Oxygen Evolution Activity In Photoautotrophs Biology Essay

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Abstract

Mn ions are essential for oxygen evolution activity in photoautotrophs. In this paper, we demonstrate the dynamic response of the photosynthetic apparatus to changes in Mn bioavailability in cyanobacteria. Cultures of the cyanobacterium Synechocystis 6803 could grow, without any observable effect on their physiology, on Mn concentrations as low as 100 nM. Below this threshold a decline in the photochemical activity of photosystem II (PSII) occurred, as evident by lower oxygen evolution rates, lower Fv/Fm values and faster QA re-oxidation rates. In 77 K chlorophyll fluorescence spectroscopy a peak at 682 nm was observed. After ruling out the contribution of phycobillisome and IsiA proteins, this band was attributed to the accumulation of partially assembled PSII. Surprisingly, the increase in the 682 nm peak was paralleled by a decrease in the 720 nm peak, dominated by photosystem I (PSI) fluorescence. The effect on PSI was confirmed by measurements of the P700 photochemical activity. The loss of activity was the result of two processes, loss of PSI core proteins and changes in the organization of PSI complexes. BN-PAGE analysis revealed a Mn limitation dependent dissociation of PSI trimers into monomers. The sensitive range for changes in the organization of the photosynthetic apparatus overlaps with the range of Mn concentrations measured in natural environments. We suggest that the ability to manipulate PSI content and organization allows cyanobacteria to balance electron transport rates between the photosystems. At naturally occurring Mn concentrations such a mechanism will provide important protection against light induced damage.

Footnotes

This work was supported by the Israeli Science Foundation (grants no. 1168/07 and 691/10).

Introduction

Manganese (Mn) is one of the most abundant transition metal in the Earth's crust and is vital for all known organisms {Frausto da Silva, 2001 #82;Hansch, 2009 #68}. Mn ions are important cofactors for a number of enzymes, many of which catalyze reactions where different oxygen species are the substrate, like the manganese superoxide dismutase {Hansch, 2009 #68}(add- J. Biol. Chem. 250: 2801-2807 2801-2807 (1975)), manganese peroxidase {Kenkebashvili, 2009 #69}, or catalase (ref- J.Biol.Chem. 258: 6015-6019 (1983)). In photosynthetic organisms Mn plays a critical role, being that this element forms a cluster of four atoms in the donor side of photosystem II (PSII) which participates in catalyzing the water splitting reaction . The cluster forms coordinative bonds with calcium and chloride ions and with residues of the D1 and CP43 proteins {Barber, 2008 #61}. Additional extrinsic proteins protect the Mn cluster from the aqueous environment of the lumen and form channels for water and oxygen diffusion. In cyanobacteria, these include the PsbO, PsbV (cytochrome c550) and PsbU proteins and potentially two more, PsbQ and PsbP {Roose, 2007 #39;Thornton, 2004 #40}.

Details on the cellular components involved in Mn transport through the outer and inner membranes of the Gram-negative cyanobacteria are limited. The cyanobacterium Synechocystis sp. strain PCC 6803 (hence Synechocystis 6803) can accumulate up to 108 Mn2+ atoms per cell in its envelope layer. This pool is used as a reservoir for intracellular Mn which is kept constant at ~106 atoms per cell. It was estimated that a large fraction of the intracellular pool is associated with PSII. Photosynthesis plays an important role in driving the envelope layer accumulation process as it does not occur in darkness and is blocked by PSII inhibitors {Keren, 2002 #16}. A periplasmic Mn binding protein, MncA, was recently discovered by Tottey and coworkers but it remains to be determined whether its function is related to envelope layer Mn accumulation {Tottey, 2008 #41}. The Mn transport route through the plasma membrane under Mn sufficient conditions is not known. Under Mn limiting conditions Mn transport is carried out by the MntABC transporter {Bartsevich, 1995 #30;Bartsevich, 1996 #29}.

In the late 1960's Cheniae and Martin demonstrated that Mn limitation in cyanobacteria resulted in a reduction in oxygen evolution capacity {Cheniae, 1967 #65;Cheniae, 1969 #66}. Addition of Mn restored oxygen evolution rates in a light dependent process termed photoactivation. This process involves the sequential oxidation and coordinative bonding of the four manganese atoms, calcium and chloride ions, to the C' terminus of the mature D1 protein and the CP43 subunit of PSII (reviewed in {Burnap, 2004 #42;Dasgupta, 2008 #44}).

