Synthesis of Fatty Acids De Novo Is Required for Photosynthetic Acclimation of Synechocystis Sp. PCC 6803 to High Temperature

Biochimica et Biophysica Acta 1797 (2010) 1483–1490

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Synthesis of fatty acids de novo is required for photosynthetic acclimation of Synechocystis sp. PCC 6803 to high temperature

Yohei Nanjo a,b,1, Naoki Mizusawa c, Hajime Wada c, Antoni R. Slabas d, Hidenori Hayashi a,b, Yoshitaka Nishiyama e,⁎ a Cell-Free Science and Technology Research Center, Ehime University, 3 Bunkyo-cho, Matsuyama 790-8577, Japan b Venture Business Laboratory, Ehime University, 3 Bunkyo-cho, Matsuyama 790-8577, Japan c Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan d School of Biological and Biomedical Sciences, University of Durham, Durham DH1 3LE, UK e Department of Biochemistry and Molecular Biology, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan article info abstract

Article history: The role of fatty acid synthesis in the acclimation of the photosynthetic machinery to high temperature was Received 11 January 2010 investigated in a mutant of the cyanobacterium Synechocystis sp. PCC 6803 that had a lower than wild-type Received in revised form 10 March 2010 level of enoyl-(acyl-carrier-protein) reductase FabI, a key component of the type-II fatty acid synthase Accepted 15 March 2010 system. The mutant exhibited marked impairment in the tolerance and acclimation of cells to high Available online 19 March 2010 temperature: photoautotrophic growth of the mutant was severely inhibited at 40 °C. Moreover, mutant cells were unable to achieve wild-type enhancement of the thermal stability of photosystem II (PSII) when the Keywords: Acclimation growth temperature was raised from 25 °C to 38 °C. Enhancement of the thermal stability of PSII was Fatty acid synthesis abolished when wild-type cells were treated with triclosan, a speciﬁc inhibitor of FabI, and the enhancement High-temperature stress of thermal stability was also blocked in darkness and in the presence of chloramphenicol. Analysis of fatty Photosystem II acids in thylakoid membranes revealed that levels of unsaturated fatty acids did not differ between mutant Synechocystis and wild-type cells, indicating that the saturation of fatty acids in membrane lipids might not be responsible Thermotolerance for the enhancement of thermal stability at elevated temperatures. Our observations suggest that the synthesis de novo of fatty acids, as well as proteins, is required for the enhancement of the thermal stability of PSII during the acclimation of Synechocystis cells to high temperature. © 2010 Elsevier B.V. All rights reserved.

1. Introduction their viability at high temperatures [5,6]. Thus, the thermal stability of PSII appears to influence the thermotolerance of cyanobacterial cells. Exposure of photosynthetic organisms to high temperatures often The thermal stability of PSII in photosynthetic organisms is results in the permanent inactivation of photosynthesis. Heat-induced affected by the growth temperature. When organisms are grown at inactivation of photosynthesis is due initially to the inactivation of moderately high temperatures, the thermal stability of PSII is photosystem II (PSII), which is the most thermally sensitive enhanced. This phenomenon has been observed in several species of component of the photosynthetic machinery [1]. Disruption of the plants [7–10]; in cyanobacteria [5,6,11–13]; and in Chlamydomonas manganese cluster in the oxygen-evolving complex of PSII is [14]. Since mutant cyanobacterial cells lacking specific extrinsic considered to be the first event in the heat-induced inactivation of proteins of PSII are unable to increase both the thermal stability of PSII [2–4]. In cyanobacteria, knockout of the genes for some of the PSII and the thermotolerance of the cells themselves [5,6], it seems extrinsic proteins of PSII, which normally stabilize the oxygen- likely that enhancement of the thermal stability of PSII might be part evolving complex, results in decreases not only in the thermal of the strategy for the acclimation of photosynthetic organisms to high stability of PSII but also in the thermotolerance of cells in terms of temperatures. Several mechanisms for the enhancement of the thermal stability of PSII have been proposed. Since a decrease in the level of unsaturated fatty acids in membrane lipids is often observed during Abbreviations: ACP, acyl-carrier-protein; Chl, chlorophyll; DM, n-dodecyl-β-D- the acclimation of plants to high temperatures, it has been suggested maltoside; PSII, photosystem II that saturated fatty acids might contribute to enhancement of the ⁎ Corresponding author. Tel.: +81 48 858 3402; fax: +81 48 858 3384. thermal stability of PSII [8,9,15–19]. However, studies with mutant E-mail address: [email protected] (Y. Nishiyama). 1 Present address: National Institute of Crop Science, NARO, 2-1-18 Kannondai, cyanobacterial cells and with transgenic plants that lack polyunsat- Tsukuba 305-8518, Japan. urated fatty acids in their membrane lipids indicate that saturation of

