Oxygen Evolution Activity In Photoautotrophs Biology Essay

Abstraction

Mn ions are indispensable for oxygen development activity in photoautotrophs. In this paper, we demonstrate the dynamic response of the photosynthetic setup to alterations in Mn bioavailability in blue-green algae. Cultures of the cyanobacterium Synechocystis 6803 could turn, without any discernible consequence on their physiology, on Mn concentrations every bit low as 100 nanometer. Below this threshold a diminution in the photochemical activity of photosystem II ( PSII ) occurred, as apparent by lower O development rates, lower Fv/Fm values and faster QA re-oxidation rates. In 77 K chlorophyll fluorescence spectroscopy a extremum at 682 nanometer was observed. After governing out the part of phycobillisome and IsiA proteins, this set was attributed to the accretion of partly assembled PSII. Surprisingly, the addition in the 682 nm extremum was paralleled by a lessening in the 720 nm extremum, dominated by photosystem I ( PSI ) fluorescence. The consequence on PSI was confirmed by measurings of the P700 photochemical activity. The loss of activity was the consequence of two procedures, loss of PSI nucleus proteins and alterations in the organisation of PSI composites. BN-PAGE analysis revealed a Mn restriction dependent dissociation of PSI trimers into monomers. The sensitive scope for alterations in the organisation of the photosynthetic setup convergences with the scope of Mn concentrations measured in natural environments. We suggest that the ability to pull strings PSI content and organisation allows blue-green algaes to equilibrate negatron conveyance rates between the photosystems. At of course happening Mn concentrations such a mechanism will supply of import protection against visible radiation induced harm.

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 passage metal in the Earth ‘s crust and is critical for all known beings { Frausto district attorney Silva, 2001 # 82 ; Hansch, 2009 # 68 } . Mn ions are of import cofactors for a figure of enzymes, many of which catalyze reactions where different O 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 beings Mn plays a critical function, being that this component forms a bunch of four atoms in the donor side of photosystem II ( PSII ) which participates in catalysing the H2O dividing reaction. The bunch signifiers coordinating bonds with Ca and chloride ions and with residues of the D1 and CP43 proteins { Barber, 2008 # 61 } . Extra extrinsic proteins protect the Mn bunch from the aqueous environment of the lms and signifier channels for H2O and O diffusion. In blue-green algae, these include the PsbO, PsbV ( cytochrome c550 ) and PsbU proteins and potentially two more, PsbQ and PsbP { Roose, 2007 # 39 ; Thornton, 2004 # 40 } .

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Detailss on the cellular constituents involved in Mn conveyance through the outer and interior membranes of the Gram-negative blue-green algae are limited. The cyanobacterium Synechocystis sp. strain PCC 6803 ( hence Synechocystis 6803 ) can roll up up to 108 Mn2+ atoms per cell in its envelope bed. This pool is used as a reservoir for intracellular Mn which is kept changeless at ~106 atoms per cell. It was estimated that a big fraction of the intracellular pool is associated with PSII. Photosynthesis plays an of import function in driving the envelope bed accretion procedure as it does non happen in darkness and is blocked by PSII inhibitors { Keren, 2002 # 16 } . A periplasmic Mn binding protein, MncA, was late discovered by Tottey and coworkers but it remains to be determined whether its map is related to envelope bed Mn accretion { Tottey, 2008 # 41 } . The Mn conveyance path through the plasma membrane under Mn sufficient conditions is non known. Under Mn restricting conditions Mn conveyance is carried out by the MntABC transporter { Bartsevich, 1995 # 30 ; Bartsevich, 1996 # 29 } .

In the late 1960 ‘s Cheniae and Martin demonstrated that Mn restriction in blue-green algae resulted in a decrease in O development capacity { Cheniae, 1967 # 65 ; Cheniae, 1969 # 66 } . Addition of Mn restored oxygen development rates in a light dependent procedure termed photoactivation. This procedure involves the consecutive oxidization and coordinating bonding of the four Mn atoms, Ca and chloride ions, to the C ‘ end point of the mature D1 protein and the CP43 fractional monetary unit of PSII ( reviewed in { Burnap, 2004 # 42 ; Dasgupta, 2008 # 44 } ) .