Despite the crucial role of manganese in the oxygen evolution process, it is not considered as a limiting factor in aqueous environments. Mn concentrations in oceans and lakes are in the nanomolar range {Sunda, 1988 #72;Sterner, 2004 #46;Morel, 2008 #75;Chester, 1974 #84}, most of it in the form of bioavailable hydrated Mn2+. In different recipes of the standard cyanobacterial growth media BG11 {Allen, 1968 #47}, Mn concentrations are in the 4-10 M range, approximately three orders of magnitude higher.

In this work we examined the response of the photosynthetic apparatus in Synechocystis 6803 to Mn concentrations in the physiologically relevant range. Our results indicate that naturally occurring Mn concentrations can limit photosynthesis. The effects exerted by this limitation were not restricted to PSII. In parallel with the influence on PSII, Mn limitation induced changes in photosystem I (PSI) content, oligomerization state and function.

Results

Effects on biomass accumulation, primary productivity and pigment composition

To examine the response of Synechocystis 6803 to Mn limitation we grew cultures in modified BG11 media (YBG11, {Shcolnick S, 2007 #35}). YBG11-0, containing no added Mn, was supplemented with MnCl2 to concentrations ranging from 10-0 M. No Significant effects on growth rate or on the physiology of the cells could be observed at the 10- 1 M range (data not shown) and our analysis focused on the 1- 0 M concentration range. Cells grown on Mn concentrations lower than 100 nM contained 60-70% lower intracellular Mn quotas (Atoms/cell) as compared to cells grown on concentrations above this threshold (Supplemental Table I).

Biomass accumulation and oxygen evolution decreased considerably below the 100 nM Mn threshold (Fig. 1). A similar Mn-dependent decrease in primary productivity was reported by Cheniae and Martin in studies of the cyanobacterium Anacystis nidulans R2 {Cheniae, 1967 #65}. Nevertheless, the cellular chlorophyll quota remained relatively constant and changed by no more than 10% in cultures over the 1 to 0 M range (Fig. 1B).

Effects on PSII function

The effect on oxygen evolution rates suggested a change in the function of PSII. To gain further insight to the response of PSII to Mn limitation we measured the rate of QA re-oxidation following a saturating flash (Fig. 2). The maximal photosynthetic yield of PSII (Fv/Fm) decreased with decreasing Mn concentrations (Fig. 2A, insert). A similar reduction in Fv/Fm values was observed in hydroxyl-amine treated cells, a procedure that removes the Mn cluster {Hwang, 2008 #43}. The rate of fluorescence relaxation, following the saturating flash, is indicative of the rate of QA re-oxidation. In the absence of DCMU, the rate of QA re-oxidation is governed by the rate of forward electron transfer from the QA to the QB quinone binding site. This rate was not affected by the reduction in intracellular Mn (Fig. 2A). In the presence of DCMU, which binds to the QB site, the rate of QA re-oxidation is governed by the rate of recombination of the electron residing in the QA site with the hole residing in the donor side of PSII. The rate of this back reaction was much slower in cells grown on Mn concentrations above the100 nM threshold than in cells grown on Mn concentrations below the threshold (Fig. 2B). QA re-oxidation in the presence of DCMU can be described in terms of double exponential decay kinetics. In Figure 2C we quantified changes in the rate of recombination as the ratio of the deconvoluted slow /fast decay phases. In the absence of an active donor, charge separation is not expected to progress beyond YZ+/QA-, an electron/hole pair that will recombine much faster than S2/ QA-. Taken together, the faster rates of charge recombination (Fig. 2) and the reduction in oxygen evolution rates (Fig. 1C), indicated an effect on the energetic properties of the donor side of PSII in Mn limited cultures. In immunoblots using an -D1 antibody we could detect a small decrease in the abundance of this reaction center protein in Mn limited cultures (Fig. 2D). Nevertheless, accumulation of unprocessed pD1 was not observed. The C-terminal extension of D1 prevents the binding of Mn and extrinsic proteins to PSII {Roose, 2004 #7}. The results presented here indicate that while binding requires processing, the processing itself is not dependent on the presence of Mn.