0005-2728/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbabio.2010.03.014 1484 Y. Nanjo et al. / Biochimica et Biophysica Acta 1797 (2010) 1483–1490 fatty acids does not enhance the thermal stability of PSII [20–23]. by PCR and the exact site of insertion of the transposon was Involvement of a variety of other factors in the thermal stability of PSII determined by DNA sequencing. One of the selected plasmids was has also been proposed. Such factors include specific lipids, such as used to transform wild-type cells of Synechocystis. sulfoquinovosyldiacylglycerol [24], phosphatidylglycerol [25,26], monoglucosyldiacylglycerol [27], and digalactosyldiacylglycerol 2.3. Assay of the thermal stability of PSII [28]; carotenoids of the xanthophyll cycle [29]; heat-shock proteins [30–34]; sigma factors [35,36]; and isoprene [37]. However, no role Thermal stability of PSII in cells was determined by the procedure for any of these compounds has been demonstrated in the described by Kimura et al. [6]. Each suspension of cells was incubated enhancement of the thermal stability of PSII that occurs during at a designated temperature for 20 min in darkness. After incubation, acclimation to high temperature. Thus, specific factors that increase the suspension was cooled rapidly, and the evolution of oxygen was the thermal stability of PSII remain to be identified. measured at 30 °C in the presence of 1 mM 1,4-benzoquinone and

In Escherichia coli, certain defects in the synthesis of fatty acids 1mM K3Fe(CN)6 with a Clark-type oxygen electrode (Hansatech result in a temperature-sensitive phenotype [38]. Mutants with Instruments, King's Lynn, UK). impaired synthesis of fatty acids are unable to grow at nonpermissive high temperatures [39]. An example of such a temperature-sensitive 2.4. Preparation of recombinant protein phenotype results from mutation of the envM gene, which encodes an enoyl-(acyl-carrier-protein) reductase (enoyl-ACP reductase), desig- The recombinant FabI protein was prepared with the pET nated FabI [40]. FabI is a component of the type-II fatty acid synthase expression system (Novagen, Madison, WI, USA). The fabI gene was system which catalyzes the reduction of enoyl-ACP to acyl-ACP, an amplified from the genomic DNA of Synechocystis by PCR with the essential step in the synthesis of fatty acids de novo [41]. Type-II fatty forward and reverse primers 5′-CATATGTTAGATCTCAGCGGC-3′ and acid synthase systems are conserved in most bacteria and in the 5′-CTCGAGCATACCCATAATTTCATA-3′, respectively. After the product plastids of various plant species [38].Inthecyanobacterium of PCR had been cloned into the pGEM-T Easy vector, the DNA Synechocystis sp. PCC 6803 (hereafter referred to as Synechocystis), fragment that contained the fabI gene was cloned between the NdeI putative genes for enzymes of the type-II fatty acid synthase system and the XhoI sites of pET-21b (Novagen). The resultant pET-fabI have been found in the genome [42]. These genes include slr1511 for plasmid was used to transform E. coli BL21 (DE3) (Novagen) for FabH (β-ketoacyl-ACP synthase III); slr1332 and sll1069 for FabF (β- expression of the recombinant protein. The recombinant protein was ketoacyl-ACP synthase); slr0886 and slr1994 for FabG (β-ketoacyl- purified on a His-Trap HP column (GE Healthcare, Piscataway, NJ, ACP reductase); sll1605 for FabZ (β-hydroxyacyl-ACP dehydratase); USA). FabI-specific antibodies were raised in rabbit against the and slr1051 for FabI. purified FabI protein. In the present study, we examined the potential role of fatty acid synthesis in the acclimation of the photosynthetic machinery to high 2.5. Electrophoresis and Western blotting analysis temperature using a mutant of Synechocystis with a lower than wild- type level of FabI that was due to insertional mutagenesis of the fabI Proteins were separated by SDS-PAGE on a 15% polyacrylamide gel gene (slr1051). Mutant cells were unable to survive at high that contained 6 M urea as described by Kashino et al. [44] and they temperatures. In addition, mutant cells failed to increase the thermal were visualized by staining with Coomassie Brilliant Blue R250. stability of PSII adequately during acclimation to high temperature. Western blotting analysis was performed with FabI-specific anti- Our observations suggest that synthesis of fatty acids de novo is bodies and with D1-specific antibodies raised against a synthetic required for stabilization of PSII against high temperature and for the oligopeptide that corresponded to the AB loop of the D1 protein [45]. thermotolerance of Synechocystis. Proteins were visualized with peroxidase-linked second antibodies and the ECL Western blotting analysis system (GE Healthcare). 2. Materials and methods Immunostained proteins were quantified with a Luminescent Image Analysis System (LAS-1000; Fuji Film, Tokyo, Japan). 2.1. Cells and culture conditions 2.6. Analysis of the fatty acid composition of lipids Cells of the wild-type strain of Synechocystis sp. PCC 6803, the fabI mutant, and the mutant that expressed histidine-tagged CP47 [43] Total lipids were extracted from thylakoid membranes as were grown photoautotrophically at designated temperatures in described by Bligh and Dyer [46]. Lipids and fatty acids were analyzed liquid BG11 medium under light at 80 µmol photons m− 2 s− 1 with as described by Wada and Murata [47]. Total lipids and lipid classes aeration by sterile air that contained 1% (v/v) CO2 [20].For were separated on thin-layer plates that had been pre-coated with maintenance of mutants, 20 µg mL− 1 kanamycin was included in silica gel (Merck, Whitehouse Station, NJ, USA), with a mixture of the culture medium. CHCl3,CH3OH, and 28% NH4OH (65:35:5, v/v) as the mobile phase, and they were then subjected to methanolysis with 5% HCl in 2.2. Construction of the fabI mutant methanol at 85 °C for 2.5 h. The resultant methyl esters were analyzed with a gas–liquid chromatograph (GC-7A; Shimadzu, Kyoto, Japan), The nucleotide sequence of the fabI gene of Synechocystis was which was equipped with a hydrogen flame-ionization detector. Fatty obtained from the CYORF website (http://cyano.genome.jp). The fabI acid methyl esters were separated at 170 °C on a capillary column gene was amplified from the genomic DNA of Synechocystis by PCR (0.25 mm i.d.×25 m; Quadrex CPS-1; Shimadzu) coated with cyano- with the forward and reverse primers 5′-AGCAAAGACAATCCTTC- propylmethyl silicone (0.25 µm thickness). Relative amounts of fatty CAAA-3′ and 5′-GGCAAAGAACTATTTTTCTCG-3′, respectively. The acid methyl esters were calculated by comparing areas under peaks amplified DNA fragment was cloned into the pGEM-T Easy vector on the chromatogram with a data processor (C-R7A; Shimadzu). (Promega, Madison, WI, USA). The resultant plasmid was used to mutate plasmids with the EZ::TN bKAN-2N Insertion Kit (Epicentre, 2.7. Isolation of thylakoid membranes and PSII complexes Madison, WI, USA), which allows random insertion of a transposon that contains a kanamycin-resistance gene cassette into target DNA Thylakoid membrane and PSII complexes were isolated at 4 °C via the Tn5 transposition system. Plasmids that contained the from mutant cells that expressed histidine-tagged CP47 as described transposon in the middle of the coding region of fabI were identified by Sakurai et al. [43] with minor modifications. Cells were broken Y. Nanjo et al. / Biochimica et Biophysica Acta 1797 (2010) 1483–1490 1485 with glass beads in a Bead-beater (BioSpec Products Inc., Bartlesville, (Fig. 1B). The incomplete disruption was probably due to the lethal OK, USA) in buffer A, which contained 50 mM MES-NaOH (pH 6.0), effect of complete absence of the fabI gene. Since fabI is located close