Despite the important function of manganese in the O development procedure, it is non considered as a restricting factor in aqueous environments. Mn concentrations in oceans and lakes are in the nanomolar scope { Sunda, 1988 # 72 ; Sterner, 2004 # 46 ; Morel, 2008 # 75 ; Chester, 1974 # 84 } , most of it in the signifier of bioavailable hydrated Mn2+ . In different formulas of the standard cyanophyte growing media BG11 { Allen, 1968 # 47 } , Mn concentrations are in the 4-10 i?­M scope, about three orders of magnitude higher.

In this work we examined the response of the photosynthetic setup in Synechocystis 6803 to Mn concentrations in the physiologically relevant scope. Our consequences indicate that of course happening Mn concentrations can restrict photosynthesis. The effects exerted by this restriction were non restricted to PSII. In analogue with the influence on PSII, Mn restriction induced alterations in photosystem I ( PSI ) content, oligomerization province and map.

Consequences

Effectss on biomass accretion, primary productiveness and pigment composing

To analyze the response of Synechocystis 6803 to Mn restriction we grew civilizations in modified BG11 media ( YBG11, { Shcolnick S, 2007 # 35 } ) . YBG11-0, incorporating no added Mn, was supplemented with MnCl2 to concentrations runing from 10-0 i?­M. No Significant effects on growing rate or on the physiology of the cells could be observed at the 10- 1 i?­M scope ( informations non shown ) and our analysis focused on the 1- 0 i?­M concentration scope. Cells adult 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 accretion and O development decreased well below the 100 nM Mn threshold ( Fig. 1 ) . A similar Mn-dependent lessening in primary productiveness was reported by Cheniae and Martin in surveies of the cyanobacterium Anacystis nidulans R2 { Cheniae, 1967 # 65 } . However, the cellular chlorophyll quota remained comparatively changeless and changed by no more than 10 % in civilizations over the 1 to 0 i?­M scope ( Fig. 1B ) .

Effectss on PSII map

The consequence on O development rates suggested a alteration in the map of PSII. To derive farther penetration to the response of PSII to Mn restriction we measured the rate of QA re-oxidation following a saturating flash ( Fig. 2 ) . The maximum photosynthetic output of PSII ( Fv/Fm ) decreased with diminishing Mn concentrations ( Fig. 2A, insert ) . A similar decrease in Fv/Fm values was observed in hydroxyl-amine treated cells, a process that removes the Mn bunch { Hwang, 2008 # 43 } . The rate of fluorescence relaxation, following the saturating flash, is declarative 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 negatron transportation from the QA to the QB quinone adhering site. This rate was non affected by the decrease 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 negatron 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 nanometer 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 footings of dual exponential decay dynamicss. In Figure 2C we quantified alterations in the rate of recombination as the ratio of the deconvoluted slow /fast decay stages. In the absence of an active giver, charge separation is non expected to come on beyond YZ+/QA- , an electron/hole brace that will recombine much faster than S2/ QA- . Taken together, the faster rates of charge recombination ( Fig. 2 ) and the decrease in O development rates ( Fig. 1C ) , indicated an consequence on the energetic belongingss of the donor side of PSII in Mn limited civilizations. In immunoblots utilizing an i??-D1 antibody we could observe a little lessening in the copiousness of this reaction centre protein in Mn limited civilizations ( Fig. 2D ) . Nevertheless, accretion of unrefined pD1 was non observed. The C-terminal extension of D1 prevents the binding of Mn and extrinsic proteins to PSII { Roose, 2004 # 7 } . The consequences presented here indicate that while adhering requires processing, the treating itself is non dependent on the presence of Mn.

Further support for the presence of PSII harbouring a non-functional donor side was provided by 77K chlorophyll fluorescence spectrometry ( Fig. 3A ) . Under Mn restricting conditions the fluorescence strength of the extremum at 682 nanometers increased significantly ( Figs. 3A and 3C ) . Fluorescence at this wavelength can originate from a figure of beginnings including the phycobillisome linker { Yamanaka, 1982 # 80 } , the IsiA protein { Burnap, 1993 # 50 } and partly assembled PSII composites { Keren, 2005 # 53 ; Seibert, 1988 # 49 } . Since the excitement wavelength was set at 430A±5 nm the part of phycobillsomes can be excluded { Hwang, 2008 # 43 } . The part of IsiA to the 682 set was examined in the i?„isiA mutation ( Fig. 3B ) . Under Fe restriction no alteration in the fluorescence at 682 nanometer was observed in the mutation, as would be expected { Burnap, 1993 # 50 } . However, under Mn restriction a distinguishable addition in fluoresce at this wavelength could be observed, bespeaking that the addition in the 682 nm set is non related to the IsiA protein. Therefore, the beginning of the signal can merely be a consequence of an accretion of partly assembled PSII composites. Similar 682 nm extremums were observed in partly assembles isolated PSII composites { Seibert, 1988 # 49 } , purified plasma membranes { Keren, 2005 # 53 } and mutations in the D1 processing peptidase CtpA in vivo { Keren, 2005 # 53 ; Shestakov, 1994 # 52 } . Surprisingly, alterations in the spectra were non limited to the PSII part. With diminishing Mn concentrations we observed a lessening in the comparative strength of fluorescence at 720 nanometer ( 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 consequences prompted us to take a closer expression at the map of PSI.

