lack of phosphatidylglycerol inhibits chlorophyll biosynthesis at … · lack of...

Lack of Phosphatidylglycerol Inhibits ChlorophyllBiosynthesis at Multiple Sites and Limits ChlorophyllideReutilization in Synechocystis sp. Strain PCC 68031

Jana Kope�cná, Jan Pilný, Vendula Krynická, Aleš Tom�cala, Mihály Kis, Zoltan Gombos, Josef Komenda,and Roman Sobotka*

Institute of Microbiology, Centre Algatech, 37981 Trebon, Czech Republic (J.Kop., J.P., V.K., J.Kom., R.S.);Faculty of Science, University of South Bohemia, 37005 Ceske Budejovice, Czech Republic (V.K., A.T., J.Kom.,R.S.); Biology Centre, Institute of Parasitology, 37005 Ceske Budejovice, Czech Republic (A.T.); and Institute ofPlant Biology, Biological Research Centre, H–6701 Szeged, Hungary (M.K., Z.G.)

ORCID IDs: 0000-0002-5232-8441 (A.T.); 0000-0001-8869-6011 (Z.G.); 0000-0003-4588-0382 (J.Kom.).

The negatively charged lipid phosphatidylglycerol (PG) constitutes up to 10% of total lipids in photosynthetic membranes, andits deprivation in cyanobacteria is accompanied by chlorophyll (Chl) depletion. Indeed, radioactive labeling of the PG-depletedDpgsA mutant of Synechocystis sp. strain PCC 6803, which is not able to synthesize PG, proved the inhibition of Chl biosynthesiscaused by restriction on the formation of 5-aminolevulinic acid and protochlorophyllide. Although the mutant accumulatedchlorophyllide, the last Chl precursor, we showed that it originated from dephytylation of existing Chl and not from the block inthe Chl biosynthesis. The lack of de novo-produced Chl under PG depletion was accompanied by a significantly weakenedbiosynthesis of both monomeric and trimeric photosystem I (PSI) complexes, although the decrease in cellular content wasmanifested only for the trimeric form. However, our analysis of DpgsA mutant, which lacked trimeric PSI because of the absenceof the PsaL subunit, suggested that the virtual stability of monomeric PSI is a result of disintegration of PSI trimers. Interestingly,the loss of trimeric PSI was accompanied by accumulation of monomeric PSI associated with the newly synthesized CP43subunit of photosystem II. We conclude that the absence of PG results in the inhibition of Chl biosynthetic pathway, whichimpairs synthesis of PSI, despite the accumulation of chlorophyllide released from the degraded Chl proteins. Based on theknowledge about the role of PG in prokaryotes, we hypothesize that the synthesis of Chl and PSI complexes are colocated in amembrane microdomain requiring PG for integrity.

Photosynthetic membrane of oxygenic phototrophshas a unique lipid composition that has been conservedduring billions of years of evolution from cyanobacteriaand algae to modern higher plants. With no knownexception, this membrane system always contains theuncharged glycolipids monogalactosyldiacylglycerol

and digalactosyldiacylglycerol (DGDG) as well as thenegatively charged lipids sulfoquinovosyldiacylglyc-erol (SQDG) and phosphatidylglycerol (PG; Murataand Siegenthaler, 1998). Interestingly, it seems that PGis the only lipid completely essential for the oxygenicphotosynthesis. The loss of DGDG has only a mildimpact on the cyanobacterial cell (Awai et al., 2007), andas shown recently in the cyanobacterium Synechocystissp. strain PCC 6803, both galactolipids can be in factreplaced by glucolipids (Awai et al., 2014). SQDG andPG are only minor lipid components, each accountingfor 5% to 12% of total lipids (Murata and Siegenthaler,1998). SQDG is dispensable, although its lack results invarious defects (Yu et al., 2002; Aoki et al., 2004), but PGplays an essential role in both cyanobacterial cells andplant chloroplasts (Hagio et al., 2000; Babiychuk et al.,2003).

The critical role of PG has been mostly connected tothe function of PSII. In both cyanobacteria and plants,lack of PG impairs the stability of PSII complexes andthe electron transport between primary and secondaryquinone acceptors inside the PSII reaction center. Asshown in Synechocystis sp., PG molecules stabilize PSIIdimers and facilitate the binding of inner antenna pro-tein CP43 within the PSII core (Laczkó-Dobos et al.,

1 This work was supported by the National Program of Sustain-ability I (identification no. LO1416), the Czech Science Foundation(project nos. Algain [EE2.3.30.0059] and P501/12/G055), the Czechand Hungarian Academy of Sciences (project no. HU/2013/06), andthe Hungarian Research Fund (project no. OTKA 108411 to M.K. andZ.G.).

* Address correspondence to sobotka@alga.cz.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Roman Sobotka (sobotka@alga.cz).

J.Kop., J.Kom., and R.S. performed most of the experiments; V.K.measured cell growth and chlorophyll content using cell counter;A.T. determined the lipid content using liquid chromatography-mass spectrometry technique; J.P. provided technical assistance forall HPLC work; M.K. prepared the pgsA/psaL strain; Z.G. designedsome of the experiments; J.Kom. and R.S. jointly wrote the article; R.S.had major responsibility for the project.

www.plantphysiol.org/cgi/doi/10.1104/pp.15.01150

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2008). Indeed, according to the PSII crystal structure,two PG molecules are located at the interface betweenCP43 and the D1-D2 heterodimer (Guskov et al., 2009).As a consequence, the PG depletion inhibits and de-stabilizes PSII complexes and also, impairs assembly ofnew PSII complexes, although all PSII subunits are stillsynthesized in the cell (Laczkó-Dobos et al., 2008).

Despite the fact that the vital link between PG andPSII is now well established, the phenotypic traits ofPG-depleted cells signal that there are other sites in thephotosynthetic membrane requiring strictly PG mole-cules. In Synechocystis sp., lack of PG triggers rapid lossof trimeric PSI complexes (Domonkos et al., 2004; Satoet al., 2004), and because PSI complexes bind more than80% of chlorophyll (Chl) in the Synechocystis sp. cell, thePG depletion is accompanied by a characteristic Chlbleaching (Domonkos et al., 2004). However, the rea-sons for this symptom are still unclear. Chl metabolismis tightly coordinated with synthesis, assembly, anddegradation of photosystem complexes (for review, seeKomenda et al., 2012b; Sobotka, 2014), and we haveshown recently that the PSI complexes are themain sinkfor de novo Chl produced in cyanobacteria (Kope�cnáet al., 2012). Given the drastic decrease in PSI content inthe PG-depleted cells, Chl biosynthesis must be directlyor indirectly affected after the PG concentration inmembranes drops below a critical value. Although itwas recently suggested that galactolipid and Chl bio-syntheses are coregulated during chloroplast biogene-sis (Kobayashi et al., 2014), a response of the Chlbiosynthetic pathway to the altered lipid content hasnot been examined.

