al., 2004; Sato et al., 2004), may indeed facilitate the transition of PSI trimers to PSI monomers in iron-stressed cells as reported here (Fig. 5, B and E). The important role of PG in this process could be understood by the presence of three negatively charged PG molecules within the cyanobacterial PSI complex (Jordan et al., 2001). Further supporting evidence for the higher abundance of PSI monomers is the blue shift (4–8 nm) in PSI fluorescence (Fig. 2; Table I), which is indicative of the presence of the monomeric form of PSI in cyanobacteria (Karapetyan et al., 1999b; Kruip et al., 1999).

In agreement with previous reports (Bibby et al., 2001a, 2001b; Boekema et al., 2001), immunoblot analysis of the Chl-protein bands separated by nondenaturating SDS-PAGE revealed that CP43' comigrates with the PSI reaction center protein PsaB (Fig. 5, C and F), thus confirming the suggestion that CP43' can be associated with both PSI-trimer and PSI-monomer Chl-protein complexes (Huner et al., 2001; Aspinwall et al., 2004; Kouil et al., 2005). The possibility that a dissociated form of CP43' comigrates with the PSI monomer cannot be excluded from our study and this possibility would be in agreement with the observation that CP43' aggregates were found unassociated with either PSI or PSII (Kouil et al., 2005). It appears also that the major effect of iron stress in cyanobacteria is the transition of trimeric PSI to its monomeric form. We suggest that, in addition to the limited dark reduction of the PQ pool, the increased proportion of PSI monomers in iron-stressed cells would also favor state I as predicted in the model explaining state transitions in cyanobacterial thylakoid membranes by reversible oligomerization of PSI (Kruip et al., 1994; Schluchter et al., 1996).

It has been shown before that a faster rate of P700 photooxidation occurs in monomeric PSI complexes as compared with PSI trimers (Karapetyan et al., 1999b). Thus, the faster kinetics of P700 photooxidation in iron-stressed cells reported in Sandström et al. (2002) could be also attributed to an increased proportion of PSI monomers under iron stress. One might argue that the faster P700 photooxidation is due to an increase in the functional absorption cross section of PSI within the CP43'-PSI supercomplex (Boekema et al., 2001). However, assuming that the CP43'-PSI supercomplex represents only a small fraction of the total PSI pool in iron-stressed cyanobacteria (Bibby et al., 2001b), this observation could not characterize the entire population of PSI centers. Indeed, our in vivo data from the same two cyanobacterial strains, Synechococcus sp. PCC 7942 (Fig. 7; Table I) and Synechocystis PCC 6803 (Table I), used by Bibby et al. (2001a) and Boekema et al. (2001) failed to demonstrate any increase of the effective absorption cross section of PSI in whole cells under iron-stress conditions. On the contrary, the in vivo PSI absorption cross section was lower in iron-stressed cells.

Thus, we conclude that the induction of CP43' under iron-limited conditions does not contribute to an increase in the effective absorption cross section of the entire population of PSI in vivo. In contrast, since the induction of CP43' is not restricted to iron stress, but has been shown to occur under a myriad of stresses in cyanobacteria (Fulda and Hagemann, 1995; Hagemann et al., 1999; Jeanjean et al., 2003; Yousef et al., 2003; Havaux et al., 2005), we favor the role of CP43' as an effective energy quencher and photoprotector for both PSII and PSI under unfavorable environmental conditions in cyanobacteria in vivo as originally proposed (Park et al., 1999; Sandström et al., 2001).

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Strains and Growth Conditions

Synechococcus sp. PCC 7942 and Synechocystis sp. PCC 6803 cells were grown axenically in liquid BG-11 inorganic medium (pH 7.5) in 80 mL of fresh growth medium in rod-shaped glass tubes bubbled with 5% CO₂ in air. Synechococcus sp. PCC 7942 cells were cultured at 35°C with continuous illumination of 50 µmol photons m^–2 s^–1 of white light (400–700 nm; Philips TLD 18 W/950 fluorescent tubes) as measured by a LI-COR quantum meter (Lambda Instruments) at the culture tube. Iron stress was achieved by culturing cells in a BG-11 medium lacking iron citrate. Synechocystis sp. PCC 6803 cells were grown in liquid BG-11 media at 30°C under constant light at 40 µmol photons m^–1 s^–2. Iron-stressed cells were grown in ironless BG-11 prepared by passing partial media (Na₂CO₃ and KH₂PO₄ only) through a Chelex column to remove trace amounts of iron. Ferric ammonium citrate (diluted by 250x or 500x) was added following retrieval from the column. All iron-limited cells used for experimentation were subcultured from previously iron-limited cells. All glassware used for iron-deficient cell growth was soaked in 1 mM EDTA to remove residual iron.