Effectss on PSI map

The capacity for PSI photochemical activity was measured in vivo by P700 optical density alterations in the presence of DCMU ( Fig. 4A ) . On a per-cell footing, the maximum P700 signal decreased with diminishing Mn concentrations ( Fig. 4B ) . As in the instance of O development and QA re-oxidation a important bead in activity was observed below the 100 nanometers threshold ( Figs. 4B-C ) . The loss of P700 photochemical activity developed bit by bit over a clip class of 3 hebdomads following the transportation of civilizations from 1 i?­M to 0 i?­M media ( Fig. 4C, insert ) .

The loss of activity was accompanied by a debasement of PSI proteins as evidenced by the loss of the nucleus PsaA protein and the accretion of a 52 kDa debasement fragment ( Fig. 4D ) . This fragment is similar in size to the PsaA fragment that accumulates as a consequence of low temperature PSI photodamge in Cucumis sativus foliages ( Kudoh and Sonoike, 2002-add ref ) .

Organization of the photosynthetic setup

In add-on to alterations in the content of PSI we detected differences in the oligomerization province of the photosynthetic composites utilizing blue-native gel cataphoresis ( BN-PAGE, Fig. 5 ) . In cells grown on 1 i?­M Mn we could detect PSI trimers, monomers and dimers. In add-on, we detected PSII monomers. At the low DM/chlorophyll ratios used here, PSII dimers were non expected { Takahashi, 2009 # 63 ; Watanabe, 2009 # 64 } .

Decreasing Mn bioavailability resulted in a little upshift in the mobility of the set incorporating PSI and PSII monomers ( Figs. 5A and B ) . Mass spectrometric analysis verified the presence of both PSI and PSII proteins in the shifted set ( Supplemental table 2 ) . The higher oligomeric provinces of PSI ( dimers, trimers and super-complexes ) changed significantly in response to Mn bioavailability. After three hebdomads of growing in Mn limited media, the degree of these PSI composites decreased to negligible sums, go forthing merely PSI monomers ( Fig. 5A ) . Similar consequences were obtained with membranes solubilized with the mild detergent digitonin ( Auxiliary figure 1 ) . As in the instance of the P700 signal, the consequence of Mn restriction developed bit by bit over clip in civilizations transferred from 1 to 0 i?­M Mn media. Over a 21 500 period most of the PSI trimers were converted into monomers ( Fig. 5B ) . Addition of Mn after 21 vitamin D resulted in the re-appearance of the trimers ( Fig. 5B ) .

The clip graduated table for recovery from Mn restriction was assessed utilizing 77K spectrometry, O development and P700 soaking up ( Fig. 6 ) . After the add-on of Mn to a limited civilization, a gradual lessening in the comparative strength of the extremum at 682 and an addition in the comparative strength of the 720 nm extremum were observed over a clip graduated table of hours ( Fig. 6 ) . In analogue, the extent of the P700 signal and the rates of whole concatenation oxygen development increased ( Fig. 6 ) . In the Mn limited province O development rates are restricted by PSII activity. Following recovery, O development rates are limited downstream of PSII, as apparent by the addition in the rate measured in the presence of unreal acceptors.

Discussion

The information presented in this paper demonstrated a conjunct response of the photosynthetic setup to restriction in intracellular Mn quotas. With worsening Mn concentrations PSII O development rates dropped ( Fig. 1C ) ; QA re-oxidation rates, in the presence of DCMU, increased ( Fig. 2B ) ; and the 682 nm set in the 77 K chlorophyll fluorescence spectra was enhanced ( Fig. 3 ) . These consequences indicated an accretion of partly assembled PSII composites missing a functional donor side. Similar consequences were reported for Mn limited A. thaliana workss where lower Fv/Fm values and a damped PSII thermoluminescence oscillation profile were observed { Lanquar, # 93 } .