To investigate Chl metabolism during PG starvation,we used the Synechocystis sp. DpgsA mutant, which isunable to synthesize PG (Hagio et al., 2000). The ad-vantage of using theDpgsA strain is in its ability to utilizeexogenous PG from growth medium, which allowsmonitoring of phenotypic changes from a wild type-likesituation to completely PG-depleted cells. Chl biosyn-thesis shares the same metabolic pathway with hemeand other tetrapyrroles. At the beginning of tetrapyrrolebiosynthesis, the initial precursor, 5-aminolevulinic acid(ALA), is made from Glu through glutamyl-tRNA andsubsequently converted in several steps to protopor-phyrin IX. The pathway branches at the point whereprotoporphyrin IX is chelated by magnesium to pro-duce Mg-protoporphyrin IX, the first intermediate onthe Chl branch. This step is catalyzed by Mg-chelatase,a multisubunit enzyme that associates relatively weaklywith the membrane; however, all following enzymesdownward in the pathway are almost exclusively boundto membranes (Masuda and Fujita, 2008; Kope�cná et al.,2012). The last enzyme of the Chl pathway, Chl syn-thase, is an integral membrane protein that attaches aphytyl chain to the last intermediate chlorophyllide(Chlide) to finalize Chl formation (Oster et al., 1997;Addlesee et al., 2000). According to current views, Chlsynthase should also be involved in reutilization ofChl molecules from degraded Chl-binding proteins,which includes dephytylation and phytylation of Chl

molecules with Chlide as an intermediate (Vavilin andVermaas, 2007).

In this study, we show a complex impact of PG de-ficiency on Chl metabolism. The lack of PG inhibitedChl biosynthesis at the two different steps: first, itdrastically reduced formation of the initial precursorALA, and second, it impaired the Mg-protoporphyrinmethyl ester IX (MgPME) cyclase enzyme catalyzingsynthesis of protochlorophyllide (Pchlide). The dimin-ished rate of Chl formation was accompanied by im-paired synthesis of both trimeric and monomeric PSIcomplexes and accumulation of a PSI monomer asso-ciated with the CP43 subunit of PSII. We also showedthat the PG-depleted cells accumulated Chlide, origi-nating from dephytylation of existing Chl, which sug-gests an inability to reutilize Chl for the PSI synthesis.We discuss a scenario that the Chl biosynthesis andsynthesis of core PSI subunits are colocated in PG-enriched membrane microdomains.

RESULTS

PG Depletion Results in Inhibition of De Novo ChlBiosynthesis in Synechocystis sp. Cells

The DpgsA cells supplemented with PG accumulatedabout 25% of PG in total lipids, which is significantlymore than a typical PG content for the Synechocystis sp.wild type (approximately 10%; Table I). However, afterreplacing PG-supplemented growth medium by freshgrowth medium lacking PG, the PG level in the mutantquickly dropped down to 2% to 3% of total lipids in2 d and about 1% in 5 d (Table I). Interestingly, evenafter this drastic decrease in PG content, cells were ableto grow, although very slowly, for an additional10 d (Fig. 1A). It indicates that the essential pool of PGis very small. In agreement with previous reports(Gombos et al., 2002; Bogos et al., 2010; Itoh et al., 2012),PG starvation triggered a decrease in Chl content; theloss of Chl per cell was already apparent after 48 hfollowed by a gradual decrease to approximately 50%of the original Chl level after 5 d (Fig. 1; Table I). After14 d of depletion, the cellular Chl content finallydropped to approximately 15%. Between days 5 and 14

Table I. Chl content in the wild type and the DpgsA mutant grown inthe presence or absence of PG in the growth medium

For the recovery, the grown medium was supplemented with20 mg mL21 PG. Each value represents the mean 6 SD from three in-dependent experiments.

Strain Depletion Chl PG

mg 108 cells21 Mol% of total lipidsWild type 4.7 6 0.2 9.6 6 0.37DpgsA +PG 3.9 6 0.4 26.5 6 5.6DpgsA 2PG 2 d 3.1 6 0.3 2.8 6 1.4DpgsA 2PG 5 d 1.9 6 0.2 1.1 6 0.14DpgsA 2PG 10 d 1.2 6 0.1 Traces (,1)DpgsA 2PG 14 d 0.6 6 0.04 Traces (,1)

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of PG starvation, cells in the culture divided roughlythree times, and the Chl content per cell decreased by afactor of 3 (Fig. 1A; Table I). In theory, in a case of zerode novo synthesis of Chl proteins, three cycles of celldividing would dilute Chl content per cell 8 times. Be-cause the cell volume did not significantly changeduring the experiment (Fig. 1A), we can conclude that,even after 2 weeks of PG starvation, Synechocystis sp.cells were still able to synthesize a limited amount ofChl proteins to partially compensate for the prolifera-tion. An intensive degradation of Chl proteins or Chlmolecules during PG starvation is thus very unlikely. Incontrast to the dramatic decrease of the total Chl

amounts, the whole-cell absorption spectra showedthat levels of phycobiliproteins remained basically un-changed (Supplemental Fig. S1). An almost identicalcourse of Chl bleaching was observed if the DpgsA cellswere supplemented by 0.5 mM Glu or 5 mM Glc (datanot shown). It suggests that the loss of Chl complexes isnot related to nitrogen/carbon deficiency or energydeprivation caused by an impaired PSII activity.

Because the result indicated that the observed Chlbleaching during PG starvation could be linked toreduced Chl production in cells, we used 14C incor-poration into Chl as a measure of the rate of Chl bio-synthesis. Cells of the wild type and DpgsA mutantwere labeled by [14C]Glu. Extracted Chl was convertedto Mg-chlorin and separated by a thin-layer chroma-tography, and the chromatographic plate was exposedto an x-ray film (Kope�cná et al., 2012). Indeed, theDpgsA strain cultivated in a PG-free medium for5 d showed a very small rate of de novo synthesis ofChl, because the signal of labeled Mg-chlorin didnot exceed 10% of that found in the wild-type strain(Fig. 1B).

PG-Depleted Cells Lack Chl Precursors But AccumulateChlide, Which Originates from Chl Dephytylation

To evaluate Chl biosynthesis in more detail and lo-calize its limiting step(s) during PG depletion, we an-alyzed intermediates of this metabolic pathway. TheDpgsA strain grown in the presence of PG accumulatedsimilar levels of Chl precursors as the wild type withexception of Pchlide, which reached only about 50% ofthat in the wild type (Fig. 2). A short 2-d PG starvationhad no detectable effect on the precursor levels,but after 5 d, a dramatic reduction of the Pchlide poolwas observed (,10% of that in the wild type); also,there was significantly less protoporphyrin IX, Mg-protoporphyrin IX, and MgPME. After 10 d of PGstarvation, only traces of Pchlide could be detected,whereas the MgPME level exhibited an increase, indi-cating a block in the conversion of MgPME to Pchlide.

Notably, the last Chl intermediate Chlide graduallyincreased during the PG depletion, although the pre-ceding intermediate Pchlide in the pathway becamebarely detectable (Fig. 2). However, Chlide is also anintermediate of Chl reutilization (Vavilin and Vermaas,2007) and can be produced by dephytylation of existingold Chl molecules. To distinguish between Chlideproduced de novo from Pchlide and Chlide producedas an intermediate of Chl recycling, we supplementedcell cultures of the wild type and the DpgsA straingrown without PG for 5 d with [14C]Glu and monitoredits incorporation into Pchlide and Chlide (Fig. 3). In thewild type, we found that, after 30 min, approximately 3timesmore [14C]Gluwas incorporated into Pchlide thaninto Chlide, which corresponded to a higher total poolof Pchlide compared with Chlide. This result indi-cated that either de novo synthesis of Chlide waslagging behind the synthesis of Pchlide and resulted

Figure 1. Changes in Chl content and rate of Chl formation during PGdepletion in the DpgsA mutant. A, Concentration of Chl per 108 cells(blue circles), cells proliferation (white diamonds), and mean of cellvolume (top x axis) during PG starvation. Data represent means fromthree independent measurements; error bars indicate SD. B, Radio-labeling of Chl molecules in the wild type (WT) and the DpgsA mutantcells starved of PG for 5 d. Chl was radiolabeled by [14C]Glu using a30-min pulse, extracted with methanol/0.2% NH4OH, and immedi-ately converted into Mg-chlorin to remove phytol chain and 13- methylgroup. Mg-chlorin was separated on a thin-layer chromatography(TLC) plate (“Materials and Methods”), and radioactivity was detectedusing an x-ray film (Autorad). The signal of labeled Mg-chlorinquantified using ImageQuant TL software (GE Healthcare) is shown inparentheses.