Further support for the presence of PSII harboring a non-functional donor side was provided by 77K chlorophyll fluorescence spectroscopy (Fig. 3A). Under Mn limiting conditions the fluorescence intensity of the peak at 682 nm increased significantly (Figs. 3A and 3C). Fluorescence at this wavelength can arise from a number of sources including the phycobillisome linker {Yamanaka, 1982 #80}, the IsiA protein {Burnap, 1993 #50} and partially assembled PSII complexes {Keren, 2005 #53;Seibert, 1988 #49}. Since the excitation wavelength was set at 430±5 nm the contribution of phycobillsomes can be excluded {Hwang, 2008 #43}. The contribution of IsiA to the 682 band was examined in the isiA mutant (Fig. 3B). Under Fe limitation no change in the fluorescence at 682 nm was observed in the mutant, as would be expected {Burnap, 1993 #50}. However, under Mn limitation a distinct increase in fluoresce at this wavelength could be observed, indicating that the increase in the 682 nm band is not related to the IsiA protein. Therefore, the origin of the signal can only be a result of an accumulation of partially assembled PSII complexes. Similar 682 nm peaks were observed in partially assembles isolated PSII complexes {Seibert, 1988 #49}, purified plasma membranes {Keren, 2005 #53} and mutants in the D1 processing protease CtpA in vivo {Keren, 2005 #53;Shestakov, 1994 #52}. Surprisingly, changes in the spectra were not limited to the PSII region. With decreasing Mn concentrations we observed a decrease in the relative intensity of fluorescence at 720 nm (Figs. 3A and 3C). At this wavelength the signal is dominated by PSI fluorescence (Add ref. Book chapter: Papageorgiou GC, Fluorescence of photosynthetic pigments in vivo and in vitro. Book: Chlorophyll a fluorescence, a signature of photosynthesis. Eds. Papageorgiou GC and Govindjee. Pages 43-63). These results prompted us to take a closer look at the function of PSI.

Effects on PSI function

The capacity for PSI photochemical activity was measured in vivo by P700 absorbance changes in the presence of DCMU (Fig. 4A). On a per-cell basis, the maximal P700 signal decreased with decreasing Mn concentrations (Fig. 4B). As in the case of oxygen evolution and QA re-oxidation a significant drop in activity was observed below the 100 nM threshold (Figs. 4B-C). The loss of P700 photochemical activity developed gradually over a time course of 3 weeks following the transfer of cultures from 1 M to 0 M media (Fig. 4C, insert).

The loss of activity was accompanied by a degradation of PSI proteins as evidenced by the loss of the core PsaA protein and the accumulation of a 52 kDa degradation fragment (Fig. 4D). This fragment is similar in size to the PsaA fragment that accumulates as a result of low temperature PSI photodamge in cucumber leaves (Kudoh and Sonoike, 2002-add ref).

Organization of the photosynthetic apparatus

In addition to changes in the content of PSI we detected differences in the oligomerization state of the photosynthetic complexes using blue-native gel electrophoresis (BN-PAGE, Fig. 5). In cells grown on 1 M Mn we could observe PSI trimers, monomers and dimers. In addition, we detected PSII monomers. At the low DM/chlorophyll ratios used here, PSII dimers were not expected {Takahashi, 2009 #63;Watanabe, 2009 #64}.

Decreasing Mn bioavailability resulted in a small upshift in the mobility of the band containing PSI and PSII monomers (Figs. 5A and B). Mass spectrometric analysis verified the presence of both PSI and PSII proteins in the shifted band (Supplemental table 2). The higher oligomeric states of PSI (dimers, trimers and super-complexes) changed significantly in response to Mn bioavailability. After three weeks of growth in Mn limited media, the level of these PSI complexes decreased to negligible amounts, leaving only PSI monomers (Fig. 5A). Similar results were obtained with membranes solubilized with the mild detergent digitonin (Supplemental figure 1). As in the case of the P700 signal, the effect of Mn limitation developed gradually over time in cultures transferred from 1 to 0 M Mn media. Over a 21 d period most of the PSI trimers were converted into monomers (Fig. 5B). Addition of Mn after 21 d resulted in the re-appearance of the trimers (Fig. 5B).

The time scale for recovery from Mn limitation was assessed using 77K spectroscopy, oxygen evolution and P700 absorption (Fig. 6). After the addition of Mn to a limited culture, a gradual decrease in the relative intensity of the peak at 682 and an increase in the relative intensity of the 720 nm peak were observed over a time scale of hours (Fig. 6). In parallel, the extent of the P700 signal and the rates of whole chain oxygen evolution increased (Fig. 6). In the Mn limited state oxygen evolution rates are restricted by PSII activity. Following recovery, oxygen evolution rates are limited downstream of PSII, as evident by the increase in the rate measured in the presence of artificial acceptors.