Sing that the overall content of D1 exhibited merely a little lessening in Mn deprived cells, we suggest that PSII composites, incorporating a processed D1, are synthesized and await completion of the assembly procedure until Mn is available. Donor side deficient PSII does non adhere phycobillisomes expeditiously { Hwang, 2008 # 43 } . Therefore, while the phycobillisome content in Mn limited cells increased ( Fig. 1B ) , partly assembled PSII will hold some protection against photoinactivation due to a smaller soaking up cross-section as compared to to the full assembled PSII.

Although Mn is non a PSI cofactor we observed a lessening in the fluorescence set at 720 nanometers, associated largely with PSI, and in P700 photochemical activity ( Figs. 3 and 4, severally ) .

Decrease in PSI activity can be the consequence of two procedures, loss of nucleus proteins ( Figure 4D ) and alterations in the oligomerization province of PSI composites. Under restricting 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 excitement spillover to PSII. As compared to trimers, PSI monomers have a much higher chance 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 transportation between PSII and PSI, a procedure frequently referred to as spillover { McConnell, 2002 # 74 } . Transportation of energy between the photosystems may supply extra defence against photo-oxidative harm to PSII under restricting Mn bioavailability. PSI monomers can slake PSII excitement and cut down the hazard of harm from reactive O species.

Trimers are considered as the dominant signifier in blue-green algae and the passage towards to the full functional monomeric PSI most likely took topographic point merely after endosymbiosis { Nelson, 2009 # 57 } . However, there are studies on an active interplay between monomers and trimers in blue-green algae. These include the work from the Rogner group { Kruip, 1994 # 60 ; El-Mohsnawy, 2010 # 92 } who were able to pull strings the PSI oligomerization province in membranes by altering the ionic strength, which led to the suggestion that changes in the oligomeric organisation are related to province passage. Ivanov and coworkers reported an accretion of PSI monomers at the early phases of Fe restriction { Ivanov, 2006 # 55 } . As in the work of Kruip et Al. ( 1994 ) , the monomerization of trimeric PSI was suggested to be associated with a decrease in the yoke with phycobilisomes, giving a smaller PSI soaking up cross subdivision and lower PSI activity.

The Mn bioavailability dependent decrease in the content of both photosystems was non accompanied by a lessening in the cellular chlorophyll content ( Fig. 1B ) . Chlorophyll is well more stable than photosystem proteins in blue-green algae. Its half life in wild type cells is longer than 200 h. The stableness is associated with the map of little cab like proteins ( SCPs ) that bind free chlorophyll and protect it from debasement ( ref- Vavilin D, Yao D, Vermaas W. 2007 ) . Chlorophyll edge to SCPs can be recycled into freshly formed photosystems in the procedure of recovery from Mn restriction. Recovery occurs on the clip graduated table of hours to yearss ( Figs. 5B and 6 ) . The rate of recovery can be compared to the rate of Mn conveyance which was calculated to be at the 105 atoms cell-1 h-1 scope { Keren, 2002 # 16 } . With 1.5 tens 106 Mn atoms per cell in full civilizations and 4.5 tens 105 atoms per cell in limited civilizations ( Supplemental tabular array I ) , it is sensible to presume that the restricting factor in the recovery procedure is the rate of Mn conveyance.

Decision

For both PSII and PSI, Mn restriction did non ensue in the initiation of extended protein debasement processes similar to those observed under drawn-out Fe restriction { Sherman, 1983 # 71 ; Yeremenko, 2004 # 78 } or nitrogen restriction { Schwarz, 1998 # 73 } . Unlike Fe or N, airting Mn from photosynthesis towards other cellular procedures will function small intent. The genome of Synechocystis 6803 does non code for a Mn superoxide-dismutase { Kaneko, 1996 # 79 } hypertext transfer protocol: //genome.kazusa.or.jp/cyanobase ) . In other cyanophyte species Mn demands for superoxide-dismutase can be offset by Fe { Morel, 2008 # 75 } .