10 mM CaCl2, 10 mM MgCl2, 1 M glycinebetaine, and 25% (w/v) to slr1050 and slr1052, both of which encode hypothetical proteins, glycerol, supplemented with 1 mM phenylmethylsulfonyl ﬂuoride, and might form an operon with these genes, we also performed 1 mM benzamidine, and 1 mM 6-amino-n-caproic acid. After cell insertional mutagenesis of slr1050 and slr1052.Wewereable debris had been removed by centrifugation at 2000×g for 10 min, completely to disrupt both of these genes and the phenotypes of the thylakoid membranes were pelleted by centrifugation at 35,000×g for resultant mutants did not differ from the phenotype of wild-type cells 30 min. Pelleted thylakoid membranes were resuspended in buffer A in terms of cell growth and the thermal stability of PSII (data not at a density of 1.0 mg chlorophyll (Chl) mL− 1. Thylakoid membranes shown). were solubilized by addition of n-dodecyl-β-D-maltoside (DM; Dojin, We examined the level of the FabI protein in the fabI mutant by Kumamoto, Japan) to a ﬁnal concentration of 1.0% (w/v) and sub- Western blotting analysis with antibodies raised against FabI. In the sequent incubation on ice for 5 min. The suspension was centrifuged mutant cells, the level of FabI was approximately 60% of that in wild- at 35,000×g for 15 min and the resulting supernatant was loaded type cells (Fig. 2). By contrast, the level of the D1 protein, which is a onto a column (1.5 cm i.d.×6 cm) of Ni-Sepharose Fast Flow (GE component of the reaction center of PSII, was unaffected by the Healthcare) that had been equilibrated with buffer A supplemented mutation. Staining with Coomassie Brilliant Blue of fractionated with 0.004% (w/v) DM. The column was washed with buffer A plus proteins in cell extracts of wild-type and fabI mutant cells failed to 0.004% (w/v) DM and 20 mM imidazole. After washing, PSII reveal any differences in the levels of any other proteins. complexes were eluted with buffer A plus 0.004% (w/v) DM and 100 mM histidine. After the eluent had been mixed with 0.8 volume of 3.2. Sensitivity of growth to high temperature 25% (w/v) polyethyleneglycol-8000 (Sigma, St. Louis. MO, USA), PSII complexes were pelleted by centrifugation at 35,000×g for 20 min We examined the photoautotrophic growth of wild-type and fabI and resuspended in buffer A. mutant cells at various temperatures in liquid medium (Fig. 3). Suspension cultures grown at 32 °C were diluted to an optical density 3. Results at 730 nm of 0.05 and were incubated at various temperatures. The growth of the fabI mutant was slightly slower than that of wild-type 3.1. Disruption of the fabI gene

To inactivate the fabI gene (slr1051)inSynechocystis, we inserted a kanamycin-resistance gene cassette into the fabI gene in the pGEM-T Easy vector, using the Tn5 transposition system. We selected a clone in which the kanamycin-resistance gene cassette was located 340 bp downstream from the initial codon of the fabI gene, and we transformed Synechocystis cells with the resultant plasmid. Fig. 1A shows a schematic illustration of the site of insertion of the cassette into the fabI gene in the Synechocystis genome and the extent of replacement of the wild-type gene in the genome by the mutated gene. Synechocystis contains approximately 13 chromosomal copies of its genome and, despite strong selective pressure, we failed to achieve complete disruption of all the genomic copies of the fabI gene

Fig. 2. Levels of the FabI protein in wild-type and the fabI mutant cells. (A) Western blotting analysis of FabI and D1 proteins in extracts of wild-type (WT) and fabI mutant (MT) cells with FabI-speciﬁc (anti-FabI) and D1-speciﬁc (anti-D1) antibodies. Extracts were prepared from cells that had been grown at 30 °C and aliquots that corresponded Fig. 1. Schematic illustration of the insertion of the kanamycin-resistance gene cassette to 20 or 1 µg of Chl were loaded onto each lane for subsequent staining with Coomassie (Kanr) into the fabI gene in the Synechocystis genome (A) and analysis by PCR of the Brilliant Blue (CBB) or Western blotting analysis, respectively. (B) Quantitation of the extent of replacement of fabI genes by the mutated gene (B). Genomic DNA was isolated FabI protein by chemiluminescence imaging. The value for mutant cells is shown as the from wild-type (WT) and fabI mutant (MT) cells. Arrows in panel A indicate the mean±S.D. of results from three independent experiments. The mean amount of FabI positions and directions of primers for PCR. protein in wild-type cells was taken as 100%. 1486 Y. Nanjo et al. / Biochimica et Biophysica Acta 1797 (2010) 1483–1490

Fig. 3. Photoautotrophic growth of wild-type and fabI mutant cells at various temperatures. Wild-type (○) and the fabI mutant (●) cells were grown at the indicated temperatures with illumination at 80 µmol photons m− 2 s− 1. The optical density of cells at 730 nm was measured at the indicated times. Values are means±S.D. (bars) of results from three independent experiments. The absence of a bar indicates that the S.D. falls within the symbol. Fig. 4. Effects of growth temperature on the profile of subsequent heat-induced inactivation of PSII in cells. Wild-type (A) and fabI mutant (B) cells were grown at 25 °C ○ ● cells at 32 °C, the optimum temperature for growth of this organism. The ( ) and 38 °C ( ). Cells were then incubated at the indicated temperatures for 20 min in darkness. The activity of PSII was measured by monitoring the evolution of oxygen at slower growth in the mutant was also observed at low temperatures, 30 °C in the presence of 1 mM 1,4-benzoquinone and 1 mM K3Fe(CN)6. The activities of such as 25 °C. At 38 °C, a moderately high temperature, the growth of PSII taken as 100% in wild-type cells grown at 25 °C and 38 °C were 874±36 and 680± − 1 − 1 the mutant was much slower than that of wild-type. At 40 °C, a 34 µmol O2 mg Chl h , respectively, and those in fabI mutant cells were 1115±53 − 1 − 1 temperature that causes heat stress in this organism, wild-type cells and 771±38 µmol O2 mg Chl h , respectively. Values are means±S.D. (bars) of results from three independent experiments. grew slightly more slowly than at 38 °C but fabI mutant cells were unable to grow. Thus, decreased levels of FabI resulted in the increased sensitivity of photoautotrophic growth to high temperature. triclosan. Triclosan binds to the catalytic site of the enzyme and forms a FabI-NAD+-triclosan ternary complex [48,49]. When wild-type cells 3.3. Impaired enhancement of the thermal stability of PSII that had been grown at 25 °C were transferred to 38 °C, with no change in the intensity of illumination, the thermal stability of PSII We examined the effects of growth temperature on the thermal increased gradually and, in 24 h, it reached the level observed in cells stability of PSII. When wild-type cells were grown at 38 °C, the that had been grown initially at 38 °C (Fig. 5A). In the fabI mutant, the thermal stability of PSII was greater than that after cells had been thermal stability of PSII increased more slowly than it did in wild-type grown at 25 °C (Fig. 4A). The temperatures for 50% inactivation of PSII cells and, after 24 h, it was 50% of that of PSII in wild-type cells in cells that had been grown at 25 °C and 38 °C were 44 °C and 48 °C, (Fig. 5A). When we added triclosan just before raising the growth respectively. These results are consistent with those in previous temperature of wild-type cells, enhancement of the thermal stability reports [5,6]. By contrast, in the fabI mutant, enhancement of the of PSII was inhibited completely (Fig. 5A). These observations indicate thermal stability of PSII was markedly reduced (Fig. 4B). There was a that the FabI-mediated synthesis of fatty acids is essential for clear difference in terms of the shapes of the curves that showed enhancement of the thermal stability of PSII. relative PSII activity plotted against incubation temperature between In darkness, the thermal stability of PSII in wild-type cells did not wild-type and mutant cells grown at 38 °C. The plateau region in the increase upon elevation of the growth temperature from 25 °C to profile of the heat inactivation of PSII in wild-type cells extended over 38 °C (Fig. 5B), suggesting that this acclimation requires light. In a considerable range after acclimation at 38 °C. By contrast, there was addition, no enhancement of thermal stability occurred in the no plateau in the case of mutant cells. The temperatures for 50% presence of chloramphenicol (Fig. 5B), suggesting that enhancement inactivation of PSII in mutant cells grown at 25 °C and 38 °C were of the thermal stability of PSII also requires the synthesis of proteins 43 °C and 45 °C, respectively. However, the absolute activity of PSII de novo. was barely affected by the mutation in fabI. The activities of PSII in wild-type cells grown at 25 °C and 38 °C were 874±36 and 680± 3.5. Saturation of fatty acids in lipids is independent of the enhancement − 1 − 1 34 µmol O2 mg Chl h , respectively, and those in fabI mutant cells of the thermal stability of PSII −1 −1 were 1115±53 and 771±38 µmol O2 mg Chl h , respectively. Thus, decreased levels of FabI resulted in marked impairment of the capacity The contribution of saturation of fatty acids to enhancement of the for enhancement of the thermal stability of PSII during acclimation. thermal stability of PSII has been the subject of controversy [8,9,20– 22]. To address this issue, we examined the effects of growth 3.4. Synthesis de novo of fatty acids and proteins is required for temperature on the composition of fatty acids in thylakoid mem- enhancement of the thermal stability of PSII during acclimation branes in wild-type and fabI mutant cells (Table 1). In wild-type cells that had been grown at 38 °C, the levels of 18:3 and 18:4 were much To confirm that FabI is involved in the enhancement of the thermal lower than those in cells that had been grown at 25 °C. The double- stability of PSII, we examined the effect of a specific inhibitor for FabI, bond index of fatty acids in cells grown at 38 °C fell to 66.7, while the Y. Nanjo et al. / Biochimica et Biophysica Acta 1797 (2010) 1483–1490 1487

acids per cell was barely affected by the mutation in fabI or by the elevation of growth temperature (Table 2).