Discussion

The data presented in this paper demonstrated a concerted response of the photosynthetic apparatus to limitation in intracellular Mn quotas. With declining Mn concentrations PSII oxygen evolution rates dropped (Fig. 1C); QA re-oxidation rates, in the presence of DCMU, increased (Fig. 2B); and the 682 nm band in the 77 K chlorophyll fluorescence spectra was enhanced (Fig. 3). These results indicated an accumulation of partially assembled PSII complexes lacking a functional donor side. Similar results were reported for Mn limited A. thaliana plants where lower Fv/Fm values and a damped PSII thermoluminescence oscillation profile were observed {Lanquar, #93}.

Considering that the overall content of D1 exhibited only a small decrease in Mn deprived cells, we suggest that PSII complexes, containing a processed D1, are synthesized and await completion of the assembly process until Mn is available. Donor side deficient PSII does not bind phycobillisomes efficiently {Hwang, 2008 #43}. Therefore, while the phycobillisome content in Mn limited cells increased (Fig. 1B), partially assembled PSII will have some protection against photoinactivation due to a smaller absorption cross-section as compared to fully assembled PSII.

Although Mn is not a PSI cofactor we observed a decrease in the fluorescence band at 720 nm, associated mostly with PSI, and in P700 photochemical activity (Figs. 3 and 4, respectively).

Reduction in PSI activity can be the result of two processes, loss of core proteins (Figure 4D) and changes in the oligomerization state of PSI complexes. Under limiting Mn concentrations PSI trimers disappeared while monomers persisted (Fig. 5, Supplemental Fig. 1 and Supplemental Table 2). The lower activity of the monomers can be attributed to two factors: loss of long wavelength chlorophylls in the monomers {El-Mohsnawy, 2010 #92} and excitation spillover to PSII. As compared to trimers, PSI monomers have a much higher probability for interactions with PSII (in trimers two out of the three membrane embedded surfaces of the monomer are locked in PSI-PSI interactions). Close interactions promote direct energy transfer between PSII and PSI, a process often referred to as spillover {McConnell, 2002 #74}. Transfer of energy between the photosystems may provide additional defense against photo-oxidative damage to PSII under limiting Mn bioavailability. PSI monomers can quench PSII excitation and reduce the risk of damage from reactive oxygen species.

Trimers are considered as the dominant form in cyanobacteria and the transition towards fully functional monomeric PSI most probably took place only after endosymbiosis {Nelson, 2009 #57}. However, there are reports on an active interplay between monomers and trimers in cyanobacteria. These include the work from the Rogner group {Kruip, 1994 #60;El-Mohsnawy, 2010 #92} who were able to manipulate the PSI oligomerization state in membranes by changing the ionic strength, which led to the suggestion that changes in the oligomeric organization are related to state transition. Ivanov and coworkers reported an accumulation of PSI monomers at the early stages of Fe limitation {Ivanov, 2006 #55}. As in the work of Kruip et al. (1994), the monomerization of trimeric PSI was suggested to be associated with a reduction in the coupling with phycobilisomes, yielding a smaller PSI absorption cross section and lower PSI activity.

The Mn bioavailability dependent reduction in the content of both photosystems was not accompanied by a decrease in the cellular chlorophyll content (Fig. 1B). Chlorophyll is considerably more stable than photosystem proteins in cyanobacteria. Its half life in wild type cells is longer than 200 h. The stability is associated with the function of small cab like proteins (SCPs) that bind free chlorophyll and protect it from degradation (ref- Vavilin D, Yao D, Vermaas W. 2007). Chlorophyll bound to SCPs can be recycled into newly formed photosystems in the process of recovery from Mn limitation. Recovery occurs on the time scale of hours to days (Figs. 5B and 6). The rate of recovery can be compared to the rate of Mn transport which was calculated to be at the 105 atoms cell-1 h-1 range {Keren, 2002 #16}. With 1.5 x 106 Mn atoms per cell in replete cultures and 4.5 x 105 atoms per cell in limited cultures (Supplemental table I), it is reasonable to assume that the limiting factor in the recovery process is the rate of Mn transport.