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Chlorophyll Metabolism under Altered Lipid Content

in accumulation of newly synthesized Pchlide or de novo-formedChlidewas quickly converted into Chlmolecules.In contrast, the PG-starved DpgsA strain accumulated 25times more Chlide than Pchlide; however, their [14C]Glulabeling result was similar, meaning that only 4% ofdetected Chlide was newly synthesized. This means thatthe vast majority of Chlide detected in the PG-depletedDpgsA strain was formed by detachment of phytol frompreviously synthesized Chl and did not originate from denovo biosynthesis.

The Chl Biosynthesis Is Blocked at Two Sites during PGDepletion, and Levels of Most Enzymes Are Decreasedwhile the Chl Synthase Overaccumulates

The attenuated total metabolite flow through the Chlpathway after PG starvation together with a transientaccumulation of MgPME and the drastic reduction inPchlide and de novo Chlide pools (Figs. 2 and 3A) in-dicated a block at the beginning of the tetrapyrrolepathway and another block at the Pchlide formation. Toassess potential bottlenecks in the Chl pathway, wesupplemented the DpgsA culture (PG depleted for 5 d)with 5 mM ALA for 1 h and detected accumulated Chlprecursors (Fig. 3B). Comparedwith controlDpgsA cellsgrown without ALA, it was obvious that the externalALA dramatically elevates levels of Chl precursorspreceding the cyclase step (approximately 7 timesfor protoporphyrin IX, approximately 25 times for

Mg-protoporphyrin, and approximately 60 times forMgPME; Fig. 3B). In contrast, the Pchlide pool was al-most unchanged (Fig. 3B). These data showed that allenzymes preceding MgPME cyclase are functional incells lacking PG but that the metabolite flow throughthe pathway is kept low because of a restriction on theALA formation step.

The monitoring of the abundance of enzymes in-volved in Chl biosynthesis by specific antibodiesshowed very different responses of individual enzymesto the PG starvation. Only the ChlD subunit of Mg-chelatase, the MgPME cyclase, and Chl synthaseshowed significantly lowered levels in DpgsA cells inthe presence of PG, whereas levels of other enzymeswere relatively similar to those of the wild type (Fig.4). Notably, after 5 d of PG starvation, the Chl syn-thase level doubled, whereas levels of other enzymesremained stable. Additional growth with no externalPG, however, affected accumulation of most enzymes;only the ferrochelatase enzyme producing heme andlight-dependent Pchlide oxidoreductase remainedstable during 10 d of PG starvation (Fig. 4). On thecontrary, the MgPME cyclase was almost lost com-pletely, which was in agreement with the increasedlevel of MgPME observed in these cells (Fig. 2). Aninteresting exception was the Chl synthase, whichexhibited the opposite trend, reaching its highest levelafter 10 d of PG depletion. No enzymes catalyzinglater steps in Chl biosynthesis accumulated in solubleprotein fraction during PG starvation (Fig. 4).

Loss of Chl in PG-Depleted Cells Parallels Inhibition ofPSI Synthesis and Formation of the PSI-CP43 Complex

Because the majority of Chl molecules in Synechocystissp. cells are present in the trimeric PSI, Chl depletioninduced by lack of PG was expected to reflect the dis-appearance of this Chl-binding complex. This wasconfirmed by analysis of membrane protein complexesusing Clear Native gel electrophoresis. This type ofseparation allowed us to visualize PSI complexes di-rectly in the gel scans and PSII complexes using fluo-rescence of Chl molecules bound to PSII proteins (Fig.5). In contrast to the trimer, the content of PSI mono-mers did not seem to be affected by a lack of PG(Supplemental Table S1). The gel also confirmed aprevious observation by Sakurai et al. (2003) that thelevel of the PSII core dimers was largely reduced inthe PG-deprivedmutant cells, whereas accumulation ofthe monomeric form of PSII was affected to a lesserextent (Supplemental Table S1). The observed stablelevel of monomeric PSI could indicate that the biogen-esis of this PSI complex is much less sensitive to PGdeficiency than the trimeric form. Alternatively, thesePSI monomers could also originate from disassembly oftrimeric complexes as proposed by Domonkos et al.(2004). To clarify this point, we inactivated the psaLgene in the DpgsA mutant to block trimerization of PSI(Chitnis and Chitnis, 1993). The course of chlorosis

Figure 2. Analysis of intermediates of Chl biosynthesis in the PG-depleted DpgsAmutant. Quantification of Chl precursors in the mutantgrown in a PG-supplemented medium (+PG) or under PG-depletedconditions (2PG) for the indicated number of days. Cells (2 mL) wereharvested at optical density at 750 nm (OD750) = 0.4, and Chl precursorswere extracted with methanol and quantified by HPLC (“Materials andMethods”). Values represent mean 6 SD from three independent mea-surements; the dotted line indicateswild-type (WT) level calculated as amean of three independent measurements. MgP, Mg-protoporphyrin IX;PPIX, protoporphyrin IX; *, significant difference tested using a pairedt test (P = 0.05).

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during PG starvation was very similar in the DpgsA andthe DpgsA/DpsaL mutants (Supplemental Fig. S1),whereas the Clear Native gel showed a gradually de-creasing level of monomeric PSI in the double mutant(Fig. 5).We can conclude that the apparently stable poolof monomeric PSI in the PG-depleted DpgsA primarilyresults from the ongoing disintegration of PSI trimers.Intriguingly, a unique pigmented complex (molecu-

lar mass of approximately 450 kD) migrating on nativeelectrophoresis between PSII dimer and PSI monomerwas detected in the PG-depleted cells of both DpgsAmutants (Fig. 5). To clarify composition of this complex,we performed two-dimensional (2D) Clear Native/SDSelectrophoresis of membrane proteins isolated fromDpgsA and DpgsA/DpsaL strains. Staining of the gel bySypro Orange revealed that the 450-kD complex is amonomeric PSI associated with CP43 (SupplementalFig. S2), and this was further confirmed by blotting of

2D gel followed by immunodetection of CP43 and PSIsubunit PsaD (Fig. 6).

To explore de novo synthesis of PSI and PSII subunitsand their assembly into high-molecular mass com-plexes in the DpgsA mutant, we monitored the incor-poration of radioactive amino acids into membraneproteins. The solubilizedmembranes from radiolabeledcells were separated by 2D Blue Native/SDS electro-phoresis, and the stained 2D gel was finally exposed toa phosphorimager to visualize labeled protein spots(Fig. 7). The DpgsA strain grown without PG for5 d accumulated very low amounts of de novo assem-bled PSII dimer, whereas the labeled PSII monomerremained still fairly abundant. Interestingly, the newlysynthesized PSII core complex lacking CP43 (RC47) as-sembly intermediate of PSII (Boehm et al., 2012) withhighly labeled D2 and D1 subunits accumulated signif-icantly more, indicating disruption of the de novo

