The crtB gene of Synechocystis sp. PCC 6803, encoding phytoene synthase, was inactivated in the ∆ crtH mutant to generate a carotenoidless ∆ crtH/B double mutant. ∆ crtH mutant cells were used because they had better transformability than wild-type cells, most probably due to their adaptation to partial carotenoid defi ciency. Cells of the ∆ crtH/B mutant were light sensitive and could grow only under light-activated heterotrophic growth conditions in the presence of glucose. Carotenoid defi ciency did not signifi cantly affect the cellular content of phycobiliproteins while the chlorophyll content of the mutant cells decreased. The mutant cells exhibited no oxygen-evolving activity, suggesting the absence of photochemically active PSII complexes. This was confi rmed by 2D electrophoresis of photosynthetic membrane complexes. Analyses identifi ed only a small amount of a non-functional PSII core complex lacking CP43, while the monomeric and dimeric PSII core complexes were absent. On the other hand, carotenoid defi ciency did not prevent formation of the cytochrome b 6 f complex and PSI, which predominantly accumulated in the monomeric form. Radioactive labeling revealed very limited synthesis of inner PSII antennae, CP47 and especially CP43. Thus, carotenoids are indispensable constituents of the photosynthetic apparatus, being essential not only for antioxidative protection but also for the effi cient synthesis and accumulation of photosynthetic proteins and especially that of PSII antenna subunits.

Keywords: Carotenoidless mutant • crtB • Membrane protein synthesis • Photosynthesis • PSII assembly • Synechocystis sp. PCC 6803 .

Abbreviations : BN , Blue Native ; Car , carotenoid ; iD1 , incompletely processed D1 intermediate ; LAHG , light-activated heterotrophic growth ; OD , optical density ; pD1 , unprocessed

D1 precursor ; RC , reaction center ; RCC(1) and RCC(2) , monomeric and dimeric PSII core complexes ; RCa , PSII reaction center subcomplex lacking both inner antennae CP47 and CP43 proteins.

Introduction

Carotenoids (Cars) are important components of photosyn-thetic complexes of aerobic phototrophs in which they are believed to play mostly a photoprotective role. This role is cor-related with their ability to quench excited Chl a triplet states which can generate toxic singlet oxygen ( Cogdell et al. 2000 ). In agreement with this function, algal mutants lacking Cars are extremely sensitive to light ( Romer et al. 1995 , McCarthy et al. 2004 ). The photoprotective role of Cars is especially important if the cells are exposed to excessive light energy. In higher plants there is a close relationship between content of a specifi c xanthophyll, zeaxanthin, and the ability of cells to dissipate excess excitation energy in antennal Chl-binding complexes ( Demmig-Adams and Adams 2006 ).

Cars together with Chls also play a role in the translation and stabilization of photosynthetic reaction center (RC) apo-proteins in Chlamydomonas reinhardtii , although they do not regulate gene transcription of RC apoproteins ( Herrin et al. 1992 ). Scenedesmus obliquus C-6E , a Car-defi cient mutant strain, is characterized by a complete lack of Chl b and by the presence of Chl a exclusively in PSI. This mutant completely lacks PSII activity while PSI activity is fully retained ( Romer et al. 1995 ).

Interestingly, carotenoidless mutants of anoxygenic pho-totrophic bacteria with type-1 or type-2 RCs were viable in the light, with normal assembly and functioning of their RCs ( Ouchane et al. 1997 , Frigaard et al. 2004 ). Even if Cars are important in photoprotection, they are not indispensable for the assembly and functioning of the RCs in these phototrophic bacteria ( Ouchane et al. 1997 ).

Involvement of Carotenoids in the Synthesis and Assembly of Protein Subunits of Photosynthetic Reaction Centers of Synechocystis sp. PCC 6803 Ozge Sozer 1 , 4 , Josef Komenda 2 , 4 , Bettina Ughy 1 , Ildikó Domonkos 1 , Hajnalka Laczkó-Dobos 1 , Przemyslaw Malec 3 , Zoltán Gombos 1 and Mihály Kis 1 , ∗ 1 Institute of Plant Biology, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary 2 Laboratory of Photosynthesis, Institute of Microbiology, Trebon, Czech Republic 3 Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland 4 These authors contributed equally to this work ∗ Corresponding author: E-mail, kism@brc.hu ; Fax, + 36-62-433-434 (Received January 25, 2010; Accepted March 7, 2010)

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In the cyanobacterium Thermosynechococcus elongatus , 22 and 12 Cars have been found by X-ray crystallographic analysis in PSI ( Jordan et al. 2001 , Fromme et al. 2001 ) and PSII ( Guskov et al. 2009 ), respectively. In the structure of the cytochrome b 6 f complex (Cyt b 6 f ) of C. reinhardtii ( Stroebel et al. 2003 ) and Mastigocladus laminosus ( Kurisu et al. 2003 ) one β -carotene has been identifi ed, and in that of Synechocystis sp. PCC 6803 one echinenone ( Boronowsky et al. 2001 ). Cars in PSI are arranged in six clusters and they are mostly bound to large PsaA and PsaB subunits; some Cars are also in contact with small membrane subunits ( Jordan et al. 2001 , Fromme et al. 2001 ). Importantly, a domain around subunits PsaI, PsaL and PsaM, which is important for formation of the PSI trimer, is especially rich in Cars ( Jordan et al. 2001 , Fromme et al. 2001 ). The Cars in PSII are bound to the periphery of all four large Chl-binding subunits D1, D2, CP43 and CP47. The initial helices of the inner antennae CP43 and CP47 are surrounded by several Cars that frequently form an interface between these helices and small PSII subunits. Finally, the molecule of β -carotene in the Cyt b 6 f complex of Mastigocladus is located in the vicinity of the last helices of subunit IV and small subunits PetL, PetM and and PetN ( Kurisu et al. 2003 ).