Conclusion

For both PSII and PSI, Mn limitation did not result in the induction of extensive protein degradation processes similar to those observed under prolonged Fe limitation {Sherman, 1983 #71;Yeremenko, 2004 #78} or nitrogen limitation {Schwarz, 1998 #73}. Unlike Fe or nitrogen, redirecting Mn from photosynthesis towards other cellular processes will serve little purpose. The genome of Synechocystis 6803 does not code for a Mn superoxide-dismutase {Kaneko, 1996 #79} http://genome.kazusa.or.jp/cyanobase). In other cyanobacterial species Mn requirements for superoxide-dismutase can be offset by Fe {Morel, 2008 #75}.

By and large, nitrogen, phosphate and iron are considered as the major limiting factors for primary productivity in water bodies. This is not the case with Mn {Morel, 2008 #75}. Nevertheless, the nM Mn concentration range of both fresh and salt water bodies {Sunda, 1988 #72;Sterner, 2004 #46;Morel, 2008 #75;Chester, 1974 #84} is within the sensitive range for changes in photosynthetic activity observed here. The 4.5-22 M Mn concentration used in standard cyanobacterial growth media (BG11, A+ and ASP2, for example) under which PSII is fully active and PSI is mostly trimeric, does not accurately represent the situation in nature. Under natural conditions the activity, the oligomeric state and the membrane distribution of the photsystems will fluctuate in response to small changes in Mn concentrations. While limitation of growth rate may result from processes downstream of the electron transfer chain altogether {Raven, 1990 #91}, the reduced activity and the changes in spatial organization should be considered in studies of the function of the photosynthetic apparatus. Furthermore, this partially active state of the photosynthetic apparatus has implications for ecological studies of primary productivity, especially when nutrient co-limitation scenarios are taken into account.

Materials and methods

Cyanobacterial strains and culture conditions

Wild type Synechocystis 6803 cultures were grown in 50-150 ml of YBG11 medium {Shcolnick S, 2007 #35} in 250 or 500 ml glass Erlenmeyer flasks, respectively. Cultures were maintained under constant shaking and illumination of 60 μmol photons m-2 s- 1 at 30o C. In YBG11 media with no added MnCl2 (YBG11-0), manganese contamination levels were below the detection limit in our ICP-MS measurements, 0.1 PPB or 1.8 nM. Starter cultures were grown on YBG11 containing 1 M Mn for 3 d, to reduce contaminations during subsequent growth in limiting media. In order to remove excess Mn, cultures were washed twice in 20 mM MES; 10mM EDTA pH = 5.0 buffer {Keren, 2002 #16}. The cells were resuspended in YBG11-0. Equal aliquots were transferred to flasks containing YBG11-0 supplemented with MnCl2 to the desired concentration. Glassware were incubated overnight in 3.7% HCl and then washed with double distilled water. The isiA mutant {Singh, 2005 #59} was grown on media containing kanamycin but was assayed in the same media as wild type cultures. Cell densities were measured as optical density at 730 nm using a Cary3000 spectrophotometer (Varian, Palo Alto, CA) as described before {Shcolnick S, 2007 #35}. Pigment content was calculated from in vivo absorption measurements as described by Arnon {Arnon, 1974 #81}. The effects of Mn limitation developed gradually as a function of media Mn concentrations and time of incubation. These parameters are reported in all of the figure legends.

Oxygen evolution measurements

Synechocystis 6803 cells grown on the various Mn concentrations were brought to the same cell density. Oxygen evolution rates were measured using a Clark type electrode (Pasco, Roseville, CA). All measurements were carried out under saturating light at 30 C. Where indicated, 5 M DMBQ and 5 mM ferricyanide were added.

Spectroscopic analysis

QA re-oxidation was measured using a Fluorowin2000 fluorometer (PSI, Berno, Czech republic). DCMU (3-[3,4-dichlorophenyl]-1,1-dimethylurea) was added to a final concentration of 10 M. Measurements of this type suffer from a fast decaying signal arising from the flash itself. To avoid this artifact the first data point following the flash was measured after a delay of 110 s.

The decay phase of the curves was deconvoluted using a double exponential decay equation:

Where:

t, time (s).

y, fluorescence intensity; yo, Fo fluorescence intensity.

A1 and A2, pre-exponential factors representing the extent of the decay phase.

t1 and t2, decay time constants.

