This Article Abstract Full Text (PDF) All Versions of this Article: 139/1/408    most recent pp.105.064782v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in Plant Physiol. Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (1) Citing Articles via Google Scholar Google Scholar Articles by Fujimori, T. Articles by Sonoike, K. Search for Related Content PubMed PubMed Citation Articles by Fujimori, T. Articles by Sonoike, K. Agricola Articles by Fujimori, T. Articles by Sonoike, K.

BIOENERGETICS AND PHOTOSYNTHESIS

The Mutant of sll1961, Which Encodes a Putative Transcriptional Regulator, Has a Defect in Regulation of Photosystem Stoichiometry in the Cyanobacterium Synechocystis sp. PCC 6803¹

Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa-shi, Chiba 277–8562, Japan (T.F., M.H., H.S., H.A., K.S.); and Department of Biochemistry and Molecular Biology, Faculty of Science, Saitama University, Saitama-shi, Saitama 338–8570, Japan (M.M., Y.H.)

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
In acclimation to changing light environments, photosynthetic organisms modulate the ratio of two photosynthetic reaction centers (photosystem I [PSI] and photosystem II). One mutant, which could not modulate photosystem stoichiometry upon the shift to high light, was isolated from mutants created by random transposon mutagenesis. Measurements of chlorophyll fluorescence and analysis of the reaction center subunits of PSI through western blotting in this mutant revealed that the content of PSI could not be suppressed under high-light condition. In the mutant, transposon was inserted to the sll1961 gene encoding a putative transcriptional regulator. DNA microarray analysis revealed that the expression of sll1773 was drastically induced in the sll1961 mutant upon exposure to high light for 3 h. Our results demonstrate that a transcriptional regulator, Sll1961, and its possible target proteins, including Sll1773, may be responsible for the regulation of photosystem stoichiometry in response to high light.

In cyanobacteria, PSI/PSII ratio is normally about 2 (Melis and Brown, 1980), and its ratio ranges from 1 to 4 depending on the light condition. Light that excites mainly PSII leads to the increase in PSI so as to bring about high PSI/PSII ratio of about 2 to 4, while light that excites mainly PSI induces the decrease in PSI, resulting in low PSI/PSII ratio as low as 1 (Fujita et al., 1985, 1988; Fujita and Murakami, 1987). The regulation of photosystem amounts by light quality is at the transcriptional and/or posttranscriptional level of PSI and PSII subunits (Glick et al., 1986). The changes in photosystem stoichiometry occur so as to balance the excitation energy distribution and maintain the efficient electron flow between PSII and PSI (for review, see Glazer and Melis, 1987). The regulation correlates with the redox state of plastoquinone pool and cytochrome b₆f complex (Fujita et al., 1987; Fujita, 1991; Murakami and Fujita, 1991). Light quantity, as well as light quality, induces the changes in PSI/PSII ratio in cyanobacteria (Kawamura et al., 1979; Murakami and Fujita, 1991). PSI/PSII ratio decreased upon the shift to high light through the selective suppression in the amount of PSI, in addition to the general decrease in the amount of both photosystems in cyanobacteria (for review, see Anderson, 1986; Hihara, 1999).

Many cyanobacterial mutants with different photosystem stoichiometry from that of the wild type were reported (Wilde et al., 1995, 2001; Mann et al., 2000; Shen et al., 2002; Kufryk and Vermaas, 2003; Yu et al., 2003; Wang et al., 2004). The deletion of genes encoding the subunits of PSI or PSII may lead to the decrease of relative PSI or PSII content. The disruption of genes involved in transcription, translation, assembly, or biogenesis of PSI or PSII may lead to the change in photosystem stoichiometry. One gene, sll0088, seems to encode a transcriptional regulator so that it may be involved in the regulation of photosystem stoichiometry (Yu et al., 2003; Wang et al., 2004). However, only a few cases have been reported for the regulatory mutant of photosystem stoichiometry under high-light condition (Hihara et al., 1998). One such case is the pmgA (sll1968) mutant. This mutant has normal amounts of PSII and PSI under low-light condition, but fails to change photosystem stoichiometry upon the shift to high light (Hihara et al., 1998). Thus, this mutant has a defect not in the process of synthesis, assembly, or degradation of photosystems, but in the process of regulating photosystem stoichiometry. The pmgA mutant is a kind of super mutant and grows faster than the wild type in exposure to high light for 24 h. In the prolonged high-light stress for 72 h, however, the growth of the pmgA mutant is severely suppressed, indicating that regulation of photosystem stoichiometry has physiological importance under high-light condition (Sonoike et al., 2001). The role of the regulation of photosystem stoichiometry during acclimation to high light is apparently not to maintain optimal photosynthesis but to protect the cells from oxidative damage through the suppression of photosynthetic electron transfer (Sonoike et al., 2001). The regulation of photosystem stoichiometry must be essential to survive under prolonged high-light condition.

The decrease in PSI contents during acclimation to high light seems to be regulated through the repression of mRNA levels of PSI genes, judging from the data of whole-genome DNA microarray (Hihara et al., 2001). Although the mRNA levels of psaA and psaAB, which encode the subunits of PSI reaction center, are similarly down-regulated in the wild type and the pmgA mutant after 1 h of exposure to high light, the continuous suppression of the transcript levels after 6 h observed in the wild type was not seen in the pmgA mutant (Muramatsu and Hihara, 2003). It was implied that the protein that the pmgA gene encodes might belong to the family of anti-sigma factors. However, it is unknown how PmgA is involved in the transcriptional regulation of psaA and psaAB under prolonged high-light condition. Moreover, it is poorly understood how sophisticated control of PSI level can be achieved under high-light condition.

In this study, we report on a putative transcriptional regulator, Sll1961, which is involved in the modulation of photosystem stoichiometry in acclimation to high light. Characterization of the disruption mutant of this gene indicates that the mutants fail to properly suppress the amount of PSI under high-light condition. Since real-time reverse transcription (RT)-PCR analysis showed that this mutation did not much affect the level of psaA and/or psaAB transcript during acclimation to high light, Sll1961 seems to function on the different signal transduction pathway from that of PmgA. The comparative study of this novel gene and the pmgA gene would lead to the breakthrough for the elucidation of the regulatory mechanism of photosystem stoichiometry.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Isolation of the Mutant with Altered Chlorophyll Fluorescence Kinetics

We created mutants by transposon-mediated random insertion of a chloramphenicol-resistant cassette into the chromosome of the wild-type strain. We could isolate several mutants that showed different chlorophyll fluorescence kinetics from that of the wild type. One such mutant, the 0205-79 mutant, showed fluorescence kinetics similar to the pmgA mutant that has a defect in the regulation of photosystem stoichiometry (Fig. 1). Although the difference between the wild type and these two mutants in fluorescence kinetics was very small under low-light (20 µmol m^–2 s^–1) condition (Fig. 1A), a large difference was observed under high-light (200 µmol m^–2 s^–1) condition (Fig. 1B).

View larger version (18K): [in this window] [in a new window]   Figure 1. Kinetics of fluorescence emitted from the wild type (bold line), the 0205-79 mutant (thin line), and the pmgA mutant (dot line) cells under low-light (A) or high-light (B) conditions. Cells on agar plates were dark adapted for 15 min. AL was applied for 3 s to monitor the fluorescence. Intensity of the fluorescence was normalized with the initial value at the start of AL.

Photosynthetic electron transport in the wild type and the mutants was characterized in detail by using pulse amplitude modulation (PAM) chlorophyll fluorometer. Changes in the intensity of chlorophyll fluorescence upon the application of multiple turnover flash or actinic light (AL) are essentially the same between low-light-acclimated cells of the wild type and the mutants (Fig. 2, A–C). When cells were grown under high-light condition, however, the sharp rise of fluorescence intensity just after exposure to AL in the wild type was not observed in the pmgA mutant and the 0205-79 mutant (Fig. 2, D–F). Upon the cessation of the AL, the fluorescence intensity rapidly decreased in high-light-acclimated cells of the wild type, while the decay of fluorescence intensity was slower and a small peak appeared in those of the pmgA mutant and the 0205-79 mutant. Slower rise of the fluorescence intensity upon application of AL as well as slower decay of that upon cessation of the AL suggested that PSI activity is higher in the mutant cells. The transient increase of the fluorescence intensity in the dark after the cessation of the AL is usually observed when the influx of electrons to plastoquinone pool by cyclic electron flow around PSI is enhanced. The results in Figure 2 suggest that the intracellular activity of PSI in the pmgA mutant and the 0205-79 mutant is higher than that in the wild type, at least in high-light-acclimated cells.

View larger version (28K): [in this window] [in a new window]   Figure 2. Fluorescence determined by a PAM fluorometer of the wild type (A, D), the 0205-79 mutant (B, E), and the pmgA mutant (C, F) under low-light (A–C) or high-light (D–F) conditions. The fluorescence measurement was initiated by applying modulated measuring light (ML) after 5 min dark adaptation. AL was turned on (AL on) to monitor the change in the level of fluorescence. Then, AL was turned off (AL off) and far-red light was turned on (FR on) to monitor the minimal level of fluorescence (Fo) with nonphotochemical quenching. During the course of fluorescence measurement, a series of multiple turnover flashes (MT flashes) was applied to obtain Fm level (in the dark) and Fm' level (under AL). Finally, dichlorophenyldimethylurea was added (+DCMU) and AL was turned on (AL on) to obtain the maximum level of fluorescence (Fm). The rise and decay of fluorescence intensity upon the application and cessation of AL were magnified in the insets.

We then directly determined photosystem stoichiometry by measuring chlorophyll fluorescence emission spectra at 77 K. The fluorescence peak at 695 nm is mainly emitted from PSII and that at 725 nm arises from PSI. Thus, the fluorescence intensity at 695 nm/fluorescence intensity at 725 nm ratio (F₆₉₅/F₇₂₅) is a good index of PSII/PSI ratio. The F₆₉₅/F₇₂₅ ratio was about 0.4 in the low-light-acclimated cells of the wild type, and it increased to 1.0 during first 24 h under high-light condition (Fig. 3, black circles). However, the increase in this ratio was not observed in the 0205-79 mutant (Fig. 3, white circles). The similar inability to increase PSII/PSI ratio upon high-light acclimation was reported earlier in the pmgA mutant (Hihara et al., 1998). The results suggest that the gene responsible for the mutation in the 0205-79 mutant is involved in the regulation of photosystem stoichiometry under high-light condition.

View larger version (11K): [in this window] [in a new window]   Figure 3. Time course of the changes in the ratio of F695/F725 (PSII/PSI) during acclimation to high light. The ratio was determined by measuring chlorophyll fluorescence emission spectra of the wild type (black circle) and the 0205-79 mutant (white circle) at 77 K. Data are the means of three samples.

The relative change in photosystem stoichiometry can be caused by the change either in PSI content or in PSII content. To estimate the amount of PSI in the mutant, we performed western analysis with an antiserum raised against PsaAB, the reaction center subunits of PSI. No differences in the amounts of PsaAB were observed between the wild type and the 0205-79 mutant under low-light condition (Fig. 4). The amounts of PsaAB proteins significantly decreased in the wild type under high-light condition. This decrease was partially suppressed in the 0205-79 mutant. The failure to modulate photosystem stoichiometry in the 0205-79 mutant during acclimation to high light must be due to the insufficient suppression of PsaAB amounts under high-light condition.

View larger version (16K): [in this window] [in a new window]   Figure 4. Western analysis of PsaAB in the wild type and the 0205-79 mutant grown under low- and high-light conditions for 24 h. Total proteins, corresponding to the cells of 3 mL culture of OD730 = 0.355, were loaded on each lane.

In order to examine whether the amount of PsaAB protein is regulated at transcriptional level, we performed real-time RT-PCR analysis of psaA and/or psaAB during acclimation to high light. Low-light-grown cells were shifted to high light and time course of change in psaA and/or psaAB transcripts was examined (Fig. 5). The transcript level of psaA and/or psaAB drastically decreased upon the shift to high light, and the level was partially recovered 12 h after the shift to high light. The level of psaA and/or psaAB transcripts in the 0205-79 mutant was not significantly higher than that in the wild type during acclimation to high light. The inability to keep the level of the transcript of psaA and psaAB under high-light condition in the pmgA mutant (Muramatsu and Hihara, 2003) was not observed in the 0205-79 mutant. The profile of the change in the level of the transcripts could not explain the difference of the protein level of PsaAB between the wild type and the 0205-79 mutant (Fig. 4). The gene responsible for the mutation of the 0205-79 mutant may have a role in the translational or posttranslational regulation of PsaAB.

View larger version (15K): [in this window] [in a new window]   Figure 5. Transcript abundance of psaA in the wild type (black circle) and the 0205-79 mutant (white circle) during acclimation to high light. The abundance of psaA transcript relative to rnpB transcript was determined by real-time RT-PCR using cDNA generated from total RNA extracts from cells collected at various time points after the shift to high light.

The growth rate of the wild type and the 0205-79 mutant was not significantly different under low-light condition (data not shown). Under the light of photon flux density at 300 µmol m^–2 s^–1, the growth of the 0205-79 mutant was better than that of the wild type during the first 24 h (Fig. 6) presumably because of the higher amounts of PSI in the mutant. The wild-type cells could continue to grow in the second and third days under the light of photon flux density at 300 µmol m^–2 s^–1, but the growth of the 0205-79 mutant was severely suppressed, implying that the failure to decrease PSI contents might result in the photodamage under prolonged high-light condition. This phenotype is very similar to that of the pmgA mutant, which was also reported to have a defect in the regulation of photosystem stoichiometry in acclimation to high light (Hihara et al., 1998; Sonoike et al., 2001).

View larger version (13K): [in this window] [in a new window]   Figure 6. Growth curve of the wild type (black circle) and the 0205-79 mutant (white circle) under photon flux density at 300 µmol m–2 s–1. Cells grown at 20 µmol m–2 s–1 were shifted to 300 µmol m–2 s–1 at time 0 and inoculated every 24 h in order to prevent the self-shading effect.

We determined the insertion site of a chloramphenicol-resistant cassette in the 0205-79 mutant by inverse PCR. The insertion site was 704 bp downstream from the start point of the open reading frame (ORF) of sll1961. In the course of screening for mutants that showed different fluorescence kinetics from that of the wild type, we isolated another mutant (the 0205-91 mutant) that showed the same phenotype in chlorophyll fluorescence kinetics as the 0205-79 mutant (data not shown). We confirmed that the 0205-91 mutant could not modulate photosystem stoichiometry in acclimation to high light by determination of fluorescence emission spectra at 77 K (data not shown). In the 0205-91 mutant, the insertion site of a chloramphenicol-resistant cassette was 714 bp downstream from the start point of the ORF of sll1961. The result suggests that the phenotype observed in the 0205-79 mutant is due to the disruption in the sll1961 gene. Furthermore, we created a new sll1961 mutant by the replacement of the 476th to 1,008th nucleotide sequence of the sll1961 gene with a spectinomycin-resistant cartridge (the sll1961-Sp^R mutant). Measurements of chlorophyll fluorescence emission spectra at 77 K confirmed that the sll1961-Sp^R mutant was unable to regulate photosystem stoichiometry in acclimation to high light (data not shown). These observations indicated that the disruption of sll1961 is really the cause of the observed phenotype.

The sll1961 gene contains an ORF of 1,032 bp, encoding a deduced protein of 343 amino acid residues. N-terminal region of Sll1961 contains a helix-turn-helix DNA-binding domain, suggesting that this protein may be a transcriptional regulator.

Search for the Putative Target Genes of Sll1961

Although Sll1961 has homology with the transcriptional regulator, the transcript level of psaA and/or psaAB was not apparently affected (Fig. 5). So, we compared genome-wide patterns of transcription between the wild type and the 0205-79 mutant (the sll1961 mutant) by DNA microarray analysis to identify the possible target genes of Sll1961. Genes whose expression level reproducibly increased or decreased in three independent experiments were defined as the genes that were affected by the disruption of the sll1961 gene. In low-light-acclimated cells, no significant differences of gene expression between the wild type and the sll1961 mutant were observed (data not shown). Under high-light condition, however, some differences were observed as presented in Figure 7. The expression of one gene, sll1773, was reproducibly higher in the sll1961 mutant than in the wild type after 3 h following the shift to high light (Table I). The expression of several genes such as slr0364, slr2076, and slr2057 was lower in the sll1961 mutant than in the wild type under high-light condition. The slr0364, slr2076, and slr2057 genes encode a hypothetical protein, 60-kD chaperonin GroEL, and water channel protein, respectively. Northern-blot analysis (Fig. 8) showed that the expression of sll1773 was very low either in the wild type or in the sll1961 mutant under low-light condition. The expression of sll1773 increased in the sll1961 mutant during 6 h after the shift to high light, while it did not change in the wild type. We also examined the reliability of microarray data of the slr0364, slr2076, and slr2057 genes by real-time RT-PCR. The transcript levels of slr0364, slr2076, and slr2057 were 0.39-, 0.47-, and 0.44-fold in the 0205-79 mutant relative to the wild type after 3 h in exposure to high light.

View larger version (26K): [in this window] [in a new window]   Figure 7. DNA microarray analysis of gene expression induced by the disruption of the sll1961 gene under high-light condition. Cells of the wild type and the 0205-79 mutant were incubated under high light for 3 h. RNA extracted from the wild-type and the 0205-79 mutant cells was used for synthesis of Cy3- and Cy5-labeled cDNAs, respectively.

View larger version (45K): [in this window] [in a new window]   Figure 8. Northern analysis of the level of mRNA of sll1773 in the wild type and in the 0205-79 mutant during acclimation to high light.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

DNA microarray analysis revealed that the expression of several genes under high-light condition was affected by the mutation of the sll1961 gene. The expression of sll1773 was significantly induced in the sll1961 mutant in exposure to high light for 3 h. The expression of the gene was very low in the wild type both under low- and high-light conditions (Fig. 8). The sll1773 gene encodes a pirin-like protein called PirA (Hihara et al., 2004). Pirin-like proteins are found in many different organisms ranging from Archaebacteria to mammals (Orzaez et al., 2001). In tomato (Lycopersicon esculentum), the gene expression of pirin was induced during apoptosis (Orzaez et al., 2001), while pirin in Arabidopsis (Arabidopsis thaliana; AtPirin) interacted with the -subunit of G protein, GPA1, and regulated seed germination and seedling development (Lapik and Kaufman, 2003). In the cyanobacterium Synechocystis sp. PCC 6803, the pirA (sll1773) gene is the only gene that encodes a pirin homolog, and its expression increased in the presence of NaCl, sorbitol, or ethanol at high concentration (Hihara et al., 2004).

Several explanations may be possible for the expression of pirA in the sll1961 mutant under high-light condition. Since the pirA gene is highly expressed under stressed condition in cyanobacteria (Hihara et al., 2004), its expression might be increased under severe stress condition caused by the combination of high light and disruption of the sll1961 gene. Secondly, PirA may have a role to suppress the decrease of PSI amounts under high-light condition. In the wild-type cells, Sll1961 may transcriptionally repress the expression of pirA under high-light condition and decrease the amounts of PSI. In the sll1961 mutant, Sll1961 cannot repress the expression of pirA and the decrease of PSI contents is suppressed by PirA. Since pirin is known to have a protein-protein interaction domain and regulate transcription (Dechend et al., 1999), PirA might interact with some proteins and inactivate transcription necessary for the modulation of photosystem stoichiometry. The exact role of PirA in cyanobacteria and its involvement in the modulation of photosystem stoichiometry should be examined in future.

Expression of three genes was suppressed in the sll1961 mutant compared with the wild type; slr0364, slr2076, and slr2057. The expression of slr0364 was most repressed in the sll1961 mutant compared with that in the wild type after 3 h of exposure to high light. The slr0364 gene encodes a hypothetical protein, which has a Thr-rich region. The gene seems to be a part of operon with at least the slr0366 gene whose product has also a Thr-rich region. It was reported that the expression of the slr0364 gene was induced at approximately 1 h after the shift to high light and remained continuously at high levels during acclimation to high light (Hihara et al., 2001). It is tempting to assume that Slr0364 has a role in the regulation of photosystem stoichiometry under high-light condition.

This study demonstrates that a transcriptional regulator Sll1961 is associated with modulation of photosystem stoichiometry in acclimation to high light. The results of genome-wide analysis of transcription between the wild type and the sll1961 mutant list up several candidates that may be related to the regulation of photosystem stoichiometry during acclimation to high light. The research to identify the role of these genes would be conducted in the near future to facilitate the understanding of the mechanism of regulating photosystem stoichiometry in exposure to high light.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Strains and Growth Conditions

Synechocystis sp. PCC 6803 wild-type and mutant strains were grown in BG-11 medium (Rippka et al., 1979) with 10 mM TES. Cells in liquid culture were grown at 30°C in 50 mL glass tubes and bubbled with air under continuous illumination provided by fluorescent lamps. Photon flux density at 20 and 200 µmol m^–2 s^–1 was regarded as low light and high light, respectively. For creating random mutants, genes in cosmid vectors were randomly disrupted in vitro by the insertion of a transposon that has a chloramphenicol-resistant cassette. The wild-type strains were then transformed by the mutated cosmid library. The cosmid libraries having random insertion are the kind gift from Dr. H. Fukuzawa in Kyoto University. The pmgA (sll1968) mutant was constructed by insertion of the spectinomycin-resistant cassette (Hihara and Ikeuchi, 1997). To generate the sll1961-Sp^R mutant, the 476th to 1,008th nucleotide sequence of the sll1961 gene was replaced with the spectinomycin-resistant cassette using HindIII and SalI sites of the sll1961 gene. The 0205-79 mutant and the 0205-91 mutant were maintained with 20 µg/mL chloramphenicol, while the sll1961-Sp^R mutant and the pmgA mutants were maintained with 20 µg/mL spectinomycin.

Monitoring of Chlorophyll Fluorescence Kinetics of Dark-Adapted Cyanobacterial Cells

Cyanobacterial cells on agar plates were dark adapted for 15 min. The plates were set in two-dimensional fluorescence imaging system (FluorCam, Photon Systems Instruments), and orange AL (160 µmol m^–2 s^–1) from light-emitting diodes was applied for 3 s to monitor the fluorescence kinetics. The fluorescence intensity was normalized with initial value at the start point of AL.

Yield of Chlorophyll Fluorescence Determined by PAM Fluorometer

Yield of chlorophyll fluorescence was determined by PAM fluorometer (PAM 101/102/103, Heinz Waltz). Cells in 3 mL liquid culture were dark adapted for 5 min and then the chlorophyll fluorescence was monitored (Sonoike et al., 2001). AL at 200 µmol m^–2 s^–1 was used both for low-light-acclimated cells and high-light-acclimated cells.

Fluorescence Emission Spectra at 77 K

Low temperature fluorescence emission spectra at 77 K were recorded using a custom-made apparatus (Sonoike and Terashima, 1994). Cells were collected and adjusted to 5 µg chlorophyll/mL in BG-11 medium. Pigments were excited with blue light passing through a filter (Corning CS 4–96). Before measurement, cells were dark adapted for more than 10 min at room temperature to eliminate the possible effects of state transition. Chlorophyll a concentrations were determined after extraction with 100% methanol (Grimme and Boardman, 1972).

Isolation of Thylakoid Membranes

Cells of Synechocystis sp. PCC 6803 were harvested and suspended in 0.4 M Suc, 10 mM NaCl, 1 mM CaCl₂, 0.2 mM phenylmethylsulfonyl fluoride, 5 mM benzamidine, and 50 mM MOPS, pH 7.0 (MOPS buffer; Sun et al., 1998). Cells were broken by a bead beater (model 1107900, Biospec Products) for three times of 30 s operation with 2 min intervals. Unbroken cells and debris were removed by low-speed centrifugation at 4,700g for 10 min. Thylakoid membranes were pelleted by centrifugation at 140,000g for 45 min. The thylakoid membranes were resuspended in the MOPS buffer.

Western Analysis

Thylakoid membranes were solubilized with 5% lithium dodecyl sulfate and 60 mM dithiothreitol for 1 h at room temperature, and subjected to SDS-PAGE. SDS gel electrophoresis was carried out using 16% to 22% polyacrylamide gel containing 7.5 M urea (Ikeuchi and Inoue, 1988). Total proteins, corresponding to the cells of 3 mL culture of OD₇₃₀ = 0.355, were loaded on each lane. Proteins were electroblotted onto poly(vinylidene difluoride) membranes (Immobilon, Millipore). The antiserum against PsaAB from Thermosynechococcus elongatus BP-1 (formerly Synechococcus elongatus BP-1) was kindly provided by Prof. Isao Enami (Tokyo Science University). Reaction with antiserum and immunodetection was performed according to Kashino et al. (1990).

Microarray Analysis

The DNA microarray analysis was performed using CyanoCHIP version 1.6 (TaKaRa) according to Hihara et al. (2001). Five micrograms of RNA was labeled using an RNA Fluorescence Labeling Core Kit (M-MLV version 2.0, TaKaRa).

Northern Analysis

Northern-blot analysis was performed according to Muramatsu and Hihara (2003).

cDNA Synthesis and Real-Time RT-PCR

RT-PCR was performed with an RT-PCR Core Kit (TaKaRa) for cDNA synthesis. Real-time RT-PCR amplifications of cDNA templates were carried out using a Smart Cycler II (Cepheid) with a SYBR Premix Taq Kit (TaKaRa). The psaA transcript accumulation was measured as the ratio of psaA RNA level to rnpB RNA level (internal control). PCR reactions were as follows: 95°C for 10 s, followed by 40 cycles at 95°C for 15 s, and 60°C for 20 s. The psaA primers are 5'-CCTTCGAGAAGTGGGGCAAGCCGGG-3' and 5'-CCACAGCGAGGTGCCCAAAGTGAGC-3'. The rnpB primers are 5'-CGCCCAGTGCGCGCGAGCGTGAGGA-3' and 5'-CCTCCGACCTTGCTTCCAACCGGGG-3'.

Identification of the Insertion Site of a Chloramphenicol-Resistant Cassette in the Mutants

Genome DNA of chloramphenicol-resistant mutant was digested with HhaI and incubated at 37°C for more than 3 h. Digested DNA fragments were self ligated using DNA Ligation Kit (Version 2, TaKaRa). To amplify the region flanking the inserted cassette, inverse PCR was performed using outward primers. First PCR was performed with primer AIB-A (5'-CAACAGTACTGCGATGAGTGGCAG-3') and AIB-B (5'-GGTAATACTAGTGTCGACCAACCAG-3'). Each sample was subjected to 35 cycles of PCR consisting of denaturation at 93°C for 30 s and annealing at 55°C for 1.5 min and elongation at 72°C for 1.5 min. The PCR product was directly used as a template of second PCR. Second PCR was performed with primer GPS21-1 (5'-CACAGCATAACTGGACTGATTTCAG-3') and GPS21-2 (5'-CGTATTAGCTTACGACGCTACACC-3'). Second PCR was carried out under the same condition as the first one.

Sequence of the amplified flanking region was determined by the dye-terminator fluorescence detection method, using a model 310 sequence analyzer (Applied Biosystems). For sequencing, PCR amplification followed by cycle sequencing with one of the primers, GPS21-1 or GPS21-2, was performed using Thermo Sequence II dye terminator cycle sequencing Kit (Amersham Pharmacia).

	ACKNOWLEDGMENTS

 
We thank Prof. Isao Enami for providing us with antiserum for PsaA/B. We are also grateful to Prof. Hideya Fukuzawa for providing cosmid libraries.

Received April 26, 2005; returned for revision June 9, 2005; accepted June 10, 2005.

	FOOTNOTES

 
¹ This work was supported by the Japan Society for the Promotion of Science (Grant-in-Aid for Scientific Research [B] no. 14340250) and the Ministry of Education, Science, Sports and Culture (Grant-in-Aid for Scientific Research on Priority Area "Genome Biology" no. 15013214).

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.064782.

^* Corresponding author; e-mail sonoike{at}k.u-tokyo.ac.jp; fax 81–4–7136–3651.

	LITERATURE CITED

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Anderson JM (1986) Photoregulation of the composition, function, and structure of thylakoid membranes. Annu Rev Plant Physiol 37: 93–136[CrossRef][ISI]

Barber J (2002) Photosystem II: a multisubunit membrane protein that oxidizes water. Curr Opin Struct Biol 12: 523–530[CrossRef][ISI][Medline]

Dechend R, Hirano F, Lehmann K, Heissmeyer V, Ansieau S, Wulczyn FG, Scheidereit C, Leutz A (1999) The Bcl-3 oncoprotein acts as a bridging factor between NF-B/Rel and nuclear co-regulators. Oncogene 18: 3316–3323[CrossRef][ISI][Medline]

Diner BA, Rappaport F (2002) Structure, dynamics, and energetics of the primary photochemistry of photosystem II of oxygenic photosynthesis. Annu Rev Plant Biol 53: 551–580[CrossRef][Medline]

Fromme P, Jordan P, Krauss N (2001) Structure of photosystem I. Biochim Biophys Acta 1507: 5–31[Medline]

Fujita Y (1991) Regulation of stoichiometry among thylakoid components in response to light regime: a story of the dynamic nature of the thylakoid system in cyanophytes. Bot Mag Tokyo (Special Issue) 2: 29–42

Fujita Y, Murakami A (1987) Regulation of electron transport composition in cyanobacterial photosynthetic system: stoichiometry among photosystem I and II complexes and their light-harvesting antennae and cytochrome b6/f complex. Plant Cell Physiol 28: 1547–1553[Abstract/Free Full Text]

Fujita Y, Murakami A, Ohki K (1987) Regulation of photosystem composition in the cyanobacterial photosynthetic system: the regulation occurs in response to the redox state of the electron pool located between the two photosystems. Plant Cell Physiol 28: 283–292[Abstract/Free Full Text]

Fujita Y, Murakami A, Ohki K, Hagiwara N (1988) Regulation of photosystem composition in cyanobacterial photosynthetic system: evidence indicating that photosystem I formation is controlled in response to the electron transport state. Plant Cell Physiol 29: 557–564[Abstract/Free Full Text]

Fujita Y, Ohki K, Murakami A (1985) Chromatic regulation of photosystem composition in the photosynthetic system red and blue-green algae. Plant Cell Physiol 26: 1541–1548[Abstract/Free Full Text]

Glazer AN, Melis A (1987) Photochemical reaction centers: structure, organization, and function. Annu Rev Plant Physiol 38: 11–45

Glick RE, McCauley SW, Grussem W, Melis A (1986) Light quality regulates expression of chloroplast genes and assembly of photosynthetic membrane complexes. Proc Natl Acad Sci USA 83: 4287–4291[Abstract/Free Full Text]

Grimme LH, Boardman NK (1972) Photochemical activities of a particle fraction P1 obtained from green-alga Chlorella fusca. Biochem Biophys Res Commun 49: 1617–1623[CrossRef][Medline]

Hihara Y (1999) The molecular mechanism for acclimation to high light in cyanobacteria. Curr Top Plant Biol 1: 37–50

Hihara Y, Ikeuchi M (1997) Mutation in a novel gene required for photomixotrophic growth leads to enhanced photoautotrophic growth of Synechocystis sp. PCC 6803. Photosynth Res 53: 243–252[CrossRef]

Hihara Y, Kamei A, Kanehisa M, Kaplan A, Ikeuchi M (2001) DNA microarray analysis of cyanobacterial gene expression during acclimation to high light. Plant Cell 13: 793–806[Abstract/Free Full Text]

Hihara Y, Muramatsu M, Nakamura K, Sonoike K (2004) A cyanobacterial gene encoding an ortholog of Pirin is induced under stress conditions. FEBS Lett 574: 101–105[CrossRef][Medline]

Hihara Y, Sonoike K, Ikeuchi M (1998) A novel gene, pmgA, specifically regulates photosystem stoichiometry in the cyanobacterium Synechocystis species PCC 6803 in response to high light. Plant Physiol 117: 1205–1216[Abstract/Free Full Text]

Ikeuchi M, Inoue Y (1988) A new 4.8-kDa polypeptide intrinsic to the PSII reaction center, as revealed by modified SDS-PAGE with improved resolution of low-molecular weight proteins. Plant Cell Physiol 29: 1233–1239[Abstract/Free Full Text]

Kashino Y, Enami I, Satoh K, Katoh S (1990) Immunological cross-reactivity among corresponding proteins of photosystem I and II from widely divergent photosynthetic organisms. Plant Cell Physiol 31: 479–488[Abstract/Free Full Text]

Kawamura M, Mimuro M, Fujita Y (1979) Quantitative relationship between two reaction centers in the photosynthetic system of blue-green algae. Plant Cell Physiol 20: 697–705[Abstract/Free Full Text]

Kufryk GI, Vermaas WF (2003) Slr2013 is a novel protein regulating functional assembly of photosystem II in Synechocystis sp. strain PCC 6803. J Bacteriol 185: 6615–6623[Abstract/Free Full Text]

Lapik YR, Kaufman LS (2003) The Arabidopsis cupin domain protein AtPirin interacts with the G protein -subunit GPA1 and regulates seed germination and early seedling development. Plant Cell 15: 1578–1590[Abstract/Free Full Text]

Mann NH, Novac N, Mullineaux CW, Newman J, Bailey S, Robinson C (2000) Involvement of an FtsH homologue in the assembly of functional photosystem I in the cyanobacterium Synechocystis sp. PCC 6803. FEBS Lett 479: 72–77[CrossRef][ISI][Medline]

Melis A, Brown JS (1980) Stoichiometry of system I and system II reaction centers and of plastoquinone in different photosynthetic membranes. Proc Natl Acad Sci USA 77: 4712–4716[Abstract/Free Full Text]

Murakami A, Fujita Y (1991) Regulation of photosystem stoichiometry in the photosynthetic system of the cyanophyte Synechocystis PCC 6714 in response to light intensity. Plant Cell Physiol 32: 223–230[Abstract/Free Full Text]

Muramatsu M, Hihara Y (2003) Transcriptional regulation of genes encoding subunits of photosystem I during acclimation to high-light conditions in Synechocystis sp. PCC 6803. Planta 216: 446–453[ISI][Medline]

Orzaez D, de Jong AJ, Woltering EJ (2001) A tomato homologue of the human protein PIRIN is induced during programmed cell death. Plant Mol Biol 46: 459–468[CrossRef][ISI][Medline]

Rippka R, Deruelles J, Waterbury JB, Herdman M, Stanier RY (1979) Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111: 1–61

Shen G, Zhao O, Reimer SK, Antonkine ML, Cai Q, Weiland SM, Golbeck JH, Bryant DH (2002) Assembly of photosystem I: inactivation of the rubA gene encoding a membrane-associated rubredoxin in the cyanobacterium Synechococcus sp. PCC 7002 causes a loss of photosystem I activity. J Biol Chem 277: 20343–20354[Abstract/Free Full Text]

Sonoike K, Hihara Y, Ikeuchi M (2001) Physiological significance of the regulation of photosystem stoichiometry upon high light acclamation of Synechocystis sp. PCC 6803. Plant Cell Physiol 42: 379–384[Abstract/Free Full Text]

Sonoike K, Terashima I (1994) Mechanism of photosystem-I photoinhibition in leaves of Cucumis sativus L. Planta 194: 287–293[CrossRef]

Sun J, Ke A, Jin P, Chitnis VP, Chitnis PR (1998) Isolation and functional study of photosystem I subunits in the cyanobacterium Synechocystis sp. PCC 6803. Methods Enzymol 297: 124–139[ISI][Medline]

Wang T, Shen G, Balasubramanian R, Mclntosh L, Bryant DA, Golbeck JH (2004) The surf gene (sll0088 in Synechocystis sp. strain PCC 6803) functions as a repressor of the sufBCDS operon in iron-sulfur cluster biogenesis in cyanobacteria. J Bacteriol 186: 956–967[Abstract/Free Full Text]

Wilde A, Hartel H, Hubschmann T, Hoffmann P, Schestakov SV, Bomer T (1995) Inactivation of a Synechocystis sp. Strain PCC 6803 gene with homology to conserved chloroplast open reading frame 184 increases the photosystem II-to-photosystem I ratio. Plant Cell 7: 649–658[Abstract]

Wilde A, Lunser K, Ossenbuhl F, Nickelsen J, Bomer T (2001) Characterization of the cyanobacterial ycf37: mutation decreases the photosystem I content. Biochem J 357: 211–216[CrossRef][ISI][Medline]

Yu J, Shen G, Wang T, Bryant DA, Golbeck JH, Mclntosh L (2003) Suppressor mutations in the study of photosystem I biogenesis: sll0088 is a previously unidentified gene involved in reaction center accumulation in Synechocystis sp. strain PCC 6803. J Bacteriol 185: 3878–3887[Abstract/Free Full Text]

Related articles in Plant Physiol.:

On the Inside

Peter V. Minorsky
Plant Physiol. 2005 139: 1-2. [Full Text]  

This article has been cited by other articles:

				H. Ozaki, M. Ikeuchi, T. Ogawa, H. Fukuzawa, and K. Sonoike Large-Scale Analysis of Chlorophyll Fluorescence Kinetics in Synechocystis sp. PCC 6803: Identification of the Factors Involved in the Modulation of Photosystem Stoichiometry Plant Cell Physiol., March 1, 2007; 48(3): 451 - 458. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 139/1/408    most recent pp.105.064782v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in Plant Physiol. Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (1) Citing Articles via Google Scholar Google Scholar Articles by Fujimori, T. Articles by Sonoike, K. Search for Related Content PubMed PubMed Citation Articles by Fujimori, T. Articles by Sonoike, K. Agricola Articles by Fujimori, T. Articles by Sonoike, K.