Figure 3. Pulse radiolabeling of Chlide and Pchlidein the DpgsA strain and assessment of Chl precursorlevels after ALA feeding. A, Wild-type (WT) andDpgsA cells grown without PG for 5 d were radio-labeled with [14C]Glu using a 30-min pulse. Chlprecursors were extracted by methanol, concen-trated by evaporation, and immediately injected intoan HPLC system equipped with a diode-array de-tector and a flow scintilator. The HPLC chromato-grams (upper) show peaks of Chlide and Pchlidedetected by A635, and the values indicate molarstoichiometries of these pigments calculated fromauthentic standards. The chromatograms (lower)show radioactive signals (autorad) of the identicalmeasurement depicted above (in this case, valuesindicate areas of integrated peaks). Note the differentscale of the y axis for the wild type and themutant. B,Content of Chl precursors inDpgsA cells PG depletedfor 5 d and then fed by 5 mM ALA for 1 h. Extractedpigments were separated by HPLC equipped by twofluorescence detectors (FLD1 and FLD2). Each de-tector was set towavelengths allowing them to detectindicated Chl precursors (“Materials and Methods”).

assembly of PSII at the step of CP43 attachment. Theassembly of PSII is obviously impaired, despite thepresence of an abundant and intensively labeled CP43assembly module, which consists of the CP43 associatedwith small PSII subunits PsbK, PsbZ, and Psb30 (Fig. 7;Komenda et al., 2004, 2012a, 2012b). In agreement withthe analysis of the DpgsA/DpsaL strain (Fig. 5), the PGdepletion strongly suppressed synthesis of both mono-meric and trimeric PSI complexes. Interestingly, the PSI-CP43 complex contained a portion of radioactivelylabelled CP43 comparable with the portion incorpo-rated into the monomeric PSII (Fig. 7). In conclusion,the synthesis of both PSI monomer and trimer wasinhibited to a similar extent by lack of PG, whereas thesynthesis of PSII Chl proteins remained intensive andthe newly synthesized CP43 associated with PSI.

DISCUSSION

In this study, we show that decrease in cellular levelof Chl in PG-depleted Synechocystis sp. cells correlateswith inhibition of the Chl biosynthesis pathway, whichwas documented by the low rate of incorporation of 14Cisotope into Chl and overall decreased levels of Chl

precursors. Although we lack information about earlyintermediates of the tetrapyrrole biosynthesis pathway,the PG depletion was accompanied by a significantdecrease in the level of protoporphyrin IX—the lastcommon substrate for the synthesis of both Chl andheme. It indicates that the lack of PG restricted totalmetabolite flow through the tetrapyrrole pathway. In-deed, levels of all monitored precursors up to Pchlidecan be strongly elevated by supplementing PG-starvedcells with the initial precursor ALA. Notably, the con-tent of phycobilisomes is much less affected by lack ofPG (Supplemental Fig. S1), implying that the remainingtraces of protoporphyrin IX are preferentially chan-neled to ferrochelatase for heme and bilin synthesis. Itmight be related to an intriguing surplus of ferroche-latase enzyme in Synechocystis sp. cells observed earlier(Sobotka et al., 2008b); a Synechocystis sp. ferrochelatasemutant possessing only about 10% of the ferrochelataseactivity produced enough heme tomaintain the cellularlevel of heme and phycobilisomes.

The MgPME oxidative cyclase seems to be the only en-zyme involved in Chl biosynthesis, activity that is mark-edly inhibited by lowPG content. It is, however, difficult tospeculate about the role of PG in the formation of Pchlide,because this enzymatic step is poorly characterized. The

Figure 5. Contents of membrane protein complexes in the wild type(WT) and DpgsA and DpgsA/psaL strains during PG depletion. Mem-brane complexes isolated from the PG-depleted cells were separated by4% to 12% Clear Native electrophoresis; R indicates 3 d of recovery.The amount of proteins loaded for 100% of each sample corresponds to200 mL of cells at OD750nm = 1. The gel was scanned in transmittancemode (scan) using an LAS 4000 Imager (Fuji), and to visualize PSIImonomer and dimer, Chl fluorescence emitted by PSII was excited byblue light and detected also by LAS 4000 (Chl fl.). Finally, the gel wasthen stained by Coomassie Blue (CBB stain). Designations of complexesare PSI[3] and PSI[1] for trimeric and monomeric PSI, respectively, RCC[2]and RCC[1] for dimeric and monomeric PSII core complexes, respec-tively, and ATPase for ATP synthase. The boxed areas and asteriskshighlight a pigmented complex accumulated during PG depletion. Forthe quantification of individual bands using ImageQuant software, seeSupplemental Table S1.

Figure 4. Quantification of enzymes of the Chl biosynthetic pathway inthe DpgsA mutant. The DpgsA mutant was grown in PG-depleted me-dium for the indicated number of days and then supplemented with PGfor 3 d for the recovery (R). Membrane and soluble protein fractionswere separated by SDS electrophoresis and blotted to a PVDF mem-brane. Enzymes were detected by specific antibodies. The amount ofproteins loaded for 100% of each sample corresponds to 100mL of cellsat OD750nm = 1. ChlH, ChlD, and ChlI are subunits of the magnesiumchelatase, and Genome Uncoupled4 protein (Gun4) is a protein re-quired for the magnesium chelatase activity. AcsF1 (Sll1214) is acomponent of the MgPME oxidative cyclase. ChlSyn, Chl synthase;DVR, 3,8-divinyl chlorophyllide 8-vinyl reductase; FeCH, ferrochela-tase; GGR, geranylgeranyl reductase; MgPMT, Mg-protoporphyrinmethyl transferase; POR, light-dependent protochlorophyllide oxido-reductase; WT, wild type.

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Aerobic cyclization system Fe-containing protein (AcsFprotein; Sll1214) is the only known component ofMgPME cyclase; however, there are probably other es-sential subunits (see Hollingshead et al., 2012 and dis-cussion therein). For the assembly of functional MgPMEcyclase complex, PG could play a critical role. It is at-tractive to speculate that a block in Pchlide synthesisturns down thewhole tetrapyrrole pathway consistentlywith some observations in tobacco (Nicotiana tabacum)AcsF-antisense plants (Peter et al., 2010). However,Synechocystis sp. mutants with low AcsF content accu-mulate huge amounts ofMgPMEandprotoporphyrin IXas well (Peter et al., 2009; Hollingshead et al., 2012),which indicates that, at least in cyanobacteria, there is notight feedback between cyclase activity and the rate ofALA synthesis.An enigmatic point is the low level of Chl synthase in

PG-supplemented DpgsA cells (Fig. 5). We can onlyspeculate that the content of this membrane enzyme issomehow negatively influenced by the aberrantly highcontent of PG (Table I). However, unlike other enzymesin the Chl pathway, the level of Chl synthase wasstrongly elevated in PG-deprived cells. We believe thatthis enzyme is essential under conditions wherede novo Chl synthesis is blocked and reutilization ofChl released from Chl proteins must be very efficient.There is solid experimental evidence provided byVavilin et al. (2005) that Chl has a much longer lifetimethan individual Chl-binding apoproteins, and it impliesthat Chl must be recycled. Moreover, Vavilin et al.(2005) showed that at least some reused Chl undergo acycle of dephytylation catalyzed by anunknownenzymeand repeated phytylation catalyzed by Chl synthase.Therefore, accumulation of increased amounts of Chl

synthase is not surprising, because dividing cells canonly rely on recycled Chl, which should be used at thehighest efficiency, and therefore, Chl synthase is re-quired. For this reason, detection of an increasing levelof Chlide during PG depletion was surprising. ThisChlide could apparently not be used for Chl formationand repeated incorporation into Chl protein. A possiblereason for this phenomenon could be localization of thispool of Chlide in a membrane region that is not avail-able for Chl synthase and/or protein synthesis ma-chinery to be incorporated into newly synthesized Chlproteins. Alternatively, the Chl reutilization could belimited by lack of isoprenoids/phytol, but in such acase, the PG depletion should also result in loweringlevels of all carotenoids. As shown in SupplementalFigure S3, a long-term PG deficiency (14 d) reducedcontent of b-carotene or zeaxanthin, but the content ofmyxoxanthophyl was more than 4 times higher than incontrol (+PG) cells. We, therefore, do not expect that thePG is required for the isoprenoid biosynthesis.

The inhibition of Chl biosynthesis during PG depri-vation was most obviously accompanied by the steepdecrease in the level of PSI trimer, which has beenshown to represent the main sink of newly synthesizedChl in Synechocystis sp. cells (Kope�cná et al., 2012). Forstrains lacking PsaL, the main sink for de novo Chl isalmost certainly the monomeric PSI, and again, PGdeficiency in the pgsA/psaLmutant triggered the loss ofthis complex. Based on ourmeasurement of Chl contentper cell correlated with cell proliferation (Fig. 1A), wecan almost rule out that the low PG content causes anextensive PSI degradation. In fact, on the contrary, it isclear that PG-depleted cells are still able to partlycompensate for dilution of complexes during cell

Figure 6. 2D analysis of the membrane proteincomplexes isolated from DpgsA (A) and DpgsA/DpsaL(B) strains grown with PG and without PG for 10 d.Membrane proteins were separated by 4% to 14%Clear-Native (CN)-PAGE and then, in the second di-mension, by 12% to 20% SDS-PAGE. The 2D gel wasstained by Sypro Orange (for stained gels, seeSupplemental Fig. S2) and then blotted to a PVDFmembrane. CP43 and PsaD proteins were detected byspecific antibodies. Protein complexes were desig-nated as in Figure 5. PSI[1]-CP43 denominates thecomplex between CP43 and monomeric PSI. Chlfluorescence emitted by PSII is also shown (Chl fl.).Designations of complexes are PSI[3] and PSI[1] fortrimeric andmonomeric PSI, respectively, and RCC[2]and RCC[1] for dimeric and monomeric PSII corecomplexes, respectively.

growth; however, the synthesis of PSI complexes issimply too weak to prevent gradual bleaching.

The origin and function of PSI monomer associatedwith CP43 as well as the abundance of this complex inPG-depleted cells are rather enigmatic. Such a complexhas been recently identified in the strain lacking CP47,which contains significant amounts of unassembledCP43 (Komenda et al., 2012a). Indeed, PG depletion hasbeen shown to impair PSII assembly process andnamely, destabilize binding of CP43 antenna within PSIIcore, resulting in an increased fraction of unassembledCP43 (Laczkó-Dobos et al., 2008). This effect was con-firmed in this study; however, significant radioactivelabeling of CP43 bound to PSI suggests that this inter-action occurs during or early after de novo synthesis ofCP43. We are tempted to speculate that a transient as-sociation of de novo CP43 with PSI before its integrationinto RC47 is a standard step of PSII biogenesis. Absenceof Chl fluorescence emitted by the PSI-CP43 complex(Fig. 5) shows an efficient energy transfer from CP43 toPSI, making, in theory, PSI monomer an effective pro-tector of the newly synthesized CP43 against photo-damage. Alternatively, PSI could also provide Chl forthe newly synthesized CP43; it has been recently pro-posed that PSI could act as a donor of Chl for a newlyformed PSII RC complex consisting of D1 and D2(Knoppová et al., 2014). It is worth noting that the

synthesis of CP43 is much less sensitive to the lack ofde novo Chl than the synthesis of the structurally similarCP47 protein (Sobotka et al., 2008a). Indeed, the CP43 aswell asD1 andD2proteins are intensively synthesized incells that produce only about 10% of de novo Chl mol-ecules (Fig. 7). These results are in line with our modelthat cyanobacteria utilizes the majority of fresh Chlmolecules to maintain a constant level of Chl-rich andlong-lived PSI complexes (Kope�cná et al., 2012). Short-lived PSIIs are produced by a different protein machin-ery, which included components for Chl recycling andpotentially, stripping Chl molecules from PSI. The factthat the syntheses of PSII and PSI subunits take place onseparated translocon systems has been shown recently(Linhartová et al., 2014).

There is a tight interconnection between Chl bio-synthesis and synthesis of Chl-binding proteins. Acomplete inhibition of Chl biosynthesis ultimatelyblocks synthesis of all Chl proteins (Kope�cná et al.,2013), most likely because the insertion of Chl mole-cules into growing polypeptides is a prerequisite for thecorrect folding of Chl-binding proteins (for review, seeSobotka, 2014). Interestingly, there is also an oppositecontrol, because we found that the inhibition of proteintranslation by chloramphenicol immediately stops Chlbiosynthesis (R. Sobotka and J. Komenda, unpublisheddata). Given this cross talk between translation of

Figure 7. Synthesis of the CP43-PSI complex in theDpgsA mutant cells grown in a PG-depleted mediumfor 5 d. Cells were radiolabeled with [35S]Met/Cysmixture using a 20-min pulse. Membrane proteinscorresponding to 5 mg of Chl were separated by BlueNative (BN) electrophoresis on a 4% to 14% lineargradient gel, and another 12% to 20% SDS electro-phoresis was used for the second dimension. The 2Dgels were stained with Coomassie Blue (CBB-stainedgels) and then dried, and the labeled proteins weredetected by a phosphoimager (Autorad). Proteincomplexes were designated as in Figure 1. The whitearrows indicate PsaA/B core subunits, and the blackarrows indicate the CP43 associated with PSI[I] (thePSI[1]-CP43 complex) on both the stained gel and theautoradiogram. Designations of complexes are PSI[3]and PSI[1] for trimeric and monomeric PSI, respec-tively, and RCC[2] and RCC[1] for dimeric andmonomeric PSII core complexes, respectively. *, PsaDsubunit of PSI; u.p., unassembled proteins.

Kope�cná et al.

apoproteins and Chl synthesis, it is difficult to distin-guish which process is actually more sensitive to lowPG content. The PSI contains three PG molecules, ofwhich two are rather peripheral, but one is buriedwithin the PsaA subunit (Jordan et al., 2001). Thissuggests that a certain level of PG could be critical forproper folding of PsaA or assembling of PSI and thatthe observed down-regulation of the tetrapyrrolepathway might simply represent a response to im-paired PSI synthesis. However, the ALA-producingenzyme in plants has been shown to be integratedinto a membrane complex (Czarnecki et al., 2011),and PG could be essential for ALA formation.Without de novo Chl, the PSI synthesis is certainlyarrested. We, however, propose another more com-plex scenario, which reflects current knowledgeabout PG function in prokaryotes.According to the available data, Chl proteins are

synthesized on membrane-bound ribosomes andinserted into membrane through SecY translocase(Zhang et al., 2001; for review, see Sobotka, 2014), andas shown recently, the Chl synthase is physically at-tached to the translocon system through YidC insertase(Chidgey et al., 2014). Although clear evidence is stillmissing, there are good reasons to expect that all en-zymes of the Chl pathway, including MgPME cyclase,are assembled into a larger metabolomic unit aroundtranslocons (Kauss et al., 2012; Chidgey et al., 2014).Such large assemblies of enzymes providing de novoChl would be particularly critical for the PSI synthesis.Interestingly, it has become apparent that PG or cardi-olipin (two linked PG molecules) domains colocalizewith SecY translocons within bacterial cells (Gold et al.,2010; Tsui et al., 2011). In addition, the functioning ofthe SecY translocase in Escherichia coli was shown to bedependent on cardiolipin—the major anionic lipid inthis bacterium (Gold et al., 2010). In another example, inBacillus subtilis, the SecY exhibits characteristic spiralpatterns of localization that are lost during PG deple-tion (Campo et al., 2004). The increasing experimentalevidence suggests that phospholipids in bacteria formmicrodomains, which are important for the transloconlocalization and function. Taking into account that thefunctional link between phospholipids and transloconapparatus is conserved in both gram-negative and-positive bacteria, it is quite likely that PG moleculescolocalize with cyanobacterial translocons as well. SuchPG- and SecY-rich microdomains could be in fact iden-tical to proposed biosynthetic centers—large proteinfactories responsible for synthesis of photosyntheticcomplexes. The presence of such centers (or zones) hasalready been shown in green alga Chlamydomonasreinhardtii (Schottkowski et al., 2012), and they are alsoexpected to occur in cyanobacteria (for review, seeKomenda et al., 2012b; Rast et al., 2015). We speculatethat lack of PG disintegrates membrane microdomainsessential for PSI synthesis. It seems completely sensi-ble that the first (ALA-producing) steps of the tetra-pyrrole pathway would be colocated in the samemembrane foci and activated only during translation

of core PSI subunits. This model needs to be tested inthe future; however, it agrees with the simultaneousinhibition of both PSI and Chl synthesis by low PGcontent observed in this work.

MATERIALS AND METHODS

Preparation of Synechocystis sp. Mutants andGrowth Conditions

The Synechocystis sp.DpgsAmutant strain is described inHagio et al., 2000. Toprepare the DpgsA/DpsaL strain, the psaL gene in the DpgsAmutant was replacedwith anv-cassette conferring spectinomycin resistance (Kłodawska et al., 2015). Ifnot stated otherwise, both Synechocystis sp. mutants were grown photoautotro-phically at 29°Cunder continuous illumination of 30mmol of photonsm22 s21 in aliquid BG11 medium supplemented with 20 mM dioleoyl-PG (Sigma). PG deple-tion was achieved bywashing the cells two times with PG-free medium followedby cultivation in a fresh PG-free medium. Wild-type cells were grown in the PG-free medium. Cultures were aerated on a rotary shaker.

Absorption Spectra and Determination of Chl Content

Absorption spectra of whole cells were measured at room temperature witha Shimadzu UV-3000 Spectrophotometer. Chl was extracted from cell pellets(2mL; OD750nm = approximately 0.4) with 100%methanol, and its concentrationwas measured spectrophotometrically according to the work by Porra et al.(1989). Numbers of cells and their sizes were determined in 0.9% (v/v) NaClelectrolyte solution with a calibrated Coulter Counter (Beckman Multisizer IV)equipped with a 20-mm aperture.

Lipid Analysis

Lipids were extracted from 1 mL of pelleted cells (OD750nm = approximately0.5) by 0.5 mL of chloroform and methanol solution using a beadbeater as de-scribed inKošťál and Šimek, 1998. A small amount (5mL) of the obtained extractwas injected into the Surveyor HPLC System connected to the Ion Trap LTQMass Spectrometer (Thermo Finnigan). Samples were separated on the Geminicolumn (250 3 2 mm, 3 mm; Phenomenex) using 5 mM ammonium acetate inmethanol as the mobile phase (A), water as B, and 2-propanol as the phase (C).The analysis was performed using the following gradient: 0 to 5 min of 92% Aand 8% B, 5 to 12 min of 100% A, 12 to 50 min of 100% to 40% A and 0% to 60%C, 50 to 65 min of 40% A and 60% C, and 65 to 80 min back to 92% A and 8% B.The separation was run with a flow rate of 250 mL min21 at 30°C. The massspectrometer was operated in the positive and negative ion detection modes at+4 and24 kV, respectively, with capillary temperature at 220°C and nitrogen asthe shielding and auxiliary gas. To obtain the full-scan electrospray ionizationmass spectra of lipids, mass range of 140 to 1,400 D was scanned every 0.5 s.Identification of particular lipids was achieved by collision-induced dissocia-tions. Peak areas of detected lipid classes (monogalactosyldiacylglycerol,DGDG, SQDG, and PG) were used for the estimation of their relative content inthe analyzed samples.

Isolation and Analysis of Membrane Protein Complexes

Membrane proteins were isolated from 50 mL of cells at OD750nm =approximately 0.4 according to Dobáková et al. (2009) using buffer A (25 mM

MES-NaOH, pH6.5, 5mMCaCl2, 10mMMgCl2, and 20% [w/v] glycerol). Isolatedmembrane complexes were solubilized in buffer A containing 1% (w/v)N-dodecyl-b-D-maltoside and analyzed by native or denaturing electrophoresis as de-scribed in Kope�cná et al., 2013. Proteins separated in the gel were stained by eitherCoomassie Blue or Sypro Orange followed by transfer onto a polyvinylidenedifluoride (PVDF) membrane. Membranes were incubated with specific primaryantibodies and then, a secondary antibody conjugated with horseradish peroxidase(Sigma). The CP43 and PsaD primary antibodies were raised in rabbits againstwhole proteins isolated from Synechocystis sp. Antibodies raised againstsubunits of Mg-chelatase, Mg-protoporphyrin methyltransferase, light-dependent Pchlide oxidoreductase, 3,8-divinyl Pchlide 8-vinyl reductase, andgeranylgeranyl reductase were provided by C. Neil Hunter (University of Shef-field). Ferrochelatase and Genome Uncoupled4 protein antibodieswere provided

byAnnegretWilde (University of Freiburg). The antibodies raised against barleyAcsF were provided by Poul Erik Jensen (University of Copenhagen). TheSynechocystis sp. Chl synthase antibodies were raised in rabbit against the syn-thetic peptide containing amino acid residues 89 to 104 of Slr0056.

Quantification of Chl Precursors

For quantitative determination of Chl precursors in the cells, 2 mL of cultureat OD750nm = approximately 0.4 was harvested, and the pellet was resuspendedin 25 mL of ultrapure water. To extract Chl precursors, cells were mixed with75 mL of methanol, and this solutionwas incubated in complete darkness and atroom temperature for 15 min. Subsequently, the sample was centrifuged, thesupernatant containing extracted pigments was collected, and the extractionwas repeated using the same volume of methanol. Obtained supernatants werecombined, and 150 mL were immediately separated by HPLC (Agilent-1200;Agilent) on an RP Column (Nova-Pak C18, 4-mm particle size, 3.9 3 150 mm;Waters) using 30% (v/v) methanol in 0.5 M ammonium acetate and 100% (v/v)methanol as solvents A and B, respectively. Porphyrins were eluted with a lineargradient of solvent B (65%–75% in 30 min) at a flow rate of 0.9 mLmin21 at 40°C.HPLC fractions containing Mg-protoporphyrin IX (retention time of approxi-mately 11 min), Chlide (approximately 12 min), Pchlide (approximately 16 min),MgPME (approximately 17 min), and protoporphyrin IX (approximately 19 min)were detected by two fluorescence detectors, with the first fluorescence detectorset to 440/650 nm (excitation/emissionwavelengths) for 0 to 17min and 400/630nm for 17 to 25min and the second set at 416/595 nm throughout the experiment.

Radioactive Labeling of Proteins and Pigments

Radioactive pulse labeling of the proteins in cells was performed using amixture of [35S]Met and [35S]Cys (Translabel; MP Biochemicals). After a short(20 min) incubation of cells with labeled amino acids, the solubilized mem-branes isolated from radiolabeled cells were separated by 2D Blue Native/SDSelectrophoresis. The stained 2D gel was finally exposed to a phosphorimagerplate, which was scanned by Storm (GEHealthcare) to visualize labeled proteinspots (Dobáková et al., 2009).

Chl was labeled by [14C]Glu (specific activity of 267 mCi mmol21; MoravekBiochemicals), and the incorporation of radioactivity was quantified essentiallyas described in Kope�cná et al., 2012. For quantitative analysis of radioactivelylabeled Chl precursors, 200mL of Synechocystis sp. cells with OD750nm = 0.4werelabeled with [14C]Glu as described above. Washed and harvested cells wereresuspended by 30 s of vortexing using 450 mL of water and 50 mL of zirconia/silica beads. Chl precursors were extracted by adding 1.1 mL of methanol toachieve a final concentration of 70% (v/v). Cell debris was then pelleted bycentrifugation for 5 min at maximal speed, and extractionwas repeated onemoretime. Supernatants fromboth extractionswere pooled and completely evaporatedunder vacuum at 35°C using a rotary evaporator. Dry extracts were then dis-solved in 200 mL of methanol, and Chl precursors were separated as describedabove. The eluted pigments were detected by a diode-array detector (Agilent1260) set at 635 nm connected to a flow scintillation analyzer (Radiomatic 150TR;Perkin Elmer) set for detection and quantification of 14C isotope.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Whole-cell spectra of the DpgsA and DpgsA/DpsaL mutants.

Supplemental Figure S2. 2D analysis of the membrane protein complexesisolated from DpgsA and DpgsA/DpsaL strains.

Supplemental Figure S3. Pigment content in the DpgsA mutant grown inmedium supplemented with PG and under PG-depleted conditions for14 d.

Supplemental Table S1. Quantification of PSI and PSII complexes in theDpgsA mutant during PG depletion.

ACKNOWLEDGMENTS

We thank C. Neil Hunter (Sheffield University, United Kingdom) andAnnegret Wilde (University of Freiburg, Germany) for the antibodies, the

Laboratory of Analytical Biochemistry and Metabolomics (Biology Centre,Czech Academy of Sciences) for free access to liquid chromatography-massspectrometry instruments, and Bára Zdvihalová (Institute of Microbiology,Trebon, Czech Republic) for technical assistance.

Received July 22, 2015; accepted August 11, 2015; published August 12, 2015.

LITERATURE CITED

Addlesee HA, Fiedor L, Hunter CN (2000) Physical mapping of bchG,orf427, and orf177 in the photosynthesis gene cluster of Rhodobactersphaeroides: functional assignment of the bacteriochlorophyll synthetasegene. J Bacteriol 182: 3175–3182

Aoki M, Sato N, Meguro A, Tsuzuki M (2004) Differing involvement ofsulfoquinovosyl diacylglycerol in photosystem II in two species ofunicellular cyanobacteria. Eur J Biochem 271: 685–693

Awai K, Ohta H, Sato N (2014) Oxygenic photosynthesis without galactolipids.Proc Natl Acad Sci USA 111: 13571–13575

Awai K,Watanabe H, Benning C, Nishida I (2007) Digalactosyldiacylglycerol isrequired for better photosynthetic growth of Synechocystis sp. PCC6803under phosphate limitation. Plant Cell Physiol 48: 1517–1523

Babiychuk E, Müller F, Eubel H, Braun HP, Frentzen M, Kushnir S (2003)Arabidopsis phosphatidylglycerophosphate synthase 1 is essential forchloroplast differentiation, but is dispensable for mitochondrial func-tion. Plant J 33: 899–909

Boehm M, Yu J, Reisinger V, Beckova M, Eichacker LA, Schlodder E,Komenda J, Nixon PJ (2012) Subunit composition of CP43-less photo-system II complexes of Synechocystis sp. PCC 6803: implications for theassembly and repair of photosystem II. Philos Trans R Soc Lond B BiolSci 367: 3444–3454

Bogos B, Ughy B, Domonkos I, Laczkó-Dobos H, Komenda J, Abasova L,Cser K, Vass I, Sallai A, Wada H, et al (2010) Phosphatidylglyceroldepletion affects photosystem II activity in Synechococcus sp. PCC 7942cells. Photosynth Res 103: 19–30

Campo N, Tjalsma H, Buist G, Stepniak D, Meijer M, Veenhuis M,Westermann M, Müller JP, Bron S, Kok J, et al (2004) Subcellular sitesfor bacterial protein export. Mol Microbiol 53: 1583–1599

Chidgey JW, Linhartová M, Komenda J, Jackson PJ, Dickman MJ,Canniffe DP, Koník P, Pilný J, Hunter CN, Sobotka R (2014) Acyanobacterial chlorophyll synthase-HliD complex associates with theYcf39 protein and the YidC/Alb3 insertase. Plant Cell 26: 1267–1279

Chitnis VP, Chitnis PR (1993) PsaL subunit is required for the formation ofphotosystem I trimers in the cyanobacterium Synechocystis sp. PCC6803. FEBS Lett 336: 330–334

Czarnecki O, Hedtke B, Melzer M, Rothbart M, Richter A, Schröter Y,Pfannschmidt T, Grimm B (2011) An Arabidopsis GluTR binding proteinmediates spatial separation of 5-aminolevulinic acid synthesis in chlo-roplasts. Plant Cell 23: 4476–4491

Dobáková M, Sobotka R, Tichý M, Komenda J (2009) Psb28 protein isinvolved in the biogenesis of the photosystem II inner antenna CP47(PsbB) in the cyanobacterium Synechocystis sp. PCC 6803. Plant Physiol149: 1076–1086

Domonkos I, Malec P, Sallai A, Kovács L, Itoh K, Shen G, Ughy B, BogosB, Sakurai I, Kis M, et al (2004) Phosphatidylglycerol is essential foroligomerization of photosystem I reaction center. Plant Physiol 134:1471–1478

Gold VA, Robson A, Bao H, Romantsov T, Duong F, Collinson I (2010)The action of cardiolipin on the bacterial translocon. Proc Natl Acad SciUSA 107: 10044–10049

Gombos Z, Várkonyi Z, Hagio M, Iwaki M, Kovács L, Masamoto K, ItohS, Wada H (2002) Phosphatidylglycerol requirement for the function ofelectron acceptor plastoquinone Q(B) in the photosystem II reactioncenter. Biochemistry 41: 3796–3802

Guskov A, Kern J, Gabdulkhakov A, Broser M, Zouni A, Saenger W(2009) Cyanobacterial photosystem II at 2.9-A resolution and the roleof quinones, lipids, channels and chloride. Nat Struct Mol Biol 16: 334–342

Hagio M, Gombos Z, Várkonyi Z, Masamoto K, Sato N, Tsuzuki M, WadaH (2000) Direct evidence for requirement of phosphatidylglycerol inphotosystem II of photosynthesis. Plant Physiol 124: 795–804

Hollingshead S, Kope�cná J, Jackson PJ, Canniffe DP, Davison PA,Dickman MJ, Sobotka R, Hunter CN (2012) Conserved chloroplast

Kope�cná et al.

open-reading frame ycf54 is required for activity of the magnesiumprotoporphyrin monomethylester oxidative cyclase in Synechocystis PCC6803. J Biol Chem 287: 27823–27833

Itoh S, Kozuki T, Nishida K, Fukushima Y, Yamakawa H, Domonkos I,Laczkó-Dobos H, Kis M, Ughy B, Gombos Z (2012) Two functionalsites of phosphatidylglycerol for regulation of reaction of plastoquinoneQ(B) in photosystem II. Biochim Biophys Acta 1817: 287–297

Jordan P, Fromme P, Witt HT, Klukas O, Saenger W, Krauss N (2001)Three-dimensional structure of cyanobacterial photosystem I at 2.5 Aresolution. Nature 411: 909–917

Kauss D, Bischof S, Steiner S, Apel K, Meskauskiene R (2012) FLU, anegative feedback regulator of tetrapyrrole biosynthesis, is physicallylinked to the final steps of the Mg(++)-branch of this pathway. FEBS Lett586: 211–216

Kłodawska K, Kovács L, Várkonyi Z, Kis M, Sozer Ö, Laczkó-Dobos H,Kóbori O, Domonkos I, Strzałka K, Gombos Z, et al (2015) Elevatedgrowth temperature can enhance photosystem I trimer formation andaffects xanthophyll biosynthesis in Cyanobacterium Synechocystis sp.PCC6803 cells. Plant Cell Physiol 56: 558–571

Knoppová J, Sobotka R, Tichý M, Yu J, Konik P, Halada P, Nixon PJ,Komenda J (2014) Discovery of a chlorophyll binding protein complexinvolved in the early steps of photosystem II assembly in Synechocystis.Plant Cell 26: 1200–1212

Kobayashi K, Fujii S, Sasaki D, Baba S, Ohta H, Masuda T, Wada H(2014) Transcriptional regulation of thylakoid galactolipid biosynthesiscoordinated with chlorophyll biosynthesis during the development ofchloroplasts in Arabidopsis. Front Plant Sci 5: 272

Komenda J, Knoppová J, Kope�cná J, Sobotka R, Halada P, Yu J, NickelsenJ, Boehm M, Nixon PJ (2012a) The Psb27 assembly factor binds to theCP43 complex of photosystem II in the cyanobacterium Synechocystis sp.PCC 6803. Plant Physiol 158: 476–486

Komenda J, Reisinger V, Müller BC, Dobáková M, Granvogl B, EichackerLA (2004) Accumulation of the D2 protein is a key regulatory step forassembly of the photosystem II reaction center complex in SynechocystisPCC 6803. J Biol Chem 279: 48620–48629

Komenda J, Sobotka R, Nixon PJ (2012b) Assembling and maintaining thePhotosystem II complex in chloroplasts and cyanobacteria. Curr OpinPlant Biol 15: 245–251

Kope�cná J, Komenda J, Bu�cinská L, Sobotka R (2012) Long-term acclima-tion of the cyanobacterium Synechocystis sp. PCC 6803 to high light is ac-companied by an enhanced production of chlorophyll that is preferentiallychanneled to trimeric photosystem I. Plant Physiol 160: 2239–2250

Kope�cná J, Sobotka R, Komenda J (2013) Inhibition of chlorophyll bio-synthesis at the protochlorophyllide reduction step results in the paralleldepletion of Photosystem I and Photosystem II in the cyanobacteriumSynechocystis PCC 6803. Planta 237: 497–508

Košťál V, Šimek P (1998) Changes in fatty acid composition of phospho-lipids and triacylglycerols after cold-acclimation of an aestivating insectprepupa. J Comp Physiol B 168: 453–460

Laczkó-Dobos H, Ughy B, Tóth SZ, Komenda J, Zsiros O, Domonkos I,Párducz A, Bogos B, Komura M, Itoh S, et al (2008) Role of phos-phatidylglycerol in the function and assembly of Photosystem II reactioncenter, studied in a cdsA-inactivated PAL mutant strain of Synechocystissp. PCC6803 that lacks phycobilisomes. Biochim Biophys Acta 1777:1184–1194

Linhartová M, Bu�cinská L, Halada P, Je�cmen T, Setlík J, Komenda J,Sobotka R (2014) Accumulation of the Type IV prepilin triggers deg-radation of SecY and YidC and inhibits synthesis of Photosystem IIproteins in the cyanobacterium Synechocystis PCC 6803. Mol Microbiol93: 1207–1223

Masuda T, Fujita Y (2008) Regulation and evolution of chlorophyll me-tabolism. Photochem Photobiol Sci 7: 1131–1149

Murata N, Siegenthaler PA (1998) Lipids in photosynthesis: an overview.In N Murata, PA Siegenthaler, eds, Lipids in Photosynthesis: Structure,Function and Genetics, Vol 6. Kluwer Academic Publishers, Dordrecht,The Netherlands, pp 1–20

Oster U, Bauer CE, Rüdiger W (1997) Characterization of chlorophyll a andbacteriochlorophyll a synthases by heterologous expression in Esche-richia coli. J Biol Chem 272: 9671–9676

Peter E, Rothbart M, Oelze ML, Shalygo N, Dietz KJ, Grimm B (2010) Mgprotoporphyrin monomethylester cyclase deficiency and effects on tet-rapyrrole metabolism in different light conditions. Plant Cell Physiol 51:1229–1241

Peter E, Salinas A, Wallner T, Jeske D, Dienst D, Wilde A, Grimm B(2009) Differential requirement of two homologous proteins encodedby sll1214 and sll1874 for the reaction of Mg protoporphyrin mono-methylester oxidative cyclase under aerobic and micro-oxic growthconditions. Biochim Biophys Acta 1787: 1458–1467

Porra RJ, Thompson WA, Kriedemann PE (1989) Determination of accu-rate extinction coefficients and simultaneous equations for assayingchlorophylls a and b extracted with four different solvents: verificationof the concentration of chlorophyll standards by atomic absorptionspectroscopy. Biochim Biophys Acta 975: 384–394

Rast A, Heinz S, Nickelsen J (2015) Biogenesis of thylakoid membranes.Biochim Biophys Acta 1847: 821–830

Sakurai I, Hagio M, Gombos Z, Tyystjarvi T, Paakkarinen V, Aro EM,Wada H (2003) Requirement of phosphatidylglycerol for maintenance ofphotosynthetic machinery. Plant Physiol 133: 1376–1384

Sato N, Suda K, Tsuzuki M (2004) Responsibility of phosphatidylglycerolfor biogenesis of the PSI complex. Biochim Biophys Acta 1658: 235–243

Schottkowski M, Peters M, Zhan Y, Rifai O, Zhang Y, Zerges W (2012)Biogenic membranes of the chloroplast in Chlamydomonas reinhardtii.Proc Natl Acad Sci USA 109: 19286–19291

Sobotka R (2014) Making proteins green; biosynthesis of chlorophyll-binding proteins in cyanobacteria. Photosynth Res 119: 223–232

Sobotka R, Dühring U, Komenda J, Peter E, Gardian Z, Tichý M, GrimmB, Wilde A (2008a) Importance of the cyanobacterial Gun4 protein forchlorophyll metabolism and assembly of photosynthetic complexes. JBiol Chem 283: 25794–25802

Sobotka R, McLean S, �Zuberová M, Hunter CN, Tichý M (2008b) TheC-terminal extension of ferrochelatase is critical for enzyme activity andfor functioning of the tetrapyrrole pathway in Synechocystis strain PCC6803. J Bacteriol 190: 2086–2095

Tsui HC, Keen SK, Sham LT, Wayne KJ, Winkler ME (2011) Dynamicdistribution of the SecA and SecY translocase subunits and septal lo-calization of the HtrA surface chaperone/protease during Streptococcuspneumoniae D39 cell division. MBio 2: e00202-11

Vavilin D, Brune DC, Vermaas W (2005) 15N-labeling to determine chlo-rophyll synthesis and degradation in Synechocystis sp. PCC 6803 strainslacking one or both photosystems. Biochim Biophys Acta 1708: 91–101

Vavilin D, Vermaas W (2007) Continuous chlorophyll degradation ac-companied by chlorophyllide and phytol reutilization for chlorophyllsynthesis in Synechocystis sp. PCC 6803. Biochim Biophys Acta 1767:920–929

Yu B, Xu C, Benning C (2002) Arabidopsis disrupted in SQD2 encodingsulfolipid synthase is impaired in phosphate-limited growth. Proc NatlAcad Sci USA 99: 5732–5737

Zhang L, Paakkarinen V, Suorsa M, Aro EM (2001) A SecY homologue isinvolved in chloroplast-encoded D1 protein biogenesis. J Biol Chem 276:37809–37814

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