Chlorophyll fluorescence at 77K was measured using a Flouromax3 spectrofluorometer (Jobin Ivon, Longjumaeu, France), with the excitation wavelength set at 430 ± 5 nm and a 5 nm emission window. The values presented were internally normalized to the maximum and minimum values of each curve. P700 photo-reduction was measured in vivo in cultures adjusted to an O.D730 = 0.5. DCMU was added to a final concentration of 10M. Measurements were performed using a Joliot type spectrophotometer JTS-10 (Bio-Logic, Claix, France) using an optical path of 10 mm. Excitation was provided by green LED's {Rappaport, 2007 #76} and the response of PSI was measured at 705 nm as described in {Joliot, 2005 #77}.

Protein analysis and mass spectrometric determination

For protein separation using BN-PAGE and SDS-PAGE techniques, Synechocystis 6803 cells were broken by rigorous bead beating and the resulting thylakoyd memebranes were collected by centrifugation as described in {Gombos, 1994 #86}. Thylakoyd membranes were resuspended in a buffer containing 330 mM mannitol, 30 mM HEPES, 2 mM EDTA and 3 mM MgCl2 pH=7.8. Linear 4.5 - 12% BN-PAGE was performed as described in {Heinemeyer, 2004 #38} using the mild detergent n-dodecylmaltoside (DM) in a ratio of 0.03:1 g/g DM/chlorophyll. Identification of the components of bands cut out of BN-PAGE gels was performed by mass-spectrometric measurements (Supplemental table 2). SDS-PAGE and immune-detection was performed as described in {Laemmli, 1970 #83} using antibodies produced by Agrisera (Vännäs, Sweden).

The elemental composition of the cells was determined as described in Scholnick et al. 2007 (ref). Extracellular Fe was removed by two subsequent 15 minutes washes in 20 mM MES, 10 mM EDTA, pH 5. The cells were digested at 100ï‚°C with distilled HNO3, evaporated to dryness, and reconstituted in double distilled water. metal quotas were also determined using a SCIEX-CDR II Inductively Coupled Plasma Mass Spectrometer (ICP-MS; Perkin Elmer, Waltham

Massachusetts).

Supplemental Material

The supplemental data includes:

Figure 1: A comparison of BN-PAGE analysis of oligomerization state of membranal protein complexes using the detergents DM and digitonin.

Table 1: intracellular metal quotas of cell grown on a range on a range of Mn media concentrations.

Table 2: Mass spectrometric identification of proteins in green BN-PAGE bands.

Literature Cited

Figure legends

Figure 1. Effects of manganese availability on biomass accumulation, pigment content and oxygen evolution rates.

Starter cultures grown on 1 M Mn medium were washed twice and transferred to YBG11-0 supplemented with 1-0 M Mn, as indicated. The cultures were analyzed after twelve days of growth.

A. Biomass accumulation (cells/ml). Cultures were inoculated at 106 cells/ml.

B. Chlorophyll and phycobillisome content, calculated on a per cell basis. In the data presented here the chlorophyll/cell value at 0 M was 90% of the value in 1 M Mn. In 4 independent repeats, measured after 18-21 d, this value was 100±14%.

C. Maximal oxygen evolution rates, calculated on a per cell basis. Measurements were carried out in the presence of DMBQ and ferricyanide at a saturating light intensity. Experiments were repeated three times with comparable results.

The reduction in biomass was strongly correlated with the reduction in intracellular Mn quotas (Supplemental table 1, correlation coefficient of 0.99). The rate of PSII dependent oxygen evolution (Panel C) was also strongly correlated to the intracellular Mn quota (correlation coefficient of 0.98).

Figure 2. Effects of media Mn availability on the activity of PSII.

Synechocystis 6803 cells, grown on a range of Mn concentrations, as described in figure 1, were analyzed for their rates of QA− re-oxidation following a single-turnover saturating flash (at the position marked by an arrow). Changes in fluorescence yield were monitored by a series of weak measuring flashes. The experiment was performed in the absence or presence of 10 M DCMU (A & B, respectively). The data were normalized to the minimal and maximal fluorescence values. Fv/Fm values are presented in the insert included in panel A. The decay of the signal following the flash in the presence of DCMU was fitted by a double exponential decay with a slow (0.9-1.5 s) and a fast (0.1-0.2 ms) phase. R2 values for the fits were >0.95. Panel C presents the ratio of the slow/fast phase, as a function of the Mn concentration, calculated from data collected in three independent experiments. The data was fitted with an exponential trendline for clarity.

Panel D: Immunoblot analysis of PsbA (D1) levels. Proteins extracted from cells grown on YBG11 containing 1 or 0 M Mn, were loaded on an equal chlorophyll basis. The results of three independent experiments were quantified using densitometry. In all three experiments D1 levels in 0 M cultures were in the range of 75% to 85% of the levels in 1 M cultures.

Figure 3. Low temperature chlorophyll fluorescence spectroscopy.

Synechocystis 6803 cells, prepared as in figure 1, were collected and frozen in liquid nitrogen for analysis. Excitation wavelength was set at 430 ± 5 nm.

Panel A: Fluorescence spectra of wild type cells grown on different manganese concentrations.

Panel B: Spectrum of the isiA cells grown on Fe or Mn depleted YBG11 media for 7 d. Longer incubation of isiA cultures in YBG11-Fe resulted in the collapse of the culture.

Panel C: Ratio of the peak intensities at 682 and 720 nm, calculated from data collected in three independent experiments performed on wild type cultures.

Figure 4. PSI photochemical activity and protein levels.

P700 photochemical activity was measured in vivo in cultures grown on a range of Mn concentrations. Data was collected on an equal cell concentration basis. 10 M DCMU were added to block PSII activity. P700 oxidation was measured as the absorbance changes at 705 nm over a range of actinic light intensities. Samples of raw data from cultures grown on 0 or 1 M Mn, measured at 590 mol photons m-2 s-1, are presented in panel A. The data presented in panels B-C are the absolute values of maximal extent of the signal recorded, Amax, as indicated in panel A.

Panel B: Amax plotted as a function of the actinic light intensity, for cultures grown on a range of Mn concentrations for 21 d.

Panel C:. Amax (measured at 590 mol photons m-2 s-1), as a function of media Mn concentrations. The results are presented as the percent of the value for 1 M. Data was collected from three independent experiments. Insert, Time course of changes in Amax following transfer from 1 to 0 M Mn media (The data was collected from two repeats). After 22 d Amax of the deficient cultures decreased to 42% of the value recorded in control Mn sufficient cultures.

Panel D: Immunoblot analysis of PsaA levels. Proteins extracted from cells grown on YBG11 containing 1 or 0 M Mn, were loaded on an equal chlorophyll basis. As a control the samples are loaded at ½ and ¼ of the original sample volume. After 14 days of incubation the PsaA content of 0 M samples was 65-75% that of 1 M samples (n=2). Extending the incubation to 21 d did result in any further change in the PsaA content of 0 M samples, as compared to 1 M samples (n=2).

Figure 5. BN-PAGE analysis of the oligomeric state of membranal protein complexes.

Panel A: Reorganization of thylakoid membrane complexes in response to changes in Mn bioavailability.

Cultures were grown on media containing 0-1 M Mn for 21 d. Membranal protein complexes were solubilized using a ratio of 0.03g DM to 1 g chlorophyll. Samples contained 5 g chlorophyll each. The identification of proteins in the different membrane complexes [PSII, PSI(1), PSI(2), PSI(3) and PSI(SC)-supercomplexes] was performed by peptide mass spectrometry. Full details of the tryptic peptides identified in each band can be found in Supplemental Table II. As a loading control, 100%, 50% and 25% of the 1 M samples were run alongside the 0 M sample. Based on these controls we could estimate that the PSI trimer content in the 0 M sample is considerably smaller than 25% of the 1 M trimer content.

Panel B: Time course of the reorganization of thylakoid membrane complexes during transition in and out of Mn limitation.

Mn sufficient cells were transferred to YBG11-0. Samples were taken and analyzed by BN-PAGE over a 0-21 d period, as indicated. Immediately after the 21st day sample was collected, MnCl2 was added to a final concentration of 1 M. The final sample was harvested 3 d after Mn repletion (3R).

Figure 6. Time course of recovery from Mn limitation.

Synechocystis 6803 cells were grown on YBG11-0 for 21 d. At time zero, MnCl2 was added to a final concentration of 1 M. Oxygen evolution rates (empty circles), P700 Amax (filled squares) and the 682/720 nm 77K chlorophyll fluorescence intensity ratio (filled circles) were monitored throughout the recovery phase. Oxygen evolution was measured in the absence of artificial acceptors. Addition of DMBQ and ferricyanide to limited cells improved the oxygen evolution rate by 5%. 24 h into the recovery process the addition of acceptors improved the rate by 65%.

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