The fi rst step in Car biosynthesis is phytoene synthesis by condensation of two molecules of geranylgeranyl pyrophos-phate (GGPP) ( Martinez-Ferez et al. 1994 ). In Synechocystis sp. PCC 6803 this reaction is catalyzed by phytoene synthase encoded by the crtB gene. Phytoene is then converted to vari-ous carotenes that are substrates for the synthesis of a variety of xanthophylls. In Synechocystis sp. PCC 6803, the major Car com-ponents are β -carotene, myxoxanthophyll, zeaxanthin and echinenone ( Takaichi et al. 2001 ). The crtO gene encoding the β -carotene ketolases was insertionally inactivated in the Synechocystis sp. PCC 6803 strain and the resulting ∆ crtO mutant was unable to synthesize echinenone. The photosyn-thetic activity of the ∆ crtO cells did not differ from that of wild-type cells. This indicates that the crtO gene is not required for normal growth under standard or high light conditions ( Fernandez Gonzalez et al. 1997 ). Inactivation of the crtR gene encoding β -carotene hydroxylase in Synechocystis sp. PCC 6803 resulted in a block of zeaxanthin synthesis and myxoxan-thophyll accumulation, although the growth ( Lagarde and Vermaas 1999 ) and the oxygen-evolving activity (from H 2 O to CO 2 ) ( Masamoto et al. 2004 ) of the ∆ crtR cells were similar to those of the wild-type cells. These fi ndings suggest that zeaxanthin and myxoxanthophyll are not required for the assembly and functions of RCs. ∆ crtRO , a double mutant of Synechocysti s sp. PCC 6803 lacking echinenone, zeaxantin and myxoxanthophyll, exhibited enhanced light sensitivity and low oxygen-evolving activity, supporting the important role of Cars in photoprotection ( Schäfer et al. 2005 ). In the caroteno-genesis of Synechocystis sp. PCC 6803, all Cars are synthesized from 15- cis phytoene via lycopene. The conversion of 15- cis phytoene to all- trans lycopene is governed by photoisomeriza-tion or by cis to trans carotene isomerase encoded by the gene crtH. Consequently, the light-grown cells of the ∆ crtH

mutant produce Cars identical to those of the wild-type strain ( Masamoto et al. 2001 ), while under light-activated het-erotrophic growth (LAHG) conditions (cells are grown in the presence of 10 mM glucose in the dark with brief, 5–10 min, daily illumination) ( Anderson and McIntosh 1991 ) the ∆ crtH cells produce primarily cis -lycopenes and a small amount of all- trans carotenes, but no xanthophylls. It was shown that the PSI activity of ∆ crtH mutant cells grown in the dark was similar to that of the wild-type cells; there was, however, no detectable PSII activity ( Masamoto et al. 2004 ). Nevertheless, these cells still contained small amounts of β -carotene even if they were grown in the dark ( Masamoto et al. 2001 ).

In the present study we constructed the fi rst oxygenic pho-tosynthetic prokaryotic mutant which is completely defi cient in Car synthesis. The mutant was produced from ∆ crtH mutant cells by inactivation of the crtB gene encoding phytoene syn-thase. In the Synechocystis sp. PCC 6803 ∆ crtH/B mutant gener-ated in this way the synthesis of Cars was blocked at the step of 15- cis phytoene synthesis. 15- cis phytoene is the fi rst commit-ted intermediate in Car biosynthesis. No complete PSII core complexes could be detected in ∆ crtH/B and the mutant cells had no oxygen-evolving activity. We could demonstrate that PSI and Cyt b 6 f complexes were assembled, while the PSII com-plex had an absolute structural and functional requirement for Cars. Furthermore, elimination of Cars remarkably suppressed the synthesis of the large PSII carotenoid-binding subunits.

Results

Inactivation of the gene crtB encoding phytoene synthase results in a light-sensitive, carotenoidless mutant of Synechocystis sp. PCC 6803 Phytoene synthase encoded by the gene crtB catalyzes the fi rst synthetic reaction of Car biosynthesis. It produces 15- cis phy-toene. While inactivation of the crtB gene in wild-type cells of Synechocystis was not successful, the fully segregated, carote-noidless mutant lacking the crtB gene was obtained by transfor-mation of the ∆ crtH mutant. Cells of ∆ crtH under LAHG conditions contain only cis -lycopenes and a small amount of all- trans carotenes ( Masamoto et al 2001 ) and they are there-fore adapted to partial Car defi ciency. As this adaptation could increase the probability of the successful survival of the mutant completely lacking Cars, the ∆ crtH strain was used for inactiva-tion of the crtB gene. This was performed by insertion of an Ω cassette that provides spectinomycin resistance ( Fig. 1A ) as a selection marker. Complete segregation of the ∆ crtH/B mutant ( Fig. 1B ) occurred only under LAHG conditions ( Anderson and McIntosh 1991 ). To prove the absence of Cars in the mutant, elution profi les of the extracted pigments of wild-type and ∆ crtH/B cells grown under LAHG conditions were compared by HPLC ( Fig. 2 ). The peaks detected at 440 nm, corresponding to Cars and Chl, were identifi ed according to their absorption spectra and retention times ( Fig. 2A ). Wild-type cells contained myxoxanthophyll, zeaxanthin, echinenone, β -carotene and

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Chl a , while in cells of ∆ crtH/B only Chl a was found; neither Cars nor any of their intermediates were detectable in the visible region ( Fig. 2B ). The same results were obtained with cells illuminated for 24 h (data not shown). When HPLC elution was monitored at 280 nm, a small peak, possibly belonging to β -carotene, was detected in the hydrophobic region of the wild-type elution profi le. Meanwhile, no peaks were observed in this region of the corresponding profi le of the mutant (data not shown). These results demonstrated that the ∆ crtH/B mutant does not contain Car derivatives, a fi nding that is in line with the inactivation of the phytoene synthase gene.

Fig. 1 Genetic inactivation of crtB . (A) Physical map of the Synechocystis sp. PCC 6803 genome fragment containing crtB . In the inactivated strain the Ω cassette replaces a 140 bp long Apa I– Hin dIII fragment. Black arrows indicate the position of the PCR primers used for checking the complete segregation. (B) PCR analysis of the wild type (W) and the ∆ crtB transformant ( ∆ crtH/B ). Sizes of the fragments containing the wild-type and mutant allele are indicated on the right and size markers (M) on the left.

Fig. 2 HPLC analysis of photosynthetic pigment extracts of wild-type (A) and ∆ crtH/B cells (B). The cells were grown under LAHG conditions. The HPLC chromatograms were recorded at 440 nm. Car derivatives were identifi ed on the basis of both their absorption spectra and their retention times. β -Car, β -carotene; Myx, myxoxanthophyll; Zea, zeaxanthin; Ech, echinenone; Chl, chlorophyll; Chl iso, chlorophyll isomers.