By and big, N, phosphate and Fe are considered as the major modification factors for primary productiveness in H2O organic structures. This is non the instance with Mn { Morel, 2008 # 75 } . However, the nM Mn concentration scope of both fresh and salt H2O organic structures { Sunda, 1988 # 72 ; Sterner, 2004 # 46 ; Morel, 2008 # 75 ; Chester, 1974 # 84 } is within the sensitive scope for alterations in photosynthetic activity observed here. The 4.5-22 i?­M Mn concentration used in standard cyanophyte growing media ( BG11, A+ and ASP2, for illustration ) under which PSII is to the full active and PSI is largely trimeric, does non accurately stand for the state of affairs in nature. Under natural conditions the activity, the oligomeric province and the membrane distribution of the photsystems will fluctuate in response to little alterations in Mn concentrations. While restriction of growing rate may ensue from procedures downstream of the negatron transportation concatenation wholly { Raven, 1990 # 91 } , the decreased activity and the alterations in spacial organisation should be considered in surveies of the map of the photosynthetic setup. Furthermore, this partly active province of the photosynthetic setup has deductions for ecological surveies of primary productiveness, particularly when alimentary co-limitation scenarios are taken into history.

Materials and methods

Cyanobacterial strains and civilization conditions

Wild type Synechocystis 6803 civilizations were grown in 50-150 milliliter of YBG11 medium { Shcolnick S, 2007 # 35 } in 250 or 500 ml glass Erlenmeyer flasks, severally. Cultures were maintained under changeless shaking and light of 60 I?mol photons m-2 s- 1 at 30o C. In YBG11 media with no added MnCl2 ( YBG11-0 ) , manganese taint degrees were below the sensing bound in our ICP-MS measurings, 0.1 PPB or 1.8 nanometers. Starter civilizations were grown on YBG11 incorporating 1 i?­M Mn for 3 vitamin D, to cut down taints during subsequent growing in restricting media. In order to take extra Mn, civilizations were washed twice in 20 millimeter MES ; 10mM EDTA pH = 5.0 buffer { Keren, 2002 # 16 } . The cells were resuspended in YBG11-0. Equal aliquots were transferred to flasks incorporating YBG11-0 supplemented with MnCl2 to the desired concentration. Glassware were incubated overnight in 3.7 % HCl and so washed with dual distilled H2O. The i?„isiA mutant { Singh, 2005 # 59 } was grown on media incorporating Kantrex but was assayed in the same media as wild type civilizations. Cell densenesss were measured as optical denseness at 730 nanometers utilizing a Cary3000 spectrophotometer ( Varian, Palo Alto, CA ) as described before { Shcolnick S, 2007 # 35 } . Pigment content was calculated from in vivo soaking up measurings as described by Arnon { Arnon, 1974 # 81 } . The effects of Mn restriction developed bit by bit as a map of media Mn concentrations and clip of incubation. These parametric quantities are reported in all of the figure legends.

Oxygen development measurings

Synechocystis 6803 cells grown on the assorted Mn concentrations were brought to the same cell denseness. Oxygen development rates were measured utilizing a Clark type electrode ( Pasco, Roseville, CA ) . All measurings were carried out under saturating visible radiation at 30i‚° C. Where indicated, 5 i?­M DMBQ and 5 millimeters ferricyanide were added.

Spectroscopic analysis

QA re-oxidation was measured utilizing a Fluorowin2000 fluorometer ( PSI, Berno, Czech democracy ) . DCMU ( 3- [ 3,4-dichlorophenyl ] -1,1-dimethylurea ) was added to a concluding concentration of 10 i?­M. Measurements of this type suffer from a fast decaying signal originating from the flash itself. To avoid this artefact the first informations point following the flash was measured after a hold of 110 i?­s.

The decay stage of the curves was deconvoluted utilizing a dual exponential decay equation:

Where:

T, clip ( s ) .

Y, fluorescence strength ; yo, Fo fluorescence strength.

A1 and A2, pre-exponential factors stand foring the extent of the decay stage.

t1 and t2, decay clip invariables.

Chlorophyll fluorescence at 77K was measured utilizing a Flouromax3 spectrofluorometer ( Jobin Ivon, Longjumaeu, France ) , with the excitement wavelength set at 430 A± 5 nanometer and a 5 nm emanation window. The values presented were internally normalized to the upper limit and minimal values of each curve. P700 photo-reduction was measured in vivo in civilizations adjusted to an O.D730 = 0.5. DCMU was added to a concluding concentration of 10iˆ i?­M. Measurements were performed utilizing a Joliot type spectrophotometer JTS-10 ( Bio-Logic, Claix, France ) utilizing an optical way of 10 millimeter. Excitement was provided by green LED ‘s { Rappaport, 2007 # 76 } and the response of PSI was measured at 705 nanometers as described in { Joliot, 2005 # 77 } .