3.6. Localization of FabI in Synechocystis cells

To investigate the localization of the FabI protein in Synechocystis cells, we performed Western blotting analysis with a mutant of Synechocystis that expressed histidine-tagged CP47, a component of PSII [43], because PSII complexes can easily be purified from this mutant. Fig. 6 shows the distribution of FabI in various subcellular compartments of cells that had been grown at 25 °C and 38 °C. FabI was detected in all fractions examined, namely, the cytosol, the thylakoid membranes, and the PSII complexes. The amount of FabI in each fraction was unaffected by growth temperature, indicating that the synthesis of FabI was not induced at high temperatures. The components of the type-II fatty acid synthase system, including FabI, are localized in the cytosol in bacterial cells and in the stroma in plastids of plants [38]. However, there are also reports of components of the type-II fatty acid synthase, such as ACP, being associated with thylakoid membranes [50]. The FabI that we detected in PSII complexes dissociated from PSII complexes when we increased the concentration of the detergent n-dodecyl-β-D-maltoside from 0.004% to 0.04% (the standard concentration) during the washing step in the purification procedure. These observations suggest that some Fig. 5. Changes in the thermal stability of PSII after an increase in the growth temperature of cells. Cells that had been grown at 25 °C were transferred to 38 °C and portion of FabI might be weakly associated with the PSII complex and grown as indicated. Triclosan and chloramphenicol were added to suspensions of cells that the synthesis of fatty acids might also occur near PSII complexes just before the transfer. Aliquots of suspensions of cells were withdrawn at the in Synechocystis cells. indicated times and incubated at 47 °C for 20 min in darkness. The activity of PSII was then measured as described in the legend to Fig. 4. The thermal stability of PSII is shown as the ratio (%) of the activity of PSII after incubation at 47 °C to the activity after 4. Discussion incubation at 30 °C for 20 min in darkness. (A) ○, Wild-type cells incubated in light; ◊, wild-type cells incubated in light in the presence of 1 µM triclosan; ▲, fabI mutant 4.1. Requirement for fatty acid synthesis during the acclimation of the cells incubated in light. (B) ●, Wild-type cells incubated in darkness; □, wild-type cells photosynthetic machinery to high temperature incubated in light in the presence of 20 µg mL− 1 chloramphenicol. Values are means ±S.D. (bars) of results from three independent experiments. In the present study, we investigated the effects of the synthesis of fatty acids on the enhancement of the thermal stability of PSII during index in cells grown at 25 °C was 96.2. These observations indicate the acclimation of Synechocystis cells to high temperature. Decreased that the extent of the desaturation of fatty acids fell when the growth levels of FabI, which is a key protein in the type-II fatty acid synthesis temperature was raised, as reported previously [8,9]. However, fabI system, by partial inactivation of the fabI gene resulted in an impaired mutant cells exhibited the same changes as wild-type cells in terms of capacity for the enhancement of the thermal stability of PSII. both the levels of polyunsaturated fatty acids and the double-bond Treatment of wild-type cells with triclosan, a specific inhibitor of index. Furthermore, transfer of wild-type cells from 25 °C to 38 °C and FabI, abolished this acclimation response completely. Our results subsequent growth for 24 h decreased the double-bond index to only show that synthesis of fatty acids de novo is required for the 50% of that in cells that had fully acclimated to 38 °C. By contrast, this enhancement of the thermal stability of PSII. However, it seems treatment resulted in full (wild-type) enhancement of the thermal unlikely that stabilization of PSII is due to the accelerated synthesis of stability of PSII (Fig. 5), suggesting that enhancement of thermal fatty acids at high temperatures since total amounts of fatty acids per stability is completed sooner than the saturation of fatty acids. In view cell were unaffected by high temperatures. of the considerable difference in terms of the thermal stability of PSII The fabI mutant was unable to grow at high temperatures under between wild-type and fabI mutant cells, it seems unlikely that the photoautotrophic conditions. It seems likely that the increased saturation of fatty acids in membrane lipids is responsible for the sensitivity to high temperature of this mutant resulted from impaired enhancement of the thermal stability of PSII. The total amount of fatty development of the thermal stability of PSII. In earlier studies, we

Table 1 Fatty acids in thylakoid membranes prepared from wild-type (WT) and fabI mutant cells after growth at two different temperatures. Cells were grown at 25 °C or 38 °C. In addition, cells that had been grown at 25 °C were transferred to 38 °C and incubated for 24 h (25 °C→38 °C). Total lipids were extracted from thylakoid membranes and fatty acids were analyzed as described in the text. The double-bond index (DBI) is the sum of percentages of unsaturated fatty acids multiplied by total number of double bonds. Positions of double bonds are indicated in parenthesis. Values are means of the results from three independent experiments.

Cell type Growth conditions Fatty acids

14:0 16:0 16:1 18:0 18:1 18:1 18:2 18:3 18:3 18:4 DBI (9) (11) (6,9,12) (9,12,15)

mol%

WT 25 °C 0.8 54.1 5.0 1.0 8.3 1.7 8.9 12.1 5.1 2.9 96.2 38 °C 1.5 61.3 3.7 1.5 10.0 0 13.6 7.6 0 0.7 66.7 25 °C→38 °C 0.2 57.8 3.0 1.4 14.8 0.2 9.7 9.9 1.9 1.2 77.5 fabI 25 °C 0.5 52.6 5.8 1.1 8.4 1.3 8.4 14.3 4.8 2.9 101.2 38 °C 0.8 57.5 3.2 2.7 15.0 0.5 12.6 7.2 0.1 0.4 67.3 25 °C→38 °C 0.6 56.2 3.5 1.8 13.9 0.9 7.2 10.0 3.6 2.4 82.9 1488 Y. Nanjo et al. / Biochimica et Biophysica Acta 1797 (2010) 1483–1490

Table 2 responsible for the enhancement of the thermal stability of PSII. This Levels of fatty acids in 10 mL of a suspension of cells before and after an increase in observation is in agreement with the previous finding that the growth temperature from 25 °C to 38 °C. Aliquots of cultures of wild-type (WT) and fabI saturation of fatty acids in membrane lipids, which was manipulated mutant cells, with an optical density at 730 nm (OD730) of 1.0, that had been grown at 25 °C were transferred to 38 °C and incubated for 24 h (25 °C→38 °C). Aliquots of by inactivation of the genes for fatty acid desaturases, did not enhance 10 mL were withdrawn and subjected to analysis of fatty acids of total lipids. Values are the thermal stability of PSII [20–22]. The physiological role of the means of the results from three independent experiments. saturation of fatty acids in the acclimation of photosynthetic organisms Cell type Growth conditions Total fatty acids Fatty acid content to high temperature remains unclear. The apparent saturation of fatty acids might result from the turnover of lipids in membranes; newly nmol nmol OD− 1 730 synthesized lipids do not undergo polyunsaturation at high tem- WT 25 °C 301.5 301.5 peratures and, consequently, the ratio of polyunsaturated fatty acids 25 °C→38 °C 745.6 298.2 decreases. fabI 25 °C 302.5 302.5 25 °C→38 °C 515.5 299.7 4.3. Possible mechanisms for the enhancement of the thermal stability of PSII during acclimation showed that photoautotrophic growth of mutant cyanobacterial cells, in which PSII had been destabilized as a result of the lack of certain The contribution of protein factors to the enhancement of the extrinsic proteins of PSII, was sensitive to high temperatures [5,6]. thermal stability of PSII has been suggested in earlier studies. Thus, the sensitivity of PSII to high temperatures might restrict the Biochemical studies with cyanobacteria and soybean showed that viability of photosynthetic organisms at such temperatures, and certain proteins, which had been dissociated from thylakoid mem- protection of PSII against such high temperatures might be essential branes by a low concentration of Triton X-100, were able to increase for the development of cellular thermotolerance. The present study the thermal stability of PSII in thylakoid membranes, suggesting that revealed for the first time, to our knowledge, that the protection of some proteins that are weakly associated with PSII might be PSII against high temperatures requires the synthesis of fatty acids de responsible for enhancing the thermal stability of PSII [10,51,52]. novo. Physiological studies with Chlamydomonas and soybean demonstrated that inhibition of the synthesis of proteins de novo prevents 4.2. Independence of the saturation of fatty acid in lipids from the increases in the thermal stability of PSII [10,14]. Essentially the same enhancement of the thermal stability of PSII result was obtained in Synechocystis in the present study (Fig. 5). Thus, it seems likely that the synthesis of both proteins and fatty acids de The present study showed that levels of saturated fatty acids in novo is required for the enhancement of the thermal stability of PSII. membrane lipids increased during the acclimation of cells to high The impaired enhancement of thermal stability in darkness (Fig. 5) temperature. This phenomenon has been observed in many photo- might be due to the suppression of the synthesis both of fatty acids synthetic organisms and the resulting rigidification of photosynthetic [53] and of proteins in the absence of light. membranes was initially considered to be the mechanism responsible Taking all these observations together, we propose a mechanism for the enhancement of the thermal stability of PSII [8,9]. However, for the enhancement of thermal stability as follows. The thermal the present study showed that levels of saturated fatty acids did not stability of PSII is enhanced by proteins that bind fatty acids, such as differ between wild-type and fabI mutant cells (Table 1), suggesting lipoproteins. These lipoproteins are newly synthesized in response to that the saturation of fatty acids in membrane lipids might not be moderately high temperatures, such as 38 °C, and are bound to the lumenal side of the PSII complex to stabilize the manganese cluster of the oxygen-evolving complex against heat-induced inactivation. In Synechocystis, there are 39 predicted proteins that contain consensus amino acid sequences specific for lipoproteins [54], and lipoproteins that are associated with the lumenal side of PSII, in particular, extrinsic lipoproteins, are likely candidates for participants in thermal stability. The PSII complex in Synechocystis includes at least six extrinsic proteins (PsbO, PsbP, PsbQ, PsbU, PsbV, and Psb27), among which PsbP, PsbQ, and Psb27 are potential lipoproteins [55–57]. Both PsbP and PsbQ act as regulators of the biogenesis of the active PSII complex in Synechocystis [58,59]. Psb27 facilitates the assembly of the manganese cluster by binding, transiently, to inactive PSII monomers during the repair of photodamaged PSII [55,60]. However, neither of PsbP nor PsbQ appears to enhance the thermal stability of PSII directly since mutants that lack these proteins are still capable of enhancing the thermal stability of PSII [61,62]. Psb27 is also unlikely to act by enhancing the thermal stability of PSII directly because Psb27 is released from PSII after repair of PSII has been completed [60], whereas the enhanced thermal stability of PSII is retained in thylakoid membranes even after isolation of the membranes [10,12]. By contrast, mutants of Synechocystis that lack PsbO, PsbU or PsbV have severely impaired capacity for enhancement of the thermal stability of PSII, suggesting that these proteins might be involved in Fig. 6. Subcellular localization of FabI in Synechocystis cells. Synechocystis cells that the protection of the oxygen-evolving complex against high temper- expressed histidine-tagged CP47 were grown at 25 °C and at 38 °C. Then cell extracts ature [5,6]. However, none of these three proteins is likely to be a (Cell), cytosolic fractions (Cytosol), thylakoid membranes (TM), and PSII complexes direct enhancer for thermal stability since their synthesis is not (PSII) were prepared as described in the text. Aliquots that corresponded to 20 µg of Chl induced or modified at high temperatures [5,6]. It is possible that as-yet- were subjected to SDS-PAGE and staining with Coomassie Brilliant Blue. Aliquots that fi corresponded to 1 µg of Chl were also subjected to Western blotting analysis with FabI- unde ned proteins, including lipoproteins, might act by directly specific (anti-FabI) antibodies. increasing the thermal stability of PSII. Such proteins might strengthen Y. Nanjo et al. / Biochimica et Biophysica Acta 1797 (2010) 1483–1490 1489 the stabilization of the oxygen-evolving complex via the actions of proteins encoded by the nuclear and chloroplast genomes, Plant Physiol. 124 (2000) 441–449. extrinsic proteins, such as PsbO, PsbU, and PsbV. However, we cannot [15] P.G. Thomas, P.J. Dominy, L. Vigh, A. Mansourian, P.J. Quinn, W.P. Williams, exclude the possibility that modification of extrinsic proteins with Increased thermal stability of pigment-protein complexes of pea thylakoids specific lipids might be altered at high temperatures. following catalytic hydrogenation of membrane lipids, Biochim. Biophys. 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