The growth rate of wild-type cells was similar to that of ∆ crtH and ∆ crtH / B cells under LAHG conditions. In 3 d the cell density of all strains gradually increased from 0.2 to 0.8–0.9 (optical density recorded at 730 nm, OD 730 ) under LAHG condi-tions ( Fig. 3 ). Following 3 d of culture the cells were exposed to light at an intensity of 35 µmol photons m − 2 s − 1 . The wild-type and ∆ crtH cells adapted to light and kept growing. Under LAHG and photomixotrophic conditions ∆ crtH and wild-type cells showed identical growth profi les, which demonstrated that disruption of the crtH gene had no effect on growth ( Masamoto et al. 2001 ). In contrast, the growth of the ∆ crtH / B cells stopped and the cells died.

WT and ∆ crtH cells have typical green coloration under LAHG and light conditions ( Fig. 4A, B ). However, ∆ crtH/B cells grown under LAHG conditions had a bluish color, indicating the presence of phycobiliproteins ( Fig. 4A ). Thus, the synthesis of phycobiliproteins was not signifi cantly affected by the muta-tion. However, following a short-term illumination by low light, the ∆ crtH/B cells were bleached and became colorless ( Fig. 4B ). These results suggested that the carotenoidless mutant cells became extremely light sensitive.

Spectroscopic properties and photosynthetic activity of the ∆ crtH/B mutant cells Absorption spectra of Synechocystis sp. PCC 6803 wild-type cells measured at room temperature showed four distinct regions. The absorption range between 435 and 450 nm corre-sponds to Soret bands of Chl. The absorption ranges of 450–550, 550–650 and 650–700 nm belong to visible bands of Cars, phycobiliproteins and Chl a , respectively. In the absorption spectra of the ∆ crtH/B mutant cells there was a sharp decrease of absorption in the 450–525 nm region corresponding to Cars, confi rming the complete absence of Cars in the cells. Furthermore, ∆ crtH / B cells exhibited a decrease in absorption, with a shoulder at about 435 and 680 nm corresponding to Chl a . One hour treatment of wild-type cells at 500 µmol photons m − 2 s − 1 had no effect on the absorption profi le of the

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Fig. 4 Effect of light intensity on the pigmentation of wild-type, ∆ crtH and ∆ crtH/B cells. (A) Synechocystis sp. PCC 6803 wild-type, ∆ crtH and ∆ crtH/B cells grown at 30 ° C at the end of the third day under LAHG conditions and (B) cells after a 48 h light treatment at 35 µmol photons m − 2 s − 1 .

Fig. 3 Growth curve of Synechocystis sp. PCC 6803 wild-type (squares), ∆ crtH (triangles) and ∆ crtH / B cells (circles) grown at 30 ° C under LAHG conditions for 3 d and then illuminated at 35 µmol photons m − 2 s − 1 . Values of a representative experiment are shown.

wild-type cells ( Fig. 5A ). However, light treatment decreased the Chl a content of ∆ crtH/B cells compared with that of the cells grown under LAHG conditions. This points to the light sensitivity of Chl a in the absence of Cars ( Fig. 5B ).

The low temperature Chl fl uorescence emission spectra of wild-type and ∆ crtH/B mutant cells (OD 730 ∼0.8) were recorded, using the 435 nm excitation wavelength of Chl a. The emission spectrum of wild-type cells showed a minor band at about 685 nm that may correspond to PSII and a major band emitted at 725 nm which corresponds to PSI-associated Chl a ( Fig. 6A ). The fl uorescence spectrum of ∆ crtH/B cells was distinctly different from that of wild-type cells. It showed three bands at 655, 685 and 723 nm ( Fig. 6A ). The peak at 655 nm may be related to free phycocyanin ( MacColl and Guard-Friar 1987 ) and the peak at 685 nm comes either from PSII-associated Chl a or terminal emitter of phycobilisomes. To distinguish between phycobilisome-specifi c and chlorophyll-specifi c fl uorescence activities, the emission spectra of isolated and well washed thylakoid membranes from wild-type and ∆ crtH/B cells were also measured ( Fig. 6B ). The majority of the large fl uorescence emission peaks at 655 and 685 nm were suppressed in the spectra of the thylakoid membranes of ∆ crtH/B cells, indicating that these bands were related to phycobiliproteins and did not come from PSII. The third, highest peak at about 720 nm belongs to PSI, and in comparison with the wild-type peak it is shifted by 4–5 nm to the blue region, most probably as a consequence of missing Cars.

Oxygen-evolving activity was detected in wild-type cells grown under LAHG conditions (220 µmol O 2 mg Chl − 1 h − 1 ). PSII activity in wild-type cells increased from 260 to 480 µmol O 2 mg Chl − 1 h − 1 following transfer to continuous light for 3 or

48 h, respectively. ∆ crtH cells grown under LAHG conditions showed no photosynthetic oxygen-evolving activity but they had active PSI-related oxygen uptake. However, after 48 h of continuous illumination, the PSII activity of ∆ crtH cells was similar to that of the wild-type cells ( Masamoto et al. 2004 ). In contrast, ∆ crtH / B cells showed no PSII activity from H 2 O to CO 2 and from H 2 O to 1,4-parabenzoquinone, an artifi cial electron acceptor, either under LAHG conditions or after 3 or 48 h of treatment. These results suggest that there are no functional PSII complexes in the ∆ crtH/B mutant, despite the presence of PSI activity.

Northern blot analysis of mRNA transcripts in Synechocystis sp. PCC 6803 ∆ crtH/B To fi nd the reason for the absence of active PSII complexes, we fi rst checked the level of transcripts of the genes encoding large PSII subunits. To this end Northern blot analysis of wild-type and ∆ crtH/B cells grown under LAHG conditions was used. Steady-state levels of psbA , psbB , psbDII and psbDIC gene transcripts encoding D1, CP47, D2 and D2 together with CP43 proteins, respectively, were detected by gene-specifi c probes. This analysis indicated that the transcription effi ciencies of these genes were similar in wild-type and ∆ crtH/B cells ( Fig. 7 ).

Effect of the absence of Cars on the biogenesis and accumulation of photosynthetic membrane complexes The effect of the absence of Cars on the accumulation and assembly of protein subunits of photosynthetic membrane complexes was characterized in ∆ crtH/B cells by 2D gel electro-phoresis. The fi rst dimension was a Blue Native PAGE, and

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Fig. 5 Absorption spectra of wild-type (A) and ∆ crtH / B (B) cells grown under LAHG conditions (LAHG, solid line) and after 1 h illumination at 500 µmol photons m − 2 s − 1 white light (1hHL, dashed line). The spectra were taken at an identical absorbancy of 750 nm.

Fig. 6 Low-temperature fl uorescence emission spectra of intact cells (A) and isolated thylakoid membranes (B) of wild-type (solid lines) and ∆ crtH/B (dashed lines) cells cultured under LAHG conditions. The excitation wavelength was 430 nm. The spectra were recorded at 77 K, corrected for the sensitivity of the photomultiplier and normalized to long wavelength maxima.

Fig. 7 Northern hybridization analysis of psbA , psbDII , psbDIC and psbB genes in wild-type and ∆ crtH/B mutant Synechocystis sp. PCC 6803 strains grown under LAHG conditions. A 10 µg aliquot of total RNA was loaded per lane. All membranes were also probed with rnpB (RNase P RNA gene) as a loading and transfer control.

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denaturing PAGE was used as the second dimension (2D-BN/SDS–PAGE). Protein bands were analyzed by autoradiography and immunoblotting. These techniques have recently been used for the characterization of the protein content of various Synechocystis sp. PCC 6803 mutants ( Dobakova et al. 2007 , Sobotka et al. 2008 , Laczko-Dobos et al. 2008 ). The protein complexes detected in wild-type cells grown under LAHG conditions were similar to those present in wild-type cells cultivated under photo-heterotrophic conditions ( Dobakova et al. 2007 ). Most wild-type PSI core complexes existed in a trimeric form and the contribution of monomeric forms

Fig. 8 2D-BN/SDS–PAGE of wild-type and ∆ crtH/B cells grown under LAHG conditions and radioactively labeled with [ 35 S]methionine and [ 35 S]cysteine. The gels were stained by Coomassie Blue and exposed to Phosphorimager plates. Designation of complexes: PSI(3) and PSI(1), trimeric and monomeric PSI complexes, respectively; and RCC(2) and RCC(1), dimeric and monomeric PSII core complexes, respectively. RC47 is a PSII core complex lacking CP43, RCa is a PSII core complex lacking both CP43 and CP47, and U.P. indicates unassembled proteins. Boxes defi ne the positions of the large subunits of the monomeric Cyt b 6 f complex. Arrow 1 designates RCC(1), arrow 2 designates RC47 and arrow 3 designates RCa complexes in the mutant samples. Each sample contained 6 µg of Chl.

did not exceed 30 % . About 90 % of PSII protein subunits of wild-type cells accumulated in monomeric and dimeric core complexes [RCC(1) and RCC(2)] and only a small amount of these proteins was present in RC47, a PSII core subcomplex that is lacking CP43 ( Komenda et al. 2004 ). Wild-type Cyt b 6 f was found exclusively in the form of monomeric complexes ( Fig. 8 ). In contrast to the wild-type cells, the monomeric form of PSI was predominant in cells of ∆ crtH/B and it had a higher mobility. Both monomeric and dimeric PSII core complexes were absent and only a negligible amount of RC47 complex was detected. On the other hand, the level of the monomeric form of the Cyt b 6 f complex in ∆ crtH/B was similar to that in the wild-type ( Fig. 8 ).

A typical labeling pattern of wild-type cells is shown on the autoradiogram of a 2D gel ( Fig. 8 , lower panels). The D1 protein was the most intensively labeled protein in both strains. In the wild-type this label was present in both PSII core complexes, RCC(1) and RCC(2), as well as in RC47, while in the mutant the majority of the labeled protein accumulated in RC47, and only a small amount was found in RCC(1) and in RCa. RCa is a PSII RC subcomplex lacking both inner antennae CP47 and CP43. It contains processed D1 as well as iD1, an incompletely processed precursor of D1 ( Komenda et al. 2004 ). In addition, the overall labeling of the ∆ crtH/B proteins was 5–10 times less intensive than that of the wild-type proteins (the ∆ crtH/B gel was exposed for fi ve times longer than the gel loaded with wild-type proteins to see the labeling).

On the autoradiograms of 2D gels of ∆ crtH/B ( Fig. 9 ) small amounts of unassembled CP43, CP47, D2 and pD1, the D1 precursor, were detected . It has been shown previously that on native gels all these proteins may exist in two forms which differ in their electrophoretic mobility ( Komenda et al. 2004 ). We have subsequently shown that the slower bands usually additionally contain the small subunit bound to the large Chl protein while the faster bands contain just the large chlorophyll protein. For instance, the slowly migrating native pD1 in fact contains not only pD1 but also a small subunit PsbI ( Dobakova et al. 2007 ), while the slower migrating CP47 band contains CP47 plus the subunit PsbH, and small proteins called Hlips or Scps ( Promnares et al. 2006 ). Hlips ( h igh l ight- i nduced p roteins) or Scps ( s mall c ap-like p roteins) are single helix pro-teins with sequence similarity to regions of the plant Chl a / b binding proteins (CAB family proteins), which are mostly induced under stress conditions, such as high irradiance or low temperature ( Funk and Vermaas 1999 , He et al. 2001 ). To evalu-ate a possible effect of the absence of Car on the binding of small subunits to PSII Chl proteins, we compared the mobilities of unassembled forms of CP47, CP43 and D2 in the BN gel in the mutants ∆ crtH/B , ∆ ycf48 and ∆ psbK . Mutant ∆ ycf48 , which is an early PSII assembly mutant, accumulates large amounts of unassembled CP47, CP43 and D2 due to the low availability of D1 caused by the absence of the assembly factor YCF48 ( Komenda et al. 2008 ). In the BN gel the majority of these unassembled Chl proteins migrated as the slower band (designated u.CP47, u.CP43 and u.D2, Fig. 9 ). This indicates

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the presence of small subunits bound to large unassembled PSII proteins in ∆ ycf48 . In contrast to ∆ ycf48 , the labeled unas-sembled band of CP43 seen in ∆ psbK ( Ikeuchi et al. 1991 ) migrated in the BN gel as the faster band (designated u.CP43 ′ ), indicating that the slower u.CP43 of ∆ ycf48 contains at least PsbK, which contributes to the decreased mobility of u.CP43 ( Fig. 9 ). Interestingly, the faster u.CP43 ′ band also prevailed in ∆ crtH/B ( Fig. 9 ). This mutant also contained the majority of unassembled D2 and CP47 in the faster migrating forms (designated u.D2 ′ and u.CP47 ′ , respectively). It again suggested the absence of small PSII subunits in these native bands of these Chl proteins. In conclusion, the lack of Cars tended to destabi-lize binding of some small subunits to large PSII Chl-binding subunits.

Taking into account the effect of Car depletion on the migration of the particular Chl-binding proteins CP43 and CP47, we were interested in whether the absence of Cars also affected the assembly of PSII core complexes. Therefore, we chased the labeled proteins in the presence of chloram-phenicol and followed their incorporation into complexes by

2D analysis ( Fig. 10 ). Most of the unassembled D2 and pD1 disappeared, with a concomitant increase in the labeling of D2 and D1 in RC47. This shows that they were effi ciently inserted into complexes ( Fig. 10 ). This partly refl ected the transformation of RCa into RC47, and partly the maturation of iD1 into D1, in the remaining RCa. Surprisingly, the amount of weakly labeled PSII proteins in RCC(1) also signifi cantly decreased during the chase. However, we could not detect labeled PSII proteins in the region of putative RCC(2). This indi-cates that RCC(1) was unstable and was rapidly converted into RC47 which was the main accumulated PSII subcomplex lacking CP43, as identifi ed by Coomassie staining ( Fig. 8 ) and immunoblot ( Fig. 10 ). Recently we have shown that PsbI, a small subunit of PSII, stabilizes binding of CP43 within PSII ( Dobakova et al. 2007 ). According to X-ray crystallographic measurements ( Loll et al. 2005 , Guskov et al. 2009 ), the PsbI is bound to D1 in the vicinity of a molecule of β -carotene. Therefore, we tested whether PsbI protein is absent from RC47 ( Fig. 10 ). Immunodetection using specifi c antibodies confi rmed the presence of PsbI in RC47. Thus, CP43 binding

Fig. 10 Autoradiograms and Western blots of membrane protein complexes of ∆ crtH/B cells separated by 2D-BN/SDS–PAGE. Cells of the ∆ crtH/B mutant grown under LAHG conditions were labeled with [ 35 S]methionine and [ 35 S]cysteine (pulse), then cold methionine, cold cysteine and chloramphenicol were added and incubation continued (pulse–chase) as described in Materials and Methods. Only the blot for the pulse experiment is shown as it was identical to that of the pulse–chase experiment. Designation of the complexes is the same as in Fig. 8 . pD1 indicates the unprocessed D1 precursor and iD1 indicates the incompletely processed D1 intermediate. The prime designates unassembled proteins with unusually fast mobility in the BN gel. Each loaded sample contained 3 µg of Chl.

Fig. 9 Autoradiograms of pulse-labeled membrane proteins of ∆ ycf48 , ∆ psbK and ∆ crtH/B mutants separated by 2D-BN/SDS–PAGE. A 6 µg aliquot of Chl was loaded onto each gel. After separation the gels were stained, dried and exposed on PhosphorImager plates. Designation of the complexes is the same as in Fig. 8 . pD1 indicates the unprocessed D1 precursor and iD1 indicates the incompletely processed D1 intermediate. u.CP47 ′ , u.CP43 ′ and u.D2 ′ designate unassembled proteins with unusually fast mobility in the BN gel most probably lacking bound small subunits.

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was not destabilized as a result of the absence of PsbI. Since no increase in the level of unassembled CP43 was observed during the chase we believe that in the absence of β -carotene, CP43 is quickly detached from RCC(1) and probably degraded. RC47 in ∆ crtH/B also contained PsbH, another small subunit of PSII which stabilizes CP47 and facilitates its binding to the D1–D2 heterodimer ( Fig. 10 ) ( Komenda et al. 2002 , Komenda et al. 2005 ).

The effect of Car defi ciency on the accumulation of PSII and PSI ( Fig. 11 ) as well as of Cyt b 6 f ( Fig. 11 ) was corroborated by semi-quantitative Western blot analysis. PSI subunit pro-teins, PsaA and PsaB, and Cyt b 6 f subunit proteins, Cyt b 6 and Cyt f , were present in similar amounts in the wild-type and the ∆ crtH/B mutant cells. In contrast, the levels of PSII subunit pro-teins, D1 and D2, in ∆ crtH/B were much lower in comparison with those in the wild type. Protein CP47 was hardly detectable and protein CP43 was completely absent, as also shown by 2D blot ( Fig. 10 ). We also tested the effect of low light on the accumulation of PSII, Cyt b 6 f and PSI proteins in wild-type and ∆ crtH/B cells. In the absence of Cars, we expected increased protein photooxidation owing to the photosensitivity

Fig. 11 Semi-quantitative Western blot analysis of the photosynthetic protein subunits of wild-type and ∆ crtH/B cells grown under LAHG conditions and after 1 h illumination at a light intensity of 50 µmol photons m − 2 s − 1 . A 1 µg aliquot of Chl per lane was loaded onto the gel. Blots were incubated with specifi c antibodies and subunits of PSII and PSI were detected by peroxidase-conjugated secondary antibodies whereas Cyt b 6 f protein subunits were detected by alkaline phosphatase-conjugated secondary antibodies. Stained bands of ATP synthase subunits AtpA and AtpB (ATPsynth) are shown to demonstrate equal protein loading.

of Chl pigments. After an exposure to 50 µmol photons m − 2 s − 1 for an hour, a further decrease in the amount of D1, D2 and CP47 was detected while the level of PSI and Cyt b 6 f protein subunits did not change signifi cantly ( Fig. 11 ). The smeared character of the D1 and D2 bands indicated photooxidation of these proteins in the absence of Cars ( Lupinkova and Komenda 2004 ).

Discussion

We studied the structural consequences of the complete lack of Cars in an oxygenic photosynthetic organism Synechocystis sp. PCC 6803. A mutant was generated in ∆ crtH mutant cells by inactivation of the crtB gene encoding phytoene synthase ( Fig. 1 ), responsible for the fi rst committed reaction of Car biosynthesis. ∆ crtH was used as a host strain because of its better transformability. Under photoautrophic conditions the ∆ crtH mutant cells have the same Car composition, growth and photosynthetic characteristics as those of the wild type ( Figs. 3 , 4 ) ( Breitenbach et al. 1998 , Masamoto et al. 2001 , Masamoto et al. 2004 ). Therefore, ∆ crtH was a suitable substitute for the wild-type in construction of the carotenoidless mutant.

Under LAHG culture conditions complete segregation of the ∆ crtH/B mutant occurred. HPLC analysis showed that ∆ crtH/B cells grown under LAHG conditions contain no Car derivatives ( Fig. 2 ). The bluish-green color of mutant cells indicated that phycobiliprotein synthesis and accumulation were not signifi cantly suppressed and phycobiliproteins were more dominant pigments than Chl a . However, under light conditions, the mutant cells were gradually bleached and died. This light susceptibility suggests that Cars are essential for the protection and functioning of the photosynthetic machin-ery and without them cells are not able to preserve their pigments.

PSI monomers assembled in the ∆ crtH/B mutant. Interest-ingly, PSI in the mutant was mostly present in the monomeric form while the prevailing form in the wild type is the trimeric form. Although we cannot exclude that trimers really exist in the mutant cells and are destabilized and decomposed during gel analysis, the result suggests an important stabilization func-tion of Car molecules in the trimerization domain ( Grotjohann and Fromme 2005 ).

Cyt b 6 f complexes possess specifi c carotenoid-binding sites, suggesting an important structural role for these pigment mol-ecules ( Wenk et al. 2005 ). Despite this expectation, ∆ crtH/B cells showed a growth rate similar to that of the wild type under LAHG conditions ( Fig. 3 ) and 2D protein analysis revealed similar accumulation of the monomeric Cyt b 6 f complex in both wild-type and ∆ crtH/B cells. This fi nding indicates that Cars are not essential for the assembly of functional Cyt b 6 f complexes.

Strikingly, among photosynthetic membrane complexes in the ∆ crtH/B mutant, PSII was the complex most severely affected by the absence of Cars. Indeed, PSII-related oxygen-evolving activity of mutant cells measured by using an artifi cial

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electron acceptor 1,4-parabenzoquinone was not detected under any conditions, pointing to the complete absence of functional PSII complexes in ∆ crtH/B mutant cells. 2D gel analy-sis even showed the inability of the mutant to accumulate PSII core complexes, indicating the importance of Cars for their biogenesis. Data from Northern blot analyses showed that the transcription of genes encoding large PSII Chl-binding subunits was not affected by elimination of Cars under LAHG conditions. Therefore, a transcription defect could not explain the changes in the carotenoidless mutant with respect to the wild type. 2D gel analyses of protein subunits in combination with their radioactive labeling provided clues to the explanation of why no active PSII complexes were assembled. Synthesis of PSII pro-teins was strongly suppressed and the most affected proteins were the Chl-binding antennae CP47 and CP43. We were able to detect a small amount of CP47 in the membrane and this protein was accumulated in the CP43-less complex termed RC47. This complex is an intermediate in the de novo PSII assembly process which accumulates in the absence of CP43 ( Komenda et al. 2004 ). The lack of CP43 in the ∆ crtH/B mutant was confi rmed by immunoblotting. The newly synthesized proteins were mostly in the unassembled state, and could be detected only transiently on the autoradiogram. An intense radioactive labeling of D1 in RC47 points to a functional D1 replacement in the ∆ crtH/B mutant, and it is in line with the selective replacement of the D1 protein during PSII repair ( Komenda et al. 2006 ). There was also an increased amount of RCa complex, which lacks both CP47 and CP43, confi rming insuffi cient availability of CP47 in the above mutant. This con-clusion is supported by the fact that protein CP47 was detected in the unassembled fraction only by radioactive labeling while specifi c antibodies detected the protein only in the RC47 complex. Similar to CP47, unassembled CP43 was transiently detected on the autoradiogram but no accumulation of this protein was seen by the use of antibodies.

The accumulation of CP43-less RC47 in ∆ crtH/B supports the view that CP43 insertion into RC47 needs Cars. Recent structural models of cyanobacterial PSII ( Ferreira et al. 2004 , Loll et al. 2005 , Guskov et al. 2009 ) have demonstrated the presence of β -carotene at the interface between initial trans-membrane helixes of large PSII subunits and small subunits outside the heterodimer D1–D2. This applies to protein D2 and subunits of Cyt b 559 , CP47 and the PsbM/PsbT pair, and above all to CP43 and the PsbK/PsbZ pair. It has been shown previously that the presence of double bands of unassembled large PSII subunits on the BN polyacrylamide gels is related to binding of some of these small PSII subunits. They seem to stabilize the unassembled large PSII Chl-binding subunits ( Komenda et al. 2005 , Dobakova et al. 2007 , Komenda et al. 2008 ). Bands with faster electrophoretic mobility of radioac-tively labeled unassembled CP47, CP43 and D2 proteins in ∆ crtH/B suggest the absence of the small subunits from these bands related to the absence of Cars. Thus, missing Cars seems to affect not only the synthesis of PSII subunits but also the stability of binding among them.

Bautista and co-workers provided important information on the structural requirements of Cars related to proper PSII formation using Synechocystis mutants with modifi ed Car con-tent. If β -carotene was replaced with linear Cars, proper PSI, but not PSII formation was observed. However, β -zeacarotene, a compound with one β -ionylidene ring that makes it resemble the structure of β -carotene, was suffi cient for PSII accumula-tion ( Bautista et al. 2005a , Bautista et al. 2005b ).

It is interesting to note that carotenoidless mutants of purple bacteria with type-2 RCs have normal photosynthesis. These RCs assembled and functioned properly, although their stability decreased compared with that of the wild-type ( Ouchane et al. 1997 ). Therefore, in these phototrophic bacteria Cars are not essential for structure and functioning of the type-2 RCs.

The elimination of Cars not only suppressed synthesis of PSII proteins, but overall synthesis of membrane proteins was also strongly diminished. This points to a general detrimental effect of the absence of Cars on the synthesis of membrane proteins. It has been shown that oxidative stress related to the action of reactive oxygen species inhibits the elongation step of D1 translation via oxidation of the elongation factor G ( Kojima et al. 2007 ). We assume that there may be a similar reason for inhibited translation in the ∆ crtH/B mutant, since lack of Cars severely decreased the ability of cells to scavenge reactive oxygen species and to prevent the inactivation of trans-lation factors.

In conclusion, complete elimination of Cars in mutant Synechocystis PCC 6803 ∆ crtH/B cells unable to synthesize phytoene causes severe light sensitivity of these cells and a strong decrease in their capacity to synthesize proteins. This is not caused by the absence of gene transcripts but rather by affecting the process of translation. The carotenoidless mutant accumulates PSI and Cyt b 6 f complexes, but fully assembled PSII core complexes are missing owing to limitations in the syn-thesis of PSII Chl-binding subunits, especially of CP47 and CP43.

Materials and Methods

Organisms and growth conditions The strains used in this study were derived from the glucose-tolerant strain of Synechocystis sp. PCC 6803 referred to as the wild type. Wild-type and ∆ crtH/B mutant cells were grown at 30 ° C in BG11 medium ( Allen 1968 ) under LAHG conditions ( Anderson and McIntosh 1991 ). The growth medium was supplemented with 5 mM HEPES buffer (pH 7.5) and 10 mM glucose. Spectinomycin (40 µg ml − 1 ) and kanamycin (20 µg ml − 1 ) were added to the medium of mutant cells. A light pulse of 15 µmol photons m − 2 s − 1 was supplied by white light lamps for 10 min once a day. Light treatment of cells grown under LAHG conditions was carried out by illumination at a light intensity of 35 µmol photons m − 2 s − 1 with white fl uorescent lamps. Cultures were aerated on a gyratory shaker at 100 r.p.m. The density of cultures was measured by the OD 730 .

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Construction of the Synechocystis sp. PCC 6803 ∆ crtH/B mutant strain Restriction enzymes Bgl II and Not I were used to cut the cosmid clone cs0798 ( http://genome.kazusa.or.jp/cyanobase/Synechocystis/map/Chr/orf16 ) provided by S. Tabata. A 7.7 kb Bgl II fragment containing the phytoene synthase-encoding crtB gene of Synechocystis sp. PCC 6803 was cloned into the Bam HI site of pMPMA2 ( Mayer 1995 ). A part of the crtB gene coding region was removed by Apa I– Hin dIII digestion and replaced with an Ω cassette. This construct was used to transform ∆ crtH mutant cells of Synechocystis sp. PCC 6803. Transformants were selected under LAHG conditions on BG11 agar plates supplemented with glucose and increasing amounts of spectinomycin by several restreakings of single colonies. Complete segregation of ∆ crtH/B mutant cells was confi rmed by PCR using the primers crtBup (5 ′ -CGGTGCCCAACTTTTACCTA-3 ′ ) and crtBdown (5 ′ -TCACCTAAGGGGAAACATCG-3 ′ ).

Determination of Chl concentration For HPLC analysis, the Chl concentration was measured by absorbance at 665 nm, using a 90 % methanol extract ( Lichtenthaler 1987 ).

Determination of Chl concentration for protein measure-ments, sedimented cells or membranes were extracted with 100 % methanol and the Chl content of the extract was calcu-lated from the absorbances at 666 and 720 nm ( Welburn and Lichtenthaler 1984 ).

Carotenoid analysis Pigments were extracted with acetone : methanol (7 : 2, v/v) and centrifuged for 10 min at 4 ° C at 20 000 × g . The pellet was discarded and the supernatant was evaporated under nitrogen gas. Finally the extracted pigments were dissolved in HPLC-grade ethanol.

Car pigments were separated on a Prostar HPLC system (Varian, Miami, FL, USA), equipped with a photodiode array spectrophotometric detector Tidas I (World Precision Instru-ments, Sarasota, FL, USA) and a Zorbax SB-C18 reversed phase column, 5 µm particle size (Agilent Technologies, Wilmington, DE, USA) using the solvent system described by Lagarde et al. (2000) . Samples (100 µl) were fi ltered through a stainless steel fi lter ( ϕ = 0.22 µm) and loaded on a column equilibrated with solvent A (acetonitrile : water : triethylamine, 9 : 1: 0.01, by vol.). The column was eluted with a one-step gradient (15 min) of solvent B (ethyl acetate 100 % ) at a constant fl ow rate of 1.5 ml min − 1 . The absorption spectra of the eluate (380–800 nm) were recorded every 0.2 s.

Absorption spectroscopy Absorption spectra of cell suspensions were recorded with a UV-3000 (Shimadzu, Japan) spectrophotometer. The cell densi-ties were adjusted to OD 730 . The absorption spectra of cell sus-pensions were scanned in the visible region from 400 to 750 nm.

All these absorption spectra were taken at room temperature and were not corrected for spectral sensitivity. For high light treatment the cells were treated at 500 µmol m − 2 s − 1 for 1 h.

Fluorescence spectroscopy Low temperature steady-state fl uorescence emission spectra (600–800 nm) were recorded at 77 K using an Aminco Bowman Series 2 luminescence spectrometer (Spectronic Unicam, Rochester, NY, USA). The cells were excited at 435 nm. Spectra were corrected for the sensitivity of the photomultiplier and normalized to the maximum of PSI emission around 725 nm.

Measurement of photosynthetic oxygen-evolving activity Photosynthetic oxygen-evolving activity in intact cells was measured with a Clark-type oxygen electrode (Hansatech Instruments, Kings Lynn, UK) as described by Gombos et al. (2002) . PSII oxygen-evolving activity was measured from H 2 O to an exogenously added artifi cial quinone, paraben-zoquinone, at a concentration of 500 µM. The cells were washed with BG11 medium and resuspended in fresh BG11 medium for the measurement of oxygen evolution. An incan-descent lamp equipped with a red optical fi lter was the light source. This arrangement was used for all the oxygen evolution measurements at a saturating light intensity of 500 µmol photons m − 2 s − 1 . The Chl concentration of the cells was adjusted to 5 µg ml − 1 .

RNA isolation and Northern blot Total RNA from Synechocystis sp. PCC 6803 wild-type and ∆ crtH/B cells grown under LAHG conditions was isolated using the hot phenol method ( Mohamed and Jansson 1989 ), with the following modifi cation: the fi rst phenol extraction was done in a boiling water bath for 3 min. Northern blot analysis was performed as described by Kis et al. (1998) . DNA probes were generated by PCR using Synechocystis sp. PCC 6803 genomic DNA and gene-specifi c primers as shown: psbA gene F, 5 ′ -GACATCGACGGTATCCGTGAG-3 ′ , psbA gene R, 5 ′ -ACAGCAGGAGCGGTCAAAG-3 ′ ; psbDII gene F, 5 ′ -TGTCCTCGACGATTGGCTAAAG-3 ′ , psbDII gene R,5 ′ -AAACCGACGATACCCACAGAAC-3 ′ ; psbDIC gene F, 5 ′ -CTTGGTGGTCGGGAAATG-3 ′ , psbDIC gene R, 5 ′ -GTGAAGGCTTGGGATTGG-3 ′ ; and psbB gene F, 5 ′ -TGCCCACATCGTTCTATC-3 ′ , psbB gene R, 5 ′ -TGCGGAATACACCATCAG-3 ′ . All membranes were probed also with the rnpB as loading and transfer control.

Preparation of thylakoid membranes Thylakoid membranes were prepared by breaking the cells using glass beads according to Komenda et al. (2005) with the following modifi cations: the cells were washed, broken and resuspended in 25 mM MES/NaOH, pH 6.5, containing 10 mM CaCl 2 , 10 mM MgCl 2 and 25 % glycerol. Glass beads were subse-quently removed by fi ltering and thylakoid membranes were obtained by differential centrifugation.

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Protein analysis Radioactive labeling of cells . For radioactive labeling, cells containing 75 µg of Chl were resuspended in 250 µl of BG11 in a microcentrifuge tube, shaken at 50 µmol photons m − 2 s − 1 for 15 min and then a mixture of [ 35 S]methionine and [ 35 S]cysteine (Trans-label, MP Biochemicals, Irvine, CA, USA) was added (fi nal specifi c activity 400 µCi ml − 1 ). The suspension was exposed to 50 µmol photons m − 2 s − 1 white light for 15 min (pulse), then chloramphenicol (1 mg ml − 1 fi nal concentration) and a mixture of cold methionine and cysteine (5 mM fi nal concentration) were added and incubation continued for an additional 15 min (pulse–chase). Afterwards the cells were frozen in liquid nitro-gen and used for isolation of thylakoid membranes.

2D-BN/SDS-PAGE. For the analysis of protein complexes, the isolated membranes were solubilized with dodecyl- β - D -maltoside (DM/Chl = 40 : 1 w/w) and analyzed by BN electro-phoresis at 4 ° C in a 5–14 % polyacrylamide gel according to Schägger et al. (1994) . Samples with 6 µg Chl content were loaded onto the gel. The protein composition of the complexes was assessed by a second electrophoresis in a denaturing 12–20 % linear gradient polyacrylamide gel containing 7 M urea ( Komenda et al. 2002 ). The lanes from the native gel were excised along their entire length, incubated for 30 min in 25 mM Tris–HCl, pH 7.5 containing 1 % SDS (w/v) and placed on top of the denaturing gel. Proteins separated in the gel were stained by Coomassie blue.

Western blot analysis of PSI and PSII protein subunits . Samples containing 1 µg of Chl were loaded onto the denatur-ating gel (described above) and the separated proteins were transferred onto a PVDF membrane. Membranes were incubated with specifi c primary antibodies and then with a secondary antibody–horseradish peroxidase conjugate (Sigma, St. Louis, MO, USA). The primary antibodies used in this study were raised in rabbits against: (i) residues 58–86 of the spinach D1 polypeptide; (ii) the last 12 residues of the D2 polypeptide from Synechocystis sp. PCC 6803; (iii) residues 380–394 of barley CP47; (iv) the whole isolated CP43 from Synechocystis sp. PCC 6803; and (v) the last 14 residues of the PsbI protein from Synechocystis sp. PCC 6803.

For autoradiography, the gel or the membrane with labeled proteins was visualized on X-ray fi lms exposed at room tem-perature for 2–3 d or on Phosphorimager plates (GE Healthcare, Vienna, Austria) overnight. Quantitation of bands was done using ImageQuant 5.2 software (GE Healthcare, Vienna, Austria).

Western blot analysis of Cyt b 6 f protein subunits . SDS–PAGE was performed according to the standard procedure, as described by Schägger and von Jagow (1987) , using 12 % gels. In each lane a sample containing 3 µg of Chl was loaded. Proteins separated by SDS–PAGE were transferred to nitrocellulose membranes (Protran BA 85; Schleicher & Schuell, Keene, NH, USA) according to Towbin et al. (1979) . Blots were probed with rabbit polyclonal antibodies raised against Cyt b 6 f subunits of Synechocystis sp. PCC 6803 (anti-cytochrome b 6 and anti-cytochrome f ). Blots were developed by using goat anti-rabbit secondary antibodies conjugated with alkaline phosphatase

according to the standard nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate staining protocol ( Ausubel et al. 1995 ).

Funding

This work was supported by the Hungarian Science Foundation [OTKA; grant Nos. T 60109 and T 68692]; the Ministry of Education, Youth and Sports of the Czech Republic [project no. MSM6007665808]; the Czech Academy of Sciences [Institutional Research Concept No. AV0Z50200510, Czech–Hungarian bilateral research priority project and project IAA400200801]; Polish–Hungarian bilateral grant [OMFB-00843/2006 TéT]; the Polish Ministry of Science and Higher Education [50/N-DFG/2007/0].

Acknowledgments

We thank Kazumori Masamoto for supplying us with the ∆ crtH mutant and for helpful discussions. The authors are grateful to Professors E.-M. Aro, L. A. Eichacker and R. Barbato for donation of specifi c antisera, J. Knoppová for help with strain cultivation and 2D gels, Anna Sallai for her technical assistance, and Professor Ferenc Solymosy for reading and correcting the manuscript.

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