Protein analysis and mass spectrometric finding

For protein separation utilizing BN-PAGE and SDS-PAGE techniques, Synechocystis 6803 cells were broken by strict bead whipping and the ensuing thylakoyd memebranes were collected by centrifugation as described in { Gombos, 1994 # 86 } . Thylakoyd membranes were resuspended in a buffer incorporating 330 mM Osmitrol, 30 millimeter HEPES, 2 millimeter EDTA and 3 millimeter MgCl2 pH=7.8. Linear 4.5 – 12 % BN-PAGE was performed as described in { Heinemeyer, 2004 # 38 } utilizing the mild detersive n-dodecylmaltoside ( DM ) in a ratio of 0.03:1 g/g DM/chlorophyll. Designation of the constituents of sets cut out of BN-PAGE gels was performed by mass-spectrometric measurings ( Supplemental table 2 ) . SDS-PAGE and immune-detection was performed as described in { Laemmli, 1970 # 83 } utilizing antibodies produced by Agrisera ( Vannas, Sweden ) .

The elemental composing of the cells was determined as described in Scholnick et Al. 2007 ( ref ) . Extracellular Fe was removed by two subsequent 15 proceedingss washes in 20 millimeter MES, 10 millimeter EDTA, pH 5. The cells were digested at 100i‚°C with distilled HNO3, evaporated to dryness, and reconstituted in dual distilled H2O. metal quotas were besides determined utilizing a SCIEX-CDR II Inductively Coupled Plasma Mass Spectrometer ( ICP-MS ; Perkin Elmer, Waltham

Massachusetts ) .

Auxiliary Material

The auxiliary informations includes:

Figure 1: A comparing of BN-PAGE analysis of oligomerization province of membranal protein composites utilizing the detergents DM and digitonin.

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

Table 2: Mass spectrometric designation of proteins in green BN-PAGE sets.

Literature Cited

Figure fables

Figure 1. Effectss of manganese handiness on biomass accretion, pigment content and O development rates.

Starter civilizations grown on 1 i?­M Mn medium were washed twice and transferred to YBG11-0 supplemented with 1-0 i?­M Mn, as indicated. The civilizations were analyzed after 12 yearss of growing.

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

B. Chlorophyll and phycobillisome content, calculated on a per cell footing. In the informations presented here the chlorophyll/cell value at 0 i?­M was 90 % of the value in 1 i?­M Mn. In 4 independent repetitions, measured after 18-21 vitamin D, this value was 100A±14 % .

C. Maximal O development rates, calculated on a per cell footing. Measurements were carried out in the presence of DMBQ and ferricyanide at a saturating light strength. Experiments were repeated three times with comparable consequences.

The decrease in biomass was strongly correlated with the decrease in intracellular Mn quotas ( Supplemental tabular array 1, correlativity coefficient of 0.99 ) . The rate of PSII dependent O development ( Panel C ) was besides strongly correlated to the intracellular Mn quota ( correlation coefficient of 0.98 ) .

Figure 2. Effectss of media Mn handiness on the activity of PSII.

Synechocystis 6803 cells, grown on a scope of Mn concentrations, as described in figure 1, were analyzed for their rates of QAa?’ re-oxidation following a single-turnover saturating flash ( at the place marked by an pointer ) . Changes in fluorescence output were monitored by a series of weak measurement flashes. The experiment was performed in the absence or presence of 10 i?­M DCMU ( A & A ; B, severally ) . The informations were normalized to the minimum and maximum 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 dual exponential decay with a slow ( 0.9-1.5 s ) and a fast ( 0.1-0.2 MS ) stage. R2 values for the tantrums were & gt ; 0.95. Panel C presents the ratio of the slow/fast stage, as a map of the Mn concentration, calculated from informations collected in three independent experiments. The information was fitted with an exponential trendline for lucidity.

Panel D: Immunoblot analysis of PsbA ( D1 ) degrees. Proteins extracted from cells grown on YBG11 incorporating 1 or 0 i?­M Mn, were loaded on an equal chlorophyll footing. The consequences of three independent experiments were quantified utilizing densitometry. In all three experiments D1 degrees in 0 i?­M civilizations were in the scope of 75 % to 85 % of the degrees in 1 i?­M civilizations.