Lipid Analysis

For lipid analysis, control and iron-stressed Synechococcus sp. PCC 7942 cells were collected by centrifugation at 4,000g_max for 15 min and frozen in liquid N₂ for later lipid extraction. Before lipid extraction, the cells were treated 10 min at 80°C in isopropanol and lipids were extracted according to Porankiewicz et al. (1998). The individual lipids were isolated by two-dimensional thin-layer chromatography and quantified from their acyl group composition as in Bligh and Dyer (1959).

Chl Fluorescence Measurements

Chl fluorescence in dark-adapted (30 min at 35°C) Synechococcus sp. PCC 7942 cells was measured at 77 K using a Jobin Yvon FluoroMax-2 spectrofluorimeter (ISA Jobin Yvon-Spex Instruments S.A.) as in Sandström et al. (2002). Chl concentration of the sample was 10 µg mL^–1. All fluorescence spectra were corrected by subtracting the medium blank and were normalized to the PSII peak centered at 695 nm. Alternatively, 77 K Chl fluorescence in Synechocystis sp. PCC 6803 cells was measured as described previously (Salehian and Bruce, 1992).

Modulated Chl a fluorescence of a dark-adapted (30 min) Synechococcus cells was measured at the growth temperature of 35°C with a PAM 101 fluorescence measuring system (Heinz Walz GmbH) equipped with an emitter-detector-cuvette assembly unit ED-101US/D (Schreiber, 1994). The AL corresponded to the growth irradiance of 50 µmol photons m^–2 s^–1.

Reduction state of the PQ pool was assessed following the postillumination transient increase of Chl fluorescence at the F_o' level (Asada et al., 1993; Mano et al., 1995).

SDS-PAGE and Immunoblotting

Total cellular protein extraction from control and iron-stressed Synechococcus cells, electrophoretic separation, and immunoblot analysis were performed as described previously (Ivanov et al., 2000; Sandström et al., 2002). The relative abundance of CP43, CP43', D1:1, NDH-H, PsaB, and PsaL proteins was detected with specific antibodies at the following dilutions: CP43, 1:1,000; CP43', 1:2,000; NDH-H, 1:750; D1:1, 1:5,000; PsaB, 1:3,000 and PsaL, 1:2,000. Density scanning of the blots from three replicate experiments was performed on a Hewlett-Packard Scan Jet 4200C desktop scanner and a Scion Image densitometry software package (Scion).

Nondenaturating PAGE

Cyanobacterial thylakoid membranes were prepared as described earlier (Giacometti et al., 1996). Electrophoretic separation of Chl-protein complexes was performed as in Komenda (2000) with some modifications. Thylakoid samples were resuspended in 0.1% (w/v) SDS, 0.45% (w/v) dodecylmalthoside, and 0.3 M Tris-HCl (pH 7.5) solubilization buffer containing 13% (v/v) glycerol. Electrophoresis was performed on an 8% (w/v) polyacrylamide resolving gel containing 150 mM Tris-HCl (pH 8.8) buffer and a 4% (w/v) stacking gel containing 50 mM Tris-HCl (pH 6.8) buffer. Running buffer contained 0.2% (v/v) Deriphat 160. Samples were loaded with an equal amount of Chl. The excised lanes were scanned at 671 nm on a Beckman DU 640 spectrophotometer (Beckman Instruments) for Chl absorbance and the relative content of each band was determined by the peak area normalized to the total area of the scan.

P700 Measurements

The effective absorption cross section () of PSI in Synechococcus sp PCC 7942 was estimated in 3-(3,4-dichlorophenyl)-1,1-dimethylurea) (10 µM) pretreated dark-adapted (30 min) whole cells. The absorption changes of P700 (A_820–860) were examined with a ED-P700DW detector (Heinz Walz GmbH) after excitation with a 10 µs flash with varying intensity from a xenon pump flash lamp/control unit XE-STC as described in Lunde et al. (2000). Relative antenna size was determined by fitting the curves with the cumulative Poisson single-hit probability distribution (Mauzerall and Greenbaum, 1989; Samson and Bruce, 1995). The antenna size of PSI in Synechocystis PCC 6803 cells were determined as described in Samson and Bruce (1995) with the following modifications. Single-turnover excitation flashes of variable wavelength were generated by an optical parametric oscillator (OPO-355 model VisIR 2 from GWU-Lasertechnik) driven by a flash-pumped YAG laser (Spectron Lasers). The excitation wavelength was 435 nm for all experiments.

	ACKNOWLEDGMENTS

 
We thank Dr P.R. Chitnis, Iowa State University, for the generous gift of antibodies against PsaL proteins.

Received April 18, 2006; returned for revision June 8, 2006; accepted June 8, 2006.

	FOOTNOTES

 
¹ This work was supported by grants from the Swedish Foundation for International Cooperation in Research and Higher Education (STINT; to G.Ö. and N.P.A.H.), the Swedish Research Council (to G.Ö.), and the Natural Science and Engineering Research Council of Canada (to N.P.A.H. and D.B.).

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Norman P.A. Huner (nhuner{at}uwo.ca).

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.106.082339.

^* Corresponding author; e-mail nhuner{at}uwo.ca; fax 519–661–3935.

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TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
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