Figure 3. Low temperature chlorophyll fluorescence spectrometry.

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

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

Panel B: Spectrum of the i?„isiA cells grown on Fe or Mn depleted YBG11 media for 7 d. Longer incubation of i?„isiA civilizations in YBG11-Fe resulted in the prostration of the civilization.

Panel C: Ratio of the peak strengths at 682 and 720 nanometer, calculated from informations collected in three independent experiments performed on wild type civilizations.

Figure 4. PSI photochemical activity and protein degrees.

P700 photochemical activity was measured in vivo in civilizations grown on a scope of Mn concentrations. Data was collected on an equal cell concentration footing. 10 i?­M DCMU were added to barricade PSII activity. P700 oxidization was measured as the optical density alterations at 705 nanometer over a scope of actinic light strengths. Samples of natural informations from civilizations grown on 0 or 1 i?­M Mn, measured at 590 i?­mol photons m-2 s-1, are presented in panel A. The information presented in panels B-C are the absolute values of maximum extent of the signal recorded, i?„Amax, as indicated in panel Angstrom.

Panel B: i?„Amax plotted as a map of the actinic visible radiation strength, for civilizations grown on a scope of Mn concentrations for 21 vitamin D.

Panel C: . i?„Amax ( measured at 590 i?­mol photons m-2 s-1 ) , as a map of media Mn concentrations. The consequences are presented as the per centum of the value for 1 i?­M. Data was collected from three independent experiments. Insert, Time class of alterations in i?„Amax following transportation from 1 to 0 i?­M Mn media ( The information was collected from two repetitions ) . After 22 500 i?„Amax of the deficient civilizations decreased to 42 % of the value recorded in control Mn sufficient civilizations.

Panel D: Immunoblot analysis of PsaA degrees. Proteins extracted from cells grown on YBG11 incorporating 1 or 0 i?­M Mn, were loaded on an equal chlorophyll footing. As a control the samples are loaded at A? and A? of the original sample volume. After 14 yearss of incubation the PsaA content of 0 i?­M samples was 65-75 % that of 1 i?­M samples ( n=2 ) . Widening the incubation to 21 vitamin D did ensue in any farther alteration in the PsaA content of 0 i?­M samples, as compared to 1 i?­M samples ( n=2 ) .

Figure 5. BN-PAGE analysis of the oligomeric province of membranal protein composites.

Panel A: Reorganization of thylakoid membrane composites in response to alterations in Mn bioavailability.

Cultures were grown on media incorporating 0-1 i?­M Mn for 21 d. Membranal protein composites were solubilized utilizing a ratio of 0.03g DM to 1 g chlorophyll. Samples contained 5 i?­g chlorophyll each. The designation of proteins in the different membrane composites [ PSII, PSI ( 1 ) , PSI ( 2 ) , PSI ( 3 ) and PSI ( SC ) -supercomplexes ] was performed by peptide mass spectroscopy. Full inside informations of the tryptic peptides identified in each set can be found in Supplemental Table II. As a burden control, 100 % , 50 % and 25 % of the 1 i?­M samples were run aboard the 0 i?­M sample. Based on these controls we could gauge that the PSI trimer content in the 0 i?­M sample is well smaller than 25 % of the 1 i?­M trimer content.

Panel B: Time class of the reorganisation of thylakoid membrane composites during passage in and out of Mn restriction.

Mn sufficient cells were transferred to YBG11-0. Samples were taken and analyzed by BN-PAGE over a 0-21 vitamin D period, as indicated. Immediately after the 21st twenty-four hours sample was collected, MnCl2 was added to a concluding concentration of 1 i?­M. The concluding sample was harvested 3 vitamin D after Mn satiety ( 3R ) .

Figure 6. Time class of recovery from Mn restriction.

Synechocystis 6803 cells were grown on YBG11-0 for 21 d. At clip zero, MnCl2 was added to a concluding concentration of 1 i?­M. Oxygen development rates ( empty circles ) , P700 i?„Amax ( filled squares ) and the 682/720 nm 77K chlorophyll fluorescence strength ratio ( filled circles ) were monitored throughout the recovery stage. Oxygen development was measured in the absence of unreal acceptors. Addition of DMBQ and ferricyanide to limited cells improved the O development rate by 5 % . 24 H into the recovery procedure the add-on of acceptors improved the rate by 65 % .

Tables: