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Environmental pH Affects Photoautotrophic Growth of Synechocystis sp. PCC 6803 Strains Carrying Mutations in the Lumenal Proteins of PSII

Environmental pH Affects Photoautotrophic Growth of Synechocystis sp. PCC 6803 Strains Carrying... Abstract Synechocystis sp. strain PCC 6803 grows photoautotrophically across a broad pH range, but wild-type cultures reach a higher density at elevated pH; however, photoheterotrophic growth is similar at high and neutral pH. A number of PSII mutants each lacking at least one lumenal extrinsic protein, and carrying a second PSII lumenal mutation, are able to grow photoautotrophically in BG-11 medium at pH 10.0, but not pH 7.5. We investigated the basis of this pH effect and observed no pH-specific change in variable fluorescence yield from PSII centers of the wild type or the pH-dependent ΔPsbO:ΔPsbU and ΔPsbV:ΔCyanoQ strains; however, 77 K fluorescence emission spectra indicated increased coupling of the phycobilisome (PBS) antenna at pH 10.0 in all mutants. DNA microarray data showed a cell-wide response to transfer from pH 10.0 to pH 7.5, including decreased mRNA levels of a number of oxidative stress-responsive transcripts. We hypothesize that this transcriptional response led to increased tolerance against reactive oxygen species and in particular singlet oxygen. This response enabled photoautotrophic growth of the PSII mutants at pH 10.0. This hypothesis was supported by increased resistance of all strains to rose bengal at pH 10.0 compared with pH 7.5. Introduction Cyanobacteria are known to be alkaliphilic microbes (Pikuta et al. 2007), although many strains are able to grow over a wide pH range. Nonetheless, a large body of research with model organisms, such as Synechocystis sp. strain PCC 6803 (hereafter Synechocystis 6803), has been conducted in buffered growth media typically around pH 7.5, which might not represent the most natural condition. Unexpectedly, several PSII-specific mutations introduced into Synechocystis 6803 were found to create strains unable to grow at pH 7.5, whereas photoautotrophic growth could be restored in these mutants at pH 10.0 (Eaton-Rye et al. 2003, Summerfield et al. 2005). The pH-sensitive strains all carried mutations targeting the lumenal proteins of PSII; these include two double mutants, ΔPsbO:ΔPsbU and ΔPsbV:ΔCyanoQ, and a third strain lacking PsbV and carrying a short segment deletion between Arg384 and Val392 in CP47 (Eaton-Rye et al. 2003, Summerfield et al. 2005). Additionally, mutation of the CP47 residue Glu364 to glutamine combined with the removal of PsbV created a pH-sensitive strain (Eaton-Rye et al. 2003). PSII is the light-driven water-plastoquinone oxidoreductase of oxygenic photosynthesis (Wydrzynski and Satoh 2005, Renger 2012). The photosystem includes a core complex of the PsbA (D1) and PsbD (D2) polypeptides that bind the P680 reaction center Chls, the Mn4CaO5 oxygen-evolving complex (OEC) and the majority of the remaining redox cofactors (Nixon et al. 2005, Kawakami et al. 2011, Müh et al. 2012). The PSII core is flanked by PsbB (CP47) and PsbC (CP43), which are Chl-binding proteins that serve as a proximal antenna transferring excitation energy from the peripheral phycobilisome (PBS) antenna to P680 (Eaton-Rye and Putnam-Evans 2005, Mullineaux 2008). Hydrophilic domains of CP47 and CP43, together with the extrinsic proteins, provide a cap over the OEC (Bricker et al. 2012). In cyanobacteria, additional lipoproteins are associated with the lumenal face of PSII; however, these proteins are not present in the available PSII crystal structures from either Thermosynechococcus elongatus or T. vulcanus (Fagerlund and Eaton-Rye 2011). The lumenal PSII extrinsic proteins play a role in protecting the OEC from bulk reductants and contribute to putative channels associated with the access of substrate H2O and the egress of O2 and H+ (Gabdulkhakov et al. 2009, Umena et al. 2011). The X-ray crystallographic structures from T. elongatus and T. vulcanus have confirmed the presence of PsbO, PsbU and PsbV as hydrophilic lumenal extrinsic proteins (Ferreria et al. 2004, Loll et al. 2005, Umena et al. 2011). In addition, the lipoprotein CyanoQ has been shown to be associated with highly active PSII preparations from Synechocystis 6803 (Roose et al. 2007). In cyanobacteria, the PsbO protein contributes to the stability of the PSII dimeric supercomplex through interactions with loop A and loop E of CP47 (De Las Rivas and Barber 2004, Sakurai et al. 2007, Bentley and Eaton-Rye 2008). Removal of PsbO increases the sensitivity of PSII to photoinactivation and results in a strict dependence on Ca2+ and Cl− for photoautotrophic growth (Bockholt et al. 1991, Burnap and Sherman 1991, Mayes et al. 1991, Philbrick et al. 1991, Burnap et al. 1996). A similar light sensitivity and a dependence on Ca2+ and Cl− for photoautotrophy has been observed when the psbV gene is deleted (Shen et al. 1995, Morgan et al. 1998, Shen et al. 1998). Susceptibility to photoinactivation is also observed when the PsbU subunit is removed (Clarke and Eaton-Rye 1999, Inoue-Kashino et al. 2005, Abasova et al. 2011). Removal of PsbU results in slower photoautotrophic growth in the absence of Ca2+ and Cl− (Shen et al. 1997); and, in the absence of both ions, the ΔPsbU mutant is not photoautotrophic (Inoue-Kashino et al. 2005, Summerfield et al. 2005). In Synechocystis 6803, the absence of PsbU affected both energy transfer and electron transfer in the PBS–PSII complex (Veerman et al. 2005). In addition, in the Synechococcus sp. PCC 7942 mutant lacking PsbU, cells exhibited an elevated resistance to oxidative stress (Balint et al. 2006, Abasova et al. 2011). Furthermore, the PsbO, PsbU and PsbV proteins are required for the thermal stability of PSII and for the development of cellular thermotolerance (Nishiyama et al. 1999, Kimura et al. 2002). It is therefore apparent that the extrinsic proteins enable cyanobacteria to adjust to a number of variable environmental parameters. Transfer of wild-type Synechocystis 6803 from pH 7.5 to pH 10.0 increased mRNA levels of genes encoding several low molecular weight intrinsic subunits as well as extrinsic proteins of PSII, including psbO and psbU (Summerfield and Sherman 2008). This was part of a cell-wide response that included mechanisms to maintain pH homeostasis in the cytosol such as the presence of monovalent cation/proton antiporters (Padan et al. 2005). Cyanobacteria exhibit additional complexity, compared with non-photosynthetic organisms, in maintaining a pH in the thylakoid lumen that is approximately 2 pH units more acid than the cytosol. Moreover, cyanobacterial cytoplasmic and lumen pHs are altered by the environmental pH, with an increase of 2 pH units in the external pH resulting in an internal increase of approximately 0.2 pH units (Belkin and Packer 1988, Ritchie, 1991). It appears that the external pH is able to influence the growth of mutants with an altered complement of PSII extrinsic proteins, even though these proteins are located in the acidic thylakoid lumen. To investigate how the different combinations of the extrinsic proteins influence the ability of Synechocystis 6803 cells to adapt to different pHs, we have investigated PSII activity in vivo using non-invasive measurements of variable Chl fluorescence yield. We have combined these PSII activity measurements with pH-dependent changes in global gene expression in the wild type and the pH-sensitive ΔPsbO:ΔPsbU mutant to investigate how modification of PSII activity by the presence or absence of specific lumenal extrinsic proteins can influence gene expression and facilitate photoautotrophic growth. Results Photoautotrophic but not photoheterotrophic growth is increased in growth medium buffered at pH 10.0 compared with pH 7.5 It has previously been observed that wild-type cultures grown at pH 10.0 and pH 7.5 have similar initial doubling times, oxygen evolution rates and number of assembled PSII centers (Eaton-Rye et al. 2003). However, following 100 h of photoautotrophic growth, wild-type cultures reached an OD730 nm of ∼3 in pH 10.0 medium, compared with an OD730 nm of <2 in medium buffered at pH 7.5 (Fig. 1A). In contrast, photoheterotrophic growth (in the presence of 20 µM atrazine and 5 mM glucose) of the wild type was similar in BG-11 buffered at either pH 10.0 or pH 7.5 (Fig. 1A). Furthermore, a strain lacking both the PsbO and PsbU proteins grew photoautotrophically at pH 10.0 but not at pH 7.5 (Eaton-Rye et al. 2003), whereas photoheterotrophic growth was similar at pH 7.5 and pH 10.0 (Fig. 1B). These growth characteristics indicate that the pH of the growth medium has a PSII-specific effect; this is supported by observations of additional PSII mutants that exhibit photoautotrophic growth at pH 10.0 but not pH 7.5 (Eaton-Rye et al. 2003, Summerfield et al. 2005). To investigate the impact of extracellular pH on PSII activity and assembly, variable Chl a fluorescence yield and 77 K fluorescence emission spectra were measured. Fig. 1 View largeDownload slide Growth of Synechocystis sp. PCC 6803 strains in BG-11 medium as measured by the optical density at 730 nm. (A) Wild type: photoautotrophic growth in media buffered at pH 7.5 (open circles) and in media buffered at pH 10.0 (filled circles); photoheterotrophic growth in the presence of 20 µM atrazine and 5 mM glucose in media buffered at pH 7.5 (open squares) and media buffered at pH 10.0 (filled squares). (B) ΔPsbO:ΔPsbU mutant: photoautotrophic growth in media buffered at pH 7.5 (open triangles) and in media buffered at pH 10.0 (filled triangles); photoheterotrophic growth in the presence of 20 µM atrazine and 5 mM glucose in media buffered at pH 7.5 (open diamonds) and media buffered at pH 10.0 (filled diamonds). In A and B, the data are the average ± SE of three independent experiments. Error bars not visible are smaller than the symbols. Fig. 1 View largeDownload slide Growth of Synechocystis sp. PCC 6803 strains in BG-11 medium as measured by the optical density at 730 nm. (A) Wild type: photoautotrophic growth in media buffered at pH 7.5 (open circles) and in media buffered at pH 10.0 (filled circles); photoheterotrophic growth in the presence of 20 µM atrazine and 5 mM glucose in media buffered at pH 7.5 (open squares) and media buffered at pH 10.0 (filled squares). (B) ΔPsbO:ΔPsbU mutant: photoautotrophic growth in media buffered at pH 7.5 (open triangles) and in media buffered at pH 10.0 (filled triangles); photoheterotrophic growth in the presence of 20 µM atrazine and 5 mM glucose in media buffered at pH 7.5 (open diamonds) and media buffered at pH 10.0 (filled diamonds). In A and B, the data are the average ± SE of three independent experiments. Error bars not visible are smaller than the symbols. Absence of extrinsic proteins of PSII alters Chl a variable fluorescence from PSII in strains grown at both pH 7.5 and pH 10.0 Chl a variable fluorescence arises from PSII and can be induced by actinic light applied to dark-adapted cells (Papageorgiou et al. 2007, Stamatakis et al. 2007, Kaňa et al. 2012). Fluorescence induction for the wild type and mutants lacking extrinsic proteins grown at pH 7.5 and pH 10.0 is presented in Fig. 2. The control strain exhibited a fluorescence induction curve typical of Synechocystis 6803 cells, with a pronounced O to J rise reflecting the photochemical reduction of QA to form QA−, followed by a slow J to I thermal phase on a millisecond time scale before undergoing a large I to P rise at around 0.5 to 1 s (Kaňa et al. 2012) (Fig. 2A). Removal of PsbU resulted in a reduction of the O to J rise, but a substantial I to P rise remained; in contrast, removal of PsbO resulted in a reduced J level and prevented any I to P rise (Fig. 2A). The ΔPsbO:ΔPsbU strain and a ΔPsbO:ΔPsbU pseudorevertant strain, that grew photoautotrophically at pH 7.5 (Summerfield et al. 2007), exhibited fluorescence induction curves similar to that observed with ΔPsbO cells (Fig. 2C). For each of the five strains, the fluorescence induction curves of cells grown at pH 10.0 and pH 7.5 were similar, except for a slight increase in the O to J rise at pH 10.0 (Fig. 2A–D). Fig. 2 View largeDownload slide Fluorescence induction kinetics of Synechocystis sp. PCC 6803 strains grown in BG-11 medium buffered at pH 7.5 (open symbols) or pH 10.0 (filled symbols). Chl a fluorescence was induced with a 455 nm, 2,800 µmol photons m−2 s−1 actinic light and probed using a non-actinic measuring light of the same wavelength. (A–D) Control (squares); ΔPsbO (circles); ΔPsbU (triangles); ΔPsbO:ΔPsbU (diamonds); ΔPsbO:ΔPsbU pseudorevertant (inverted triangles). (E and F) Wild type (squares); ΔCyanoQ (triangles); ΔPsbV (circles); ΔCyanoQ:ΔPsbV (diamonds). Traces were normalized to (F – Fo)/Fo. Fig. 2 View largeDownload slide Fluorescence induction kinetics of Synechocystis sp. PCC 6803 strains grown in BG-11 medium buffered at pH 7.5 (open symbols) or pH 10.0 (filled symbols). Chl a fluorescence was induced with a 455 nm, 2,800 µmol photons m−2 s−1 actinic light and probed using a non-actinic measuring light of the same wavelength. (A–D) Control (squares); ΔPsbO (circles); ΔPsbU (triangles); ΔPsbO:ΔPsbU (diamonds); ΔPsbO:ΔPsbU pseudorevertant (inverted triangles). (E and F) Wild type (squares); ΔCyanoQ (triangles); ΔPsbV (circles); ΔCyanoQ:ΔPsbV (diamonds). Traces were normalized to (F – Fo)/Fo. Fluorescence induction was investigated in the pH-sensitive ΔPsbV:ΔCyanoQ mutant (Fig. 2E, F) as this strain exhibited a similar phenotype to theΔPsbO:ΔPsbU strain (Summerfield et al. 2005). The wild type and the ΔCyanoQ strain possessed a similar O–J–I–P transient, whereas the ΔPsbV and ΔPsbV:ΔCyanoQ strains exhibited suppressed fluorescence induction. The results in Fig. 2 indicate that the absence of an O–J–I–P transient does not preclude photoautotrophic growth, and the ability of the ΔPsbO:ΔPsbU or ΔPsbV:ΔCyanoQ strains to grow at pH 10.0 was not accompanied by restoration of an I to P rise. Since in all strains the O to J rise was slightly enhanced at pH 10.0, and this might correlate with an increased absorption cross-section for energy transfer to PSII from the peripheral antenna or PBS, energy transfer was investigated using 77 K fluorescence emission spectra. Growth medium pH alters 77 K fluorescence emission spectra of PSII mutants We measured 77 K fluorescence emission using 580 nm excitation of the PBS (Fig. 3). Compared with the control strain, the ΔPsbU and ΔPsbO strains had increased emission at 685 nm, suggesting an enhanced emission from the terminal emitter of the PBS in cultures grown at pH 7.5 (Fig. 3A). Increased fluorescence yield was previously reported for the ΔPsbU strain grown in unbuffered BG-11 (Veerman et al. 2005). Emission at 695 nm from the CP47 Chl a core antenna was not increased in either the ΔPsbU or ΔPsbO strains, supporting the interpretation that the increased fluorescence results from the terminal emitters of the PBS and not the CP43 Chl a core antenna which also emits at 685 nm. The ΔPsbO:ΔPsbU strain had an emission spectrum similar to that of the ΔPsbO strain, whereas the ΔPsbO:ΔPsbU pseudorevertant had decreased emission at ∼685 nm compared with the ΔPsbO and ΔPsbO:ΔPsbU strains, but this was still increased compared with the wild type (Fig. 3C). Notably, the fluorescence emission at 685 nm was markedly decreased in strains lacking PsbO and PsbU when grown at pH 10.0 (Fig. 3B, D). Fig. 3 View largeDownload slide 77 K fluorescence emission spectra of Synechocystis sp. PCC 6803 strains grown in BG-11 medium buffered at pH 7.5 (A, C) or pH 10.0 (B, D). Spectra were collected using excitation at 580 nm and normalized to a PSI emission peak at 725 nm. (A and B) Control (black); ΔPsbO (red); ΔPsbU (green). (C and D) ΔPsbO:ΔPsbU (blue); ΔPsbO:ΔPsbU pseudorevertant (green). (E and F) Time course following transition from BG-11 medium at pH 7.5 to pH 10.0 (E) and from BG-11 medium at pH 10.0 to pH 7.5 (F) in a ΔPsbO:ΔPsbU strain, pre-transfer (black); 2 h after transfer (blue); 6 h after transfer (green); and 12 h after transfer (red). The pre-transfer (black line) and 2 h after transfer (blue line) values are almost overlapping in E and F. Fig. 3 View largeDownload slide 77 K fluorescence emission spectra of Synechocystis sp. PCC 6803 strains grown in BG-11 medium buffered at pH 7.5 (A, C) or pH 10.0 (B, D). Spectra were collected using excitation at 580 nm and normalized to a PSI emission peak at 725 nm. (A and B) Control (black); ΔPsbO (red); ΔPsbU (green). (C and D) ΔPsbO:ΔPsbU (blue); ΔPsbO:ΔPsbU pseudorevertant (green). (E and F) Time course following transition from BG-11 medium at pH 7.5 to pH 10.0 (E) and from BG-11 medium at pH 10.0 to pH 7.5 (F) in a ΔPsbO:ΔPsbU strain, pre-transfer (black); 2 h after transfer (blue); 6 h after transfer (green); and 12 h after transfer (red). The pre-transfer (black line) and 2 h after transfer (blue line) values are almost overlapping in E and F. At pH 7.5, compared with the wild type, the ΔCyanoQ strain showed slightly increased emission at 648, 665 and 685 nm corresponding to phycocyanin, allophycocyanin and the terminal emitter of the PBS, respectively (Supplementary Fig. S1A). The ΔPsbV and ΔPsbV:ΔCyanoQ strains exhibited an increased emission at 685 nm when grown at pH 7.5 (Supplementary Fig. S1A), and this was reversed when these strains were grown at pH 10.0 (Supplementary Fig. S1B), similar to the changes observed in the 77 K fluorescence emission spectra of the ΔPsbU, ΔPsbO and ΔPsbO:ΔPsbU strains. Confirmation that the elevated fluorescence resulted from excitation of the PBS and not the core antenna pigments was obtained by measuring the Fo fluorescence level when probed with a red light (625 nm) compared with blue light (455 nm) at pH 7.5. The Fo fluorescence emission was elevated following excitation with red light which excited the PBS compared with when blue light (455 nm) was used to excite the core antenna pigments directly (Supplementary Fig. S1C). At pH 10.0, the Fo fluorescence levels probed with red light or blue light were similar (Supplementary Fig. S1D), consistent with quenching of fluorescence from the PBS at pH 10.0. In Synechocystis 6803, non-photochemical quenching (NPQ) involves interaction of the orange carotenoid protein (OCP) with the PBS to increase energy dissipation, therefore decreasing the amount of energy arriving at PSII (Wilson et al. 2006). To assess whether the decreased fluorescence at pH 10.0 was due to increased NPQ, we induced NPQ in the wild type and ΔPsbO:ΔPsbU strains grown at both pH 7.5 and pH 10.0. Induction of NPQ using blue light resulted in a small decrease in fluorescence at 648, 665 and 685 nm, but did not substantially reduce the elevated fluorescence at 685 nm in the ΔPsbO:ΔPsbU strain grown at pH 7.5 (Supplementary Fig. S2A, B). This demonstrates that NPQ is not the cause of fluorescence quenching in the PSII mutant strains at pH 10.0 and is consistent with increased coupling of the PBS to PSII at pH 10.0. Our measurements of 77 K fluorescence emission, using excitation at 580 nm, in ΔPsbO:ΔPsbU cells transferred from pH 7.5 to pH 10.0 also showed that decreased fluorescence did not occur until several hours after transfer to elevated pH (Fig. 3E). In addition, cells grown at pH 10.0 and transferred to pH 7.5 showed no increase in fluorescence at 685 nm even 12 h after transfer to the lower pH (Fig. 3F). The kinetics of these changes in fluorescence emission suggest that the altered coupling of the PBS to PSII was not a primary response to the change in pH of the growth medium. Furthermore, the pH-dependent coupling of the PBS by itself does not explain the photoautotrophic growth of the ΔPsbO:ΔPsbU and ΔPsbV:ΔPsbQ strains at pH 10.0, but not pH 7.5, as the ΔPsbO, ΔPsbU and ΔPsbV strains showed similar increased fluorescence emission at pH 7.5 and grew photoautotrophically at this pH (Supplementary Fig S1A). To identify mechanisms involved in recovery of photoautotrophic growth of pH-sensitive mutants at pH 10.0, we investigated the cell-wide response to altered growth medium pH by examining the transcriptional response following the transition from pH 10.0 to pH 7.5 in the wild type and the ΔPsbO:ΔPsbU strain. Global transcriptional response to transition from pH 10.0 to pH 7.5 in the wild type and ΔPsbO:ΔPsbU strain Synechocystis 6803 cultures were grown in continuous light in BG-11 medium buffered at pH 10.0. Samples were taken for RNA isolation at 0 h (t0), and 2 h (t2) following transfer from pH 10.0 to pH 7.5. As indicated in the Materials and Methods, we considered genes to be differentially regulated if they showed a fold change of ≥1.5 with a false discovery rate (FDR) = 0.05. A similar number of genes met these criteria in the two strains; 467 and 413 genes in the wild type and ΔPsbO:ΔPsbU strain, respectively. Increased transcript abundance at pH 7.5 was observed for 209 and 242 genes for the wild type and ΔPsbO:ΔPsbU strain, respectively. Genes were divided into functional categories according to the Cyanobase designation (http://genome.microbedb.jp/cyanobase), and the number of differentially expressed genes in each category is shown in Table 1. Changes in transcript level, across a range of different functional categories, indicated a cell-wide response to external pH; categories containing large numbers of genes exhibiting differential transcript abundance included: photosynthesis and respiration (20 and 42 genes in the wild type and the ΔPsbO:ΔPsbU strain, respectively); transport and binding proteins (36 and 28 genes in the wild type and the ΔPsbO:ΔPsbU cells, respectively); regulatory functions (20 genes in both strains); and cellular processes (14 and 17 genes in the wild type and the ΔPsbO:ΔPsbU strain, respectively). Table 1 Functional categories of pH-responsive genesa in wild-type and ΔPsbO:ΔPsbU Synechocystis sp. PCC 6803 strains Gene category  No. of genesb  Differentially regulated genes       WT (Up)c  OU (Up)c  OU/WT pH 7.5d  OU/WT pH 10.0d  Amino acid biosynthesis  97  17 (10)  11 (6)  11 (4)d  5 (1)d  Biosynthesis of cofactors, prosthetic groups and carriers  124  13 (5)  15 (7)  13 (6)  8 (4)  Cell envelope  67  7 (6)  19 (19)  15 (15)  2 (1)  Cellular processes  76  14 (2)  17 (10)  11 (8)  9 (9)  Central intermediary metabolism  31  4 (3)  4 (3)  1 (1)  1 (0)  DNA replication, restriction, recombination and repair  60  5 (2)  6 (4)  4 (1)  3 (3)  Energy metabolism  132  19 (14)  17 (12)  12 (7)  7 (4)  Hypothetical  1076  169 (53)  133 (64)  92 (51)  70 (52)  Other categories  306  38 (22)  21 (9)  16 (7)  8 (3)  Photosynthesis and respiration  141  20 (13)  42 (37)  18 (14)  12 (11)  Purines, pyrimidines, nucleosides and nucleotides  41  5 (2)  3 (1)  2 (1)  1 (1)  Regulatory functions  146  20 (8)  20 (11)  9 (4)  6 (4)  Transcription  30  8 (6)  6 (3)  2 (0)  2 (2)  Translation  168  23 (15)  17 (12)  8 (3)  4 (3)  Transport and binding proteins  196  36 (27)  28 (13)  28 (9)  16 (4)  Unknown  474  69 (21)  54 (31)  37 (31)  34 (22)  Total number  3165b  467 (209)  413 (242)  279 (162)  188 (124)  Gene category  No. of genesb  Differentially regulated genes       WT (Up)c  OU (Up)c  OU/WT pH 7.5d  OU/WT pH 10.0d  Amino acid biosynthesis  97  17 (10)  11 (6)  11 (4)d  5 (1)d  Biosynthesis of cofactors, prosthetic groups and carriers  124  13 (5)  15 (7)  13 (6)  8 (4)  Cell envelope  67  7 (6)  19 (19)  15 (15)  2 (1)  Cellular processes  76  14 (2)  17 (10)  11 (8)  9 (9)  Central intermediary metabolism  31  4 (3)  4 (3)  1 (1)  1 (0)  DNA replication, restriction, recombination and repair  60  5 (2)  6 (4)  4 (1)  3 (3)  Energy metabolism  132  19 (14)  17 (12)  12 (7)  7 (4)  Hypothetical  1076  169 (53)  133 (64)  92 (51)  70 (52)  Other categories  306  38 (22)  21 (9)  16 (7)  8 (3)  Photosynthesis and respiration  141  20 (13)  42 (37)  18 (14)  12 (11)  Purines, pyrimidines, nucleosides and nucleotides  41  5 (2)  3 (1)  2 (1)  1 (1)  Regulatory functions  146  20 (8)  20 (11)  9 (4)  6 (4)  Transcription  30  8 (6)  6 (3)  2 (0)  2 (2)  Translation  168  23 (15)  17 (12)  8 (3)  4 (3)  Transport and binding proteins  196  36 (27)  28 (13)  28 (9)  16 (4)  Unknown  474  69 (21)  54 (31)  37 (31)  34 (22)  Total number  3165b  467 (209)  413 (242)  279 (162)  188 (124)  WT, wild type; OU, ΔPsbO:ΔPsbU mutant. a Genes were considered differentially regulated when fold change was >1.5 fold. b Total number of genes based on Kazusa annotation prior to May, 2002. c Number of genes with increased mRNA levels at pH 7.5 compared with pH 10.0 in a functional category. d Number of genes with increased mRNA levels in the mutant compared with the wild type in a functional category. View Large There were 198 genes up-regulated both on transfer from pH 7.5 to pH 10.0 and on transfer from pH 10.0 to pH 7.5: these had previously been identified and designated pH independent (Summerfield and Sherman 2008), and these genes are shown in Supplementary Table S1. The majority of these genes (157/198) had increased mRNA levels following transfer and represented functional categories including: translation (51 genes, 43 encoding ribosomal proteins); respiration and photosynthesis (23 genes, including genes encoding inducible bicarbonate transporters, carboxysome components and ATP synthase); biosynthesis of cofactors, prosthetic groups and carriers (11 genes); energy metabolism (10 genes); and hypothetical (22 genes). The majority of genes with decreased transcript abundance following transfer (34/41 genes) belonged to categories hypothetical, unknown or other. All except four of these genes were similarly regulated or unchanged in the ΔPsbO:ΔPsbU strain on transition from pH 10.0 to pH 7.5. Differences in the transcript level response to transfer from pH 10.0 to pH 7.5 in the wild type and the ΔPsbO:ΔPsbU strain Large numbers of genes involved in photosynthesis and respiration were increased in the ΔPsbO:ΔPsbU strain but not in the wild type at pH 7.5. These included genes encoding Cyt c oxidase (slr1136–slr1138); this is the major terminal oxidase in Synechocystis 6803 with a role in both the thylakoid and plasma membranes (Howitt and Vermaas 1998). This up-regulation probably reflects increased respiration in this strain due to the inability of the mutant to grow photoautotrophically at pH 7.5. In addition, genes encoding several components of the electron transport chain had increased transcript abundance in the ΔPsbO:ΔPsbU mutant at pH 7.5 compared with pH 10.0, but were unchanged in the wild type. This included transcripts encoding: PSI components (PsaL, PsaK1 and PsaE); core subunits of NADH dehydrogenase (NdhB and NdhD1); the Cyt b559 subunits together with other low molecular weight PSII proteins from the same operon (PsbEFLJ); and three genes (ssl0020, sll1584 and slr1828) encoding ferredoxin or ferredoxin-like proteins, including the most highly expressed ferredoxin gene, ssl0020, that is essential for viability (Poncelet et al. 1998) (Table 2). Ferredoxin is the final electron acceptor of the photosynthetic electron transport chain, interacting with both regulatory and metabolic polypeptides (Hanke et al. 2011). Table 2 Selected genesa showing altered transcript abundance on transition from pH 10.0 to pH 7.5 in Synechocystis sp. PCC 6803 wild type and a ΔPsbO:ΔPsbU strain No.  Gene Name  H2O2b  WTc  OUc  WT/OUd             pH 10.0  pH 7.5  Biosysthesis of cofactors, prosthetic groups      slr0233  trxQ  I  −2.3  −1.6  1.2  −1.2      slr0600  ntr  –  −1.5  −1.6  −1.3  −1.3      slr1562  grxX1  –  −1.5  −1.4  1.0  1.1  Cellular processes      sll0170  dnaK2  I  −2.1  −1.4  1.3  −1.1      sll0430  htpG  I  −2.3  −2.2  1.0  1.0      sll1514  hspA  I  −6.0  −1.7  1.6  −2.2      sll1933  dnaJ  –  −1.6  −1.1  1.4  1.1      sll0093  dnaJ  I  −1.7  −1.2  1.0  −1.3      sll0755  tpx  I  −1.3  −1.5  −1.2  1.0  Hypothetical      sll0939  hypo  I  −4.0  −2.9  1.0  −1.4      slr1128  hypo  –  −2.2  −1.5  1.0  −1.4      slr1963  ocp  I  −1.7  −1.1  1.8  1.2  Other      sll0550  flv3  I  −1.7  −1.3  1.2  −1.1      sll1621  ahpC  I  −1.1  −1.9  −1.4  −1.3      ssl2542  hliA  I  −1.4  −1.5  −1.6  −1.5      ssr2595  hliB  I  −1.7  −4.1  −2.6  −1.1  Photosynthesis and respiration   Respiration      slr1136  ctaD1  –  1.0  2.1  1.0  −2.0      slr1137  ctaC1  –  −1.1  1.5  1.2  −1.5      slr1138  ctaE1  –  1.0  1.6  1.0  −1.6   NADH dehydrogenase      sll0026  ndhF4  R  1.8  2.0  1.1  1.0      sll0027  ndhD4  R  2.1  1.8  1.2  1.4      sll0223  ndhB  –  −1.3  1.6  1.6  −1.3      slr0331  ndhD1  R  −1.5  2.9  2.4  −1.8      PSI                  slr1655  psaL  R  1.0  1.9  1.8  −1.1      ssr0390  psaK1  −  1.1  1.9  1.7  1.0      ssr2381  psaE  R  1.0  1.5  1.0  −1.6   PSII      sll0258  psbV  R  1.7  1.6  −1.1  1.0      sll1398  psb28  −  1.6  1.2  −1.2  1.1      slr1739  psb28-2  I  −3.2  −2.4  1.1  −1.2      Phycobilisomes                  sll1577  cpcB  –  1.2  1.7  1.4  1.0      sll1579  cpcC2  R  1.8  2.6  1.2  −1.1      sll1580  cpcC1  R  1.4  1.7  1.2  1.0      slr0335  apcE  R  1.0  2.1  2.0  −1.1      ssr3383  apcC  R  1.8  3.2  1.6  −1.2      slr1687  nblB  I  −2.3  −2.0  −1.4  −1.6      ssl0452  nblA1  I  −1.2  −1.5  −1.4  −1.1      ssl0453  nblA2  I  −2.1  −2.4  −1.1  1.0      Soluble electron carriers                  sll1584  fdl  –  1.3  1.7  1.1  −1.3      slr1828  petF  R  1.3  1.7  −1.4  −1.8      ssl0020  petF  –  1.1  2.1  1.6  −1.2  Regulatory functions      sll0797  rppA  –  1.5  1.5  −1.1  −1.1      sll0798  rppB  –  1.6  2.2  1.1  −1.2      sll1392  pfsR  –  −3.9  −3.3  1.0  −1.1      slr0311  hik29  –  1.5  2.0  1.0  −1.3      slr0533  hik10  –  1.5  2.2  1.0  −1.3      slr0947  rpaB  I  −1.7  −2.3  −1.2  −1.1      slr1285  hik34  I  −2.8  −1.7  1.2  −1.3      slr1529  ntrX  –  1.5  1.9  −1.1  −1.4  Transcription      sll0184  sigC  I  −1.9  −1.2  1.1  −1.4      sll0306  sigB  I  −2.5  −1.7  1.0  −1.5      sll1818  rpoA  –  2.9  1.4  −2.1  1.0      slr1129  rne  –  2.7  1.5  −1.4  1.3  Translation      sll0020  clpC  I  −2.0  −1.2  1.1  −1.5      slr0257  ctpB  –  −1.7  −1.2  1.3  −1.1      slr1641  clpB1  I  −1.6  −1.2  1.1  −1.2      slr1751  ctpC  –  −3.1  −1.3  1.9  −1.2  Gene clusters      sll0788  hypo  I  −4.0  −5.8  1.1  1.5      sll0789  rre34  I  −3.5  −11.5  −2.1  1.6      sll0790  hik31  R  −1.3  −12.5  −6.2  1.5      slr0074  ycf24  I  −2.0  −2.3  −1.1  1.0      slr0075  ycf16  I  −1.5  −2.1  1.0  1.4  No.  Gene Name  H2O2b  WTc  OUc  WT/OUd             pH 10.0  pH 7.5  Biosysthesis of cofactors, prosthetic groups      slr0233  trxQ  I  −2.3  −1.6  1.2  −1.2      slr0600  ntr  –  −1.5  −1.6  −1.3  −1.3      slr1562  grxX1  –  −1.5  −1.4  1.0  1.1  Cellular processes      sll0170  dnaK2  I  −2.1  −1.4  1.3  −1.1      sll0430  htpG  I  −2.3  −2.2  1.0  1.0      sll1514  hspA  I  −6.0  −1.7  1.6  −2.2      sll1933  dnaJ  –  −1.6  −1.1  1.4  1.1      sll0093  dnaJ  I  −1.7  −1.2  1.0  −1.3      sll0755  tpx  I  −1.3  −1.5  −1.2  1.0  Hypothetical      sll0939  hypo  I  −4.0  −2.9  1.0  −1.4      slr1128  hypo  –  −2.2  −1.5  1.0  −1.4      slr1963  ocp  I  −1.7  −1.1  1.8  1.2  Other      sll0550  flv3  I  −1.7  −1.3  1.2  −1.1      sll1621  ahpC  I  −1.1  −1.9  −1.4  −1.3      ssl2542  hliA  I  −1.4  −1.5  −1.6  −1.5      ssr2595  hliB  I  −1.7  −4.1  −2.6  −1.1  Photosynthesis and respiration   Respiration      slr1136  ctaD1  –  1.0  2.1  1.0  −2.0      slr1137  ctaC1  –  −1.1  1.5  1.2  −1.5      slr1138  ctaE1  –  1.0  1.6  1.0  −1.6   NADH dehydrogenase      sll0026  ndhF4  R  1.8  2.0  1.1  1.0      sll0027  ndhD4  R  2.1  1.8  1.2  1.4      sll0223  ndhB  –  −1.3  1.6  1.6  −1.3      slr0331  ndhD1  R  −1.5  2.9  2.4  −1.8      PSI                  slr1655  psaL  R  1.0  1.9  1.8  −1.1      ssr0390  psaK1  −  1.1  1.9  1.7  1.0      ssr2381  psaE  R  1.0  1.5  1.0  −1.6   PSII      sll0258  psbV  R  1.7  1.6  −1.1  1.0      sll1398  psb28  −  1.6  1.2  −1.2  1.1      slr1739  psb28-2  I  −3.2  −2.4  1.1  −1.2      Phycobilisomes                  sll1577  cpcB  –  1.2  1.7  1.4  1.0      sll1579  cpcC2  R  1.8  2.6  1.2  −1.1      sll1580  cpcC1  R  1.4  1.7  1.2  1.0      slr0335  apcE  R  1.0  2.1  2.0  −1.1      ssr3383  apcC  R  1.8  3.2  1.6  −1.2      slr1687  nblB  I  −2.3  −2.0  −1.4  −1.6      ssl0452  nblA1  I  −1.2  −1.5  −1.4  −1.1      ssl0453  nblA2  I  −2.1  −2.4  −1.1  1.0      Soluble electron carriers                  sll1584  fdl  –  1.3  1.7  1.1  −1.3      slr1828  petF  R  1.3  1.7  −1.4  −1.8      ssl0020  petF  –  1.1  2.1  1.6  −1.2  Regulatory functions      sll0797  rppA  –  1.5  1.5  −1.1  −1.1      sll0798  rppB  –  1.6  2.2  1.1  −1.2      sll1392  pfsR  –  −3.9  −3.3  1.0  −1.1      slr0311  hik29  –  1.5  2.0  1.0  −1.3      slr0533  hik10  –  1.5  2.2  1.0  −1.3      slr0947  rpaB  I  −1.7  −2.3  −1.2  −1.1      slr1285  hik34  I  −2.8  −1.7  1.2  −1.3      slr1529  ntrX  –  1.5  1.9  −1.1  −1.4  Transcription      sll0184  sigC  I  −1.9  −1.2  1.1  −1.4      sll0306  sigB  I  −2.5  −1.7  1.0  −1.5      sll1818  rpoA  –  2.9  1.4  −2.1  1.0      slr1129  rne  –  2.7  1.5  −1.4  1.3  Translation      sll0020  clpC  I  −2.0  −1.2  1.1  −1.5      slr0257  ctpB  –  −1.7  −1.2  1.3  −1.1      slr1641  clpB1  I  −1.6  −1.2  1.1  −1.2      slr1751  ctpC  –  −3.1  −1.3  1.9  −1.2  Gene clusters      sll0788  hypo  I  −4.0  −5.8  1.1  1.5      sll0789  rre34  I  −3.5  −11.5  −2.1  1.6      sll0790  hik31  R  −1.3  −12.5  −6.2  1.5      slr0074  ycf24  I  −2.0  −2.3  −1.1  1.0      slr0075  ycf16  I  −1.5  −2.1  1.0  1.4  WT, wild type; OU,ΔPsbO:ΔPsbU mutant. a Genes were considered differentially regulated when fold change was >1.5 fold. b Genes previously shown to have altered mRNA levels following exposure to H2O2 (Li et al. 2004, Kanesaki et al. 2007). R, repressed; I, induced. c Genes with altered mRNA levels at pH 7.5 vs. pH 10.0. d Genes with altered mRNA levels in the WT vs. OU. View Large Oxidative stress-responsive genes exhibit decreased transcript abundance in the wild type and the ΔPsbO:ΔPsbU strain on transfer to pH 7.5 Several genes with increased mRNA levels following exposure to hydrogen peroxide had decreased mRNA levels at pH 7.5; conversely, genes with decreased mRNA levels following hydrogen peroxide treatment were increased at pH 7.5 (Table 2). Genes that exhibited decreased transcript abundance in the wild type included dnaK2, dnaJ, hspA and htpG (Table 2) (cf. Li et al. 2004, Kanesaki et al. 2007). Two of these genes (htpG and hspA) had decreased mRNA levels in the mutant strain at pH 7.5. Increased transcript levels of these genes form part of a global response to numerous environmental factors, including oxidative, heat, UV light, high light and osmotic stresses (Hihara et al. 2001, Li et al. 2004, Singh et al. 2006, Rupprecht et al. 2007). Genes with roles in scavenging reactive oxygen species (ROS) exhibited decreased transcript levels at pH 7.5 compared with pH 10.0 in both strains. This included trxQ (slr0233) encoding one of the four thioredoxins in Synechocystis 6803 that is up-regulated in response to hydrogen peroxide (Perez-Perez et al. 2009a), and ntr encoding a putative NADP+ thioredoxin reductase (NTR); it has been suggested that NTR donates electrons to TrxQ (Perez-Perez et al. 2009a). Deletion of either trxQ or ntr increased sensitivity to oxidative stress (Hishiya et al. 2008, Perez-Perez et al. 2009a). One of the two genes encoding glutaredoxin (grx1, slr1562) showed decreased transcript abundance at pH 7.5 in the wild type: the Grx1 protein has been shown to accept electrons from NTR (Marteyn et al. 2009). In the ΔPsbO:ΔPsbU strain, the detoxification gene tpx (sll0755) encoding a peroxiredoxin shown to accept electrons from thioredoxin showed decreased mRNA levels at pH 7.5; this gene is also up-regulated under high light and heat exposure (Perez-Perez et al. 2009b). Similarly, the gene ahpC (sll1621) exhibited decreased mRNA levels at pH 7.5 in the ΔPsbO:ΔPsbU strain; this gene encodes a protein with sequence identity to type 2 peroxiredoxin-like hypothetical proteins, and may play a critical role in adapting to photooxidative stress (Kobayashi et al. 2004). Oxidative stress-responsive genes with roles in maintaining photosynthetic performance under stress conditions had decreased mRNA levels in both strains at pH 7.5. These included the hli genes encoding high-light-inducible polypeptides (HLIPs), also known as small CAB (Chl a/b-binding)-like proteins or SCPs (Dolganov et al. 1995, Funk et al. 1999), that are up-regulated under various stress conditions, and are thought to maintain photosynthetic performance by absorbing excess excitation energy or enabling the cells to cope with elevated ROS (He et al. 2001). Two of the four hli genes, hliA and hliB, had decreased mRNA levels at pH 7.5 and were up-regulated following hydrogen peroxide treatment. The HLIPs interact with Slr1128, a protein of unknown function (Wang et al. 2008): slr1128 transcript levels are decreased at pH 7.5 in both strains. The gene encoding OCP is induced following hydrogen peroxide treatment and has decreased mRNA levels in the wild type at pH 7.5. Several genes involved in photosynthesis that exhibited altered transcript levels following exposure to hydrogen peroxide were inversely affected by transfer to pH 7.5 (Table 2). In the wild type, decreased mRNA levels were observed for sll0550, encoding a flavoprotein (Flv3) involved in the Mehler reaction (Helman et al. 2003). The genes encoding NADH dehydrogenase subunits NdhD4 and NdhF4 had increased mRNA levels in both strains at pH 7.5; these were decreased following hydrogen peroxide treatment. These subunits are involved in CO2 uptake (Ogawa et al. 2000) and more recently they have been shown to be involved in PSI-mediated cyclic electron flow (Bernat et al. 2011). The gene encoding the PsbV PSII extrinsic protein had increased mRNA levels at pH 7.5 and decreased mRNA levels following hydrogen peroxide treatment, and the psb28-2 gene, which has similarity to psb28, had decreased mRNA levels at pH 7.5 and was up-regulated under stress including exposure to hydrogen peroxide, UV light and osmotic stress (see Table 2; Huang et al. 2002, Li et al. 2004, Paithoonrangsarid et al. 2004). Increased transcript levels of PBS structural genes (apcABC and cpcBA), and decreased transcription of genes nblA1, nblA2 and nblB, that encode proteins involved in PBS degradation, were observed at pH 7.5 in the wild type and to a greater extent in the ΔPsbO:ΔPsbU strain (Table 2). These PBS structural genes are repressed by hydrogen peroxide treatment while the nblA and nblB genes are induced. Response regulator RppA (Sll0797) is involved in regulation of the PBS structural genes and the nblA genes (Li and Sherman 2000). At pH 7.5, genes encoding this response regulator and neighboring histidine kinase (sll0798, rppB) exhibited increased transcript levels. Genes involved in transcriptional regulation that were induced by hydrogen peroxide treatment exhibited decreased mRNA levels in both strains at pH 7.5: hik34 encoding a histidine kinase and sigB encoding a sigma factor. In addition, the sigma factor-encoding gene, sigC, had decreased mRNA levels in the wild type. The gene cluster sll0788–sll0790 encodes a hypothetical protein, a response regulator (Rre34) and a histidine kinase (Hik31), respectively. Two of these genes, sll0789 and sll0788, were repressed by hydrogen peroxide treatment and exhibited decreased transcript levels at pH 7.5 in both strains. The gene encoding Hik31 was decreased in the mutant but not in the wild type at pH 7.5, and this gene was repressed by hydrogen peroxide treatment (Li et al. 2004, Kanesaki et al. 2007; see Table 2). Hik31 is involved in glucose metabolism (Kahlon et al. 2006) and has been shown to be involved in acclimation to low oxygen conditions, where it plays a role in down-regulating transcripts involved in photosynthesis (including the PBS components), chaperones and ribosomal proteins (Summerfield et al. 2011). Regulatory gene sll1392 (pfsR), which is involved in photosynthesis and an Fe-homeostasis stress response, had decreased mRNA levels at pH 7.5. In addition, transcriptional regulator slr0947 exhibited a decreased mRNA level at pH 7.5. Down-regulation of this gene has been shown to decrease energy transfer from the PBS to PSII (Ashby and Mullineaux, 1999); this is consistent with the decoupling of the PBS observed at pH 7.5 (Fig. 3). Under normal light conditions, Slr0947 (RpaB) binds upstream of the stress-responsive hliB gene and this binding is weaker under high light conditions (Kappell and Van Waasbergen 2007). Under low light conditions, RpaB acts a repressor of stress-responsive genes in Synechococcus elongatus strain PCC 7942, and these include hliA, although immunoprecipitation experiments indicate that additional regulatory mechanisms may be involved in this response (Seki et al., 2007, Hanaoka and Tanaka 2008). The down-regulation of rpaB at pH 7.5 is consistent with previous reports of altered energy transfer from the PBS to PSII, but is not consistent with the decreased levels of hliB under these conditions. Overall, transcriptional changes following transition from pH 10.0 to pH 7.5 indicated that growth at pH 10.0 stimulated a response with numerous similarities to the oxidative stress response in both strains. Expression of stress-responsive genes is decreased in the ΔPsbV:ΔCyanoQ strain at pH 7.5 To determine whether decreased mRNA levels of the stress responsive genes were shared by other pH-sensitive mutants, semi-quantitative reverse transcription–PCR (RT–PCR) was performed on RNA extracted from the ΔPsbV:ΔCyanoQ strain. The mRNA levels of a subset of genes that showed differential transcript abundance in the wild type and the ΔPsbO:ΔPsbU strain were examined. Genes encoding the heat shock protein HspA and sigma factor SigB are up-regulated following peroxide stress, and these genes showed decreased mRNA levels in the ΔPsbV:ΔCyanoQ strain on transfer from pH 10.0 to pH 7.5 (Fig. 4). The gene encoding Rre34 that is in an operon with the gene encoding the histidine kinase Hik31 had decreased transcript levels at pH 7.5, whereas sll1577 encoding a PBS subunit was unchanged. This is similar to the response for the wild type and the ΔPsbO:ΔPsbU strain. Hence, the wild type and the ΔPsbO:ΔPsbU and ΔPsbV:ΔCyanoQ mutants showed decreased mRNA levels of stress-responsive genes following transfer to pH 7.5. Based on our observations, we hypothesized that increased transcript abundance of oxidative stress-responsive genes at pH 10.0 improves resistance to oxidative stress. Fig. 4 View largeDownload slide Semi-quantitative RT–PCR of Synechocystis sp. PCC 6803 wild type and a ΔPsbV:ΔCyanoQ strain grown photoautotrophically at pH 10.0 and transferred to pH 7.5. Wild-type and ΔPsbV:ΔCyanoQ cells were grown in BG-11 at pH 10.0 and samples were harvested at t0; the remaining cells were transferred to pH 7.5 and samples were harvested at 1 and 2 h following transfer, as indicated above the lane. Equal amounts of RNA were used for each time point. Transcripts amplified were: sll0306, sigB; sll0789, rre34; sll1514, hspA; sll1577, cpcB; and rnpB. Fig. 4 View largeDownload slide Semi-quantitative RT–PCR of Synechocystis sp. PCC 6803 wild type and a ΔPsbV:ΔCyanoQ strain grown photoautotrophically at pH 10.0 and transferred to pH 7.5. Wild-type and ΔPsbV:ΔCyanoQ cells were grown in BG-11 at pH 10.0 and samples were harvested at t0; the remaining cells were transferred to pH 7.5 and samples were harvested at 1 and 2 h following transfer, as indicated above the lane. Equal amounts of RNA were used for each time point. Transcripts amplified were: sll0306, sigB; sll0789, rre34; sll1514, hspA; sll1577, cpcB; and rnpB. The wild type is more sensitive to rose bengal when grown in BG-11 medium at pH 7.5 compared with pH 10.0 To test whether the cells have increased resistance to oxidative stress at pH 10.0 compared with pH 7.5, we exposed wild-type cultures grown at pH 10.0 and at pH 7.5 to the singlet oxygen generator rose bengal. Photoautotrophic wild-type cultures at pH 7.5 exhibited increased sensitivity to rose bengal compared with cultures grown at pH 10.0 (Fig. 5A). The addition of either 5 or 2.5 µM rose bengal to BG-11 medium at pH 7.5 prevented photoautotrophic growth of the wild type (Fig. 5A). However, addition of 2.5 µM rose bengal to the wild type at pH 10.0 had no impact on doubling time, and the presence of 5 µM rose bengal only slightly decreased the growth rate (Fig. 5A). Absorbance at 543 nm was used to demonstrate the stability of rose bengal; this was similar in BG-11 at pH 7.5 and pH 10.0 (Fig. 5B). Photomixotrophic growth of the wild type in BG-11 medium buffered at pH 7.5 or pH 10.0 in the presence of 5 mM glucose was similar for the first 50 h, but by 100 h the pH 10.0 culture had reached a much higher OD730 nm than the pH 7.5 culture (almost 5 compared with ∼2.7), indicating a pH effect similar to that observed in photoautotrophically grown cultures (cf. Figs. 1A and 6A). Photomixotrophically grown cultures also showed pH-dependent sensitivity to rose bengal, with 2.5 µM rose bengal preventing photomixotrophic growth of the wild type at pH 7.5 but not pH 10.0 (Fig. 6A). Fig. 5 View largeDownload slide (A) Photoautotrophic growth of the wild-type Synechocystis sp. PCC 6803 strains in BG-11 medium as measured by the optical density at 730 nm. Growth of the wild-type strain plus 5 µM rose bengal (squares) and 2.5 µM rose bengal (circles), in BG-11 medium buffered at pH 7.5 (open symbols) or pH 10.0 (filled symbols). (B) Absorbance at 543 nm of 5 µM rose bengal in BG-11 medium buffered at pH 7.5 (open symbols) or pH 10.0 (filled symbols). Fig. 5 View largeDownload slide (A) Photoautotrophic growth of the wild-type Synechocystis sp. PCC 6803 strains in BG-11 medium as measured by the optical density at 730 nm. Growth of the wild-type strain plus 5 µM rose bengal (squares) and 2.5 µM rose bengal (circles), in BG-11 medium buffered at pH 7.5 (open symbols) or pH 10.0 (filled symbols). (B) Absorbance at 543 nm of 5 µM rose bengal in BG-11 medium buffered at pH 7.5 (open symbols) or pH 10.0 (filled symbols). Fig. 6 View largeDownload slide Photomixotrophic growth of the Synechocystis sp. PCC 6803 strains in BG-11 medium in the presence of 5 mM glucose as measured by the optical density at 730 nm. Cells were incubated in BG-11 medium buffered at pH 7.5 (open symbols) or pH 10.0 (filled symbols). (A) Growth of the wild-type strain (diamonds), plus 2.5 µM rose bengal (circles). (B) Growth of theΔPsbO:ΔPsbU strain (diamonds) and with the addition of 1 µM rose bengal (triangles). (C) Growth of the ΔPsbV:ΔCyanoQ strain (diamonds) and with the addition of 1 µM rose bengal (triangles). (D) Growth of the ΔPsbO:ΔPsbU pseudorevertant strain (diamonds) and with the addition of 1 µM rose bengal (triangles). (E) Growth of the wild-type strain plus 0.5 µM (diamonds), 1 µM (squares) or 2 µM methyl viologen (circles). (F) Growth of the ΔPsbO:ΔPsbU strain (diamonds) and with the addition of 0.1 µM (triangles), 0.5 µM (diamonds) or 2 µM methyl viologen (circles). Fig. 6 View largeDownload slide Photomixotrophic growth of the Synechocystis sp. PCC 6803 strains in BG-11 medium in the presence of 5 mM glucose as measured by the optical density at 730 nm. Cells were incubated in BG-11 medium buffered at pH 7.5 (open symbols) or pH 10.0 (filled symbols). (A) Growth of the wild-type strain (diamonds), plus 2.5 µM rose bengal (circles). (B) Growth of theΔPsbO:ΔPsbU strain (diamonds) and with the addition of 1 µM rose bengal (triangles). (C) Growth of the ΔPsbV:ΔCyanoQ strain (diamonds) and with the addition of 1 µM rose bengal (triangles). (D) Growth of the ΔPsbO:ΔPsbU pseudorevertant strain (diamonds) and with the addition of 1 µM rose bengal (triangles). (E) Growth of the wild-type strain plus 0.5 µM (diamonds), 1 µM (squares) or 2 µM methyl viologen (circles). (F) Growth of the ΔPsbO:ΔPsbU strain (diamonds) and with the addition of 0.1 µM (triangles), 0.5 µM (diamonds) or 2 µM methyl viologen (circles). The ΔPsbO:ΔPsbU and ΔPsbV:ΔCyanoQ strains are more sensitive to rose bengal than the wild type The ΔPsbO:ΔPsbU strain grew photomixotrophically at both pH 10.0 and pH 7.5, although cultures grew faster at pH 10.0 (Fig. 6B). The mutant was more sensitive than the wild type to rose bengal, with the mutant showing no growth in the presence of 2.5 µM rose bengal at pH 10.0 or pH 7.5 (data not shown). The presence of 1 µM rose bengal prevented growth of the ΔPsbO:ΔPsbU strain at pH 7.5 but not pH 10.0, although growth was reduced at pH 10.0 (Fig. 6B). Addition of 1 µM rose bengal had a similar impact on photomixotrophic growth of the ΔPsbV:ΔCyanoQ strain, with no growth of this strain at pH 7.5 and decreased growth at pH 10.0 (Fig. 6C). This strain appeared more sensitive than the ΔPsbO:ΔPsbU strain to the presence of rose bengal at pH 10.0 (Fig. 6B, C). The ΔPsbO:ΔPsbU pseudorevertant grew photomixotrophically at both pH 10.0 and pH 7.5, with a similar doubling time for the first ∼50 h, similar to the wild type (Fig. 6D). However, this strain was unable to grow photomixotrophically in the presence of 1 µM rose bengal at pH 7.5 (Fig. 6D). Growth was retarded under photomixotrophic conditions at pH 10.0 in the presence of 1 µM rose bengal to a similar extent to the ΔPsbO:ΔPsbU strain (Fig. 6B, D). The pseudorevertant’s increased sensitivity to rose bengal at pH 7.5 compared with pH 10.0 indicates that the mechanism enabling photoautotrophic growth of the pseudorevertant at pH 7.5 is not sufficient to confer resistance to rose bengal and is not the same mechanism enabling growth of the mutants at pH 10.0. To examine whether the increased sensitivity at pH 7.5 was observed in the presence of other ROS generators, methyl viologen was added to growth media as this generates superoxide. In both the wild type and ΔPsbO:ΔPsbU strain, sensitivity to methyl viologen was similar at pH 7.5 and pH 10.0 (Fig. 6E, F). At pH 7.5 and pH 10.0, growth of the ΔPsbO:ΔPsbU strain and the wild type was slightly retarded by the addition of 0.5 and 1 µM methyl viologen, respectively and neither strain grew in the presence of 2 µM methyl viologen. These data indicate that the observed pH sensitivity is not induced by all ROS generators. Discussion Altered Chl a variable fluorescence from PSII in pH-sensitive PSII mutants grown at pH 10.0 is not sufficient to explain recovery of photoautotrophic growth At elevated pH, we observed improved growth of the wild-type strain and growth of PSII mutants under photoautotrophic conditions, but there was no pH effect on photoheterotrophic growth, indicating a PSII-specific pH effect. Fluorescence induction measurements showed that the PSII mutants differed from the wild type, but growth under elevated pH did not restore a typical O–J–I–P transient as observed with wild-type cells. The 77 K fluorescence showed an increased emission at ∼685 nm in cells grown at pH 7.5; this may reflect partial decoupling of the PBS resulting in increased fluorescence from the terminal phycobilin emitters in the PSII mutants and to a lesser extent in the wild type. At pH 10.0, the increased fluorescence emission is reduced; this was not due to NPQ but is probably due to improved energy transfer from the PBS to PSII, and this may account in part for the pH-induced recovery of photoautotrophy in these strains. However, increased coupling of the PBS to PSII at pH 10.0 is not sufficient to explain fully the restoration of photoautotrophic growth in the PSII mutants as the ΔPsbO and ΔPsbV mutants exhibited increased fluorescence emission at pH 7.5, but grew photoautotrophically at this pH. Furthermore, the time course experiment (Fig. 3E, F) indicated that the decoupling of the PBS at pH 7.5 was a secondary effect. Transcript level changes on transition from pH 10.0 to pH 7.5 indicate maintenance of cellular homeostasis in the wild type and ΔPsbO:ΔPsbU strain Cell-wide transcriptional changes were observed in both the wild type and the ΔPsbO:ΔPsbU strain on transfer from growth medium buffered at pH 10.0 to pH 7.5. These changes were consistent with the results of a previous pH 7.5 to pH 10.0 transition experiment with the wild type (Summerfield and Sherman 2008), reflecting maintenance of cellular homeostasis, as genes with altered mRNA levels were involved in osmotic, pH and ion homeostasis. However, the kinetics of transcript level changes differ markedly depending on whether the transition is from pH 7.5 to pH 10.0 or from pH 10.0 to pH 7.5. The pH-independent differential regulation of 198 genes following both transfer from pH 7.5 to pH 10.0 and transfer from pH 10.0 to pH 7.5 identified further genes involved in maintenance of cellular homeostasis including adjusting to changes such as an altered bicarbonate/carbon dioxide ratio (Summerfield and Sherman 2008). In the ΔPsbO:ΔPsbU mutant, increased transcript levels of genes involved in photosynthesis may result from perturbation of the photosynthetic electron transport chain A major difference in the response of the two strains was increased transcript abundance of photosynthesis genes in the ΔPsbO:ΔPsbU mutant; these changes may result from perturbation of the photosynthetic electron transport chain due to the absence of PsbO and PsbU. The impaired PSII centers in the ΔPsbO:ΔPsbU and ΔPsbV:ΔCyanoQ mutants may increase ROS generation preventing photoautotrophic growth at pH 7.5. Balint et al. (2006) observed up-regulation of antioxidative mechanisms in their ΔpsbU mutant compared with wild-type Synechococcus sp. PCC 7942 and attributed this to an increased production of ROS in their ΔpsbU cells. Furthermore, the removal of PsbU in Synechocystis 6803 was shown to increase PBS fluorescence and to alter primary photochemistry of PSII (Veerman et al. 2005); here we have demonstrated a similar increase in PBS fluorescence in the absence of PsbO and PsbV but not CyanoQ. These findings are suggestive of a role for the extrinsic proteins in moderating light entering PSII and are consistent with the decreased psbO and psbU transcript levels observed by Tucker et al. (2001) in the unicellular diazotrophic cyanobacterium Cyanothece sp. ATCC 51142 in the dark, and the hypothesis that alterations on the oxidizing side of PSII mediate PSII activity as Cyanothece sp. ATCC 51142 proceeds through a 12 h light−12 h dark diurnal cycle (Meunier et al. 1998). Increased abundance of transcripts encoding oxidative stress-responsive genes at pH 10.0 may facilitate photoautotrophic growth of the pH-sensitive PSII mutants A proposed mechanism for the recovery of the pH-sensitive ΔPsbO:ΔPsbU and ΔPsbV:ΔCyanoQ mutants at pH 10.0 involves increased abundance of transcripts encoding oxidative stress-responsive genes at pH 10.0 (Fig. 7). In the mutant strains, we suggest increased mRNA levels of stress-responsive genes protect the impaired PSII centers, as well as the rest of the cell, from excessive ROS damage and facilitate photoautotrophic growth. Many genes exhibiting increased transcript abundance under oxidative stress also showed increased mRNA levels in the wild type following transfer from pH 7.5 to pH 10.0 (Summerfield and Sherman 2008). In addition, increased inorganic carbon availability at elevated pH may act to mitigate oxidative damage. However, there was no evidence to support inorganic carbon limitation at pH 7.5 compared with pH 10.0, as wild-type cells grown and measured at pH 7.5 and pH 10.0 without the addition of electron donors or acceptors had similar oxygen evolution rates (data not shown). Furthermore, comparison of DNA microarray data from our study with those of Wang et al. (2004) showed that none of the genes that exhibited increased transcript levels following a shift to lower carbon dioxide (up to 1–3 h) was increased on transfer to pH 7.5, and only three of the 23 genes with decreased mRNA levels following a shift to increased carbon dioxide were decreased on transfer to pH 7.5. Increased inorganic carbon availability may increase carbon fixation and reduce damage to PSII, contributing to the recovery of the strains at pH 10.0, but this does not appear sufficient to explain fully the recovery of the PSII mutants at elevated pH. Fig. 7 View largeDownload slide Model of the response to transition from pH 10.0 to pH 7.5 of Synechocystis sp. PCC 6803 wild type (WT) and a ΔPsbO:ΔPsbU mutant that is unable to grow photoautotrophically at pH 7.5, and comparison of a ΔPsbO:ΔPsbU mutant with a ΔPsbO:ΔPsbU pseudorevertant strain that is able to grow photoautotrophically at pH 7.5. Selected differentially abundant genes (≥1.5-fold change) at pH 10.0 compared with pH 7.5 in the wild type and the ΔPsbO:ΔPsbU strain are listed. The results are presented in the form of a Venn diagram that highlights the overlap among oxidative stress-induced genes and genes involved in scavenging ROS that exhibit increased mRNA levels at pH 10.0. In addition, genes with increased transcript levels in the pseudorevertant compared with the ΔPsbO:ΔPsbU strain at pH 7.5 are shown (Summerfield et al. 2007). Genes designated with a superscript 1 were reported previously to exhibit elevated transcripts following exposure to oxidative stress, and genes designated with a superscript 2 exhibited decreased transcripts following exposure to oxidative stress (Li et al. 2004, Kanesaki et al. 2007). We suggest that global stress-induced gene expression changes are sufficient to account for restoration of photoautotrophic growth in the ΔPsbO:ΔPsbU strain and the increased resistance to rose bengal (RB) observed in all strains at pH 10.0 and directly or indirectly led to changes in light harvesting. Fig. 7 View largeDownload slide Model of the response to transition from pH 10.0 to pH 7.5 of Synechocystis sp. PCC 6803 wild type (WT) and a ΔPsbO:ΔPsbU mutant that is unable to grow photoautotrophically at pH 7.5, and comparison of a ΔPsbO:ΔPsbU mutant with a ΔPsbO:ΔPsbU pseudorevertant strain that is able to grow photoautotrophically at pH 7.5. Selected differentially abundant genes (≥1.5-fold change) at pH 10.0 compared with pH 7.5 in the wild type and the ΔPsbO:ΔPsbU strain are listed. The results are presented in the form of a Venn diagram that highlights the overlap among oxidative stress-induced genes and genes involved in scavenging ROS that exhibit increased mRNA levels at pH 10.0. In addition, genes with increased transcript levels in the pseudorevertant compared with the ΔPsbO:ΔPsbU strain at pH 7.5 are shown (Summerfield et al. 2007). Genes designated with a superscript 1 were reported previously to exhibit elevated transcripts following exposure to oxidative stress, and genes designated with a superscript 2 exhibited decreased transcripts following exposure to oxidative stress (Li et al. 2004, Kanesaki et al. 2007). We suggest that global stress-induced gene expression changes are sufficient to account for restoration of photoautotrophic growth in the ΔPsbO:ΔPsbU strain and the increased resistance to rose bengal (RB) observed in all strains at pH 10.0 and directly or indirectly led to changes in light harvesting. The wild type and mutant strains are more resistant to singlet oxygen at pH 10.0 than at pH 7.5 All strains exhibited decreased sensitivity to rose bengal when grown at pH 10.0 compared with pH 7.5, thus demonstrating an increased resistance of these cells to oxidative stress at elevated pH. This pH 10.0 acclimation to rose bengal is similar to the acclimation response to low levels of singlet oxygen observed in Chlamydomonas reinhardtii (Ledford et al. 2007) where exposure to low levels of rose bengal resulted in increased transcript abundance of genes involved in the oxidative stress response and led to increased tolerance to exposure to higher levels of rose bengal. In our model, at pH 10.0, increased resistance to exogenous singlet oxygen from rose bengal arises due to changes in transcript levels of stress-responsive genes (Fig. 7). It is also noteworthy that singlet oxygen can give rise to other ROS such as hydrogen peroxide and superoxide through reactions with ascorbate (Miyake et al. 1991, Kramarenko et al. 2006). Moreover, since the primary site of singlet oxygen production in oxygenic photosynthesis is the antenna of PSII, our observed down-regulation of energy transfer from the PBS to the PSII reaction center in our mutants would be consistent with a mechanism to reduce singlet oxygen production at the non-permissive pH for photoautotrophic growth of these strains. The wild type and ΔPsbO:ΔPsbU strain did not show increased resistance to methyl viologen at pH 10.0. The different impact of the two ROS generators may be explained by their different stabilities. Methyl viologen is more stable than rose bengal, and methyl viologen stability and redox properties are not altered by pH (Bird and Kuhn, 1981). The stability of methyl viologen coupled with the fact that exposure to this herbicide stimulates a rapid transcriptional response in Synechocystis 6803 (Kobayashi et al., 2004) suggests that cultures grown at both pH 7.5 and pH 10.0 in the presence of methyl viologen may exhibit similar transcriptional profiles and this induces similar tolerance of ROS at both pH values. A subset of stress-responsive genes with elevated transcripts in the wild type or ΔPsbO:ΔPsbU strain at pH 10.0 compared with pH 7.5 also exhibited increased mRNA levels in an experiment comparing the ΔPsbO:ΔPsbU pseudorevertant with the ΔPsbO:ΔPsbU strain at pH 7.5 (Summerfield et al. 2007; Fig. 7), and we suggest that these transcripts may be involved in photoautotrophic growth of the pseudorevertant at pH 7.5. The fact that only a subset of the stress-responsive genes that had increased transcript levels in the wild type and ΔPsbO:ΔPsbU at pH 10.0 were increased in the pseudorevertant at pH 7.5 may account for the ability of this strain to grow photoautotrophically but still exhibit increased sensitivity to rose bengal at pH 7.5. Furthermore, additional stress-responsive genes such as tpx and sll1615 exhibited elevated transcript levels at pH 10.0 compared with pH 7.5 in the ΔPsbO:ΔPsbU mutant and these were not changed in the wild type and were not elevated in the pseudorevertant compared with the ΔPsbO:ΔPsbU strain at pH 7.5 (Fig. 7). Materials and Methods Cyanobacterial strains and growth conditions The glucose-tolerant variant of Synechocystis 6803 (Williams 1988) was used in this study. Cultures were maintained on BG-11 plates containing 5 mM glucose, 20 µM atrazine and appropriate antibiotics. In both solid and liquid media, chloramphenicol was present at a concentration of 15 µg ml−1, and erythromycin, kanamycin and spectinomycin were present at 25 µg ml−1. The BG-11 solid media were supplemented with 10 mM TES-NaOH (pH 8.2) and 0.3% sodium thiosulfate. Liquid cultures were bubbled with air and grown in BG-11 media containing either 25 mM HEPES (pH 7.5) or 25 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS; pH 10.0). Photomixotrophic and photoheterotrophic cultures contained 5 mM glucose and appropriate antibiotics; in addition, photoheterotrophic grown cultures contained 20 µM atrazine. Cultures were maintained at 30°C under constant illumination at 30 µmol photons m−2 s−1 in an MLR-350 growth cabinet (Sanyo Electric Biomedical Co. Ltd.). Growth curves were initiated from cultures harvested at log phase (OD730 nm between 0.4 and 0.8, measured with a Jasco V-550 UV/vis spectrophotometer; Jasco International) Mutants lacking PsbO and/or PsbU were produced as described in Eaton-Rye et al. (2003); these strains were constructed in the background of a control strain that has a kanamycin-resistance cassette located downstream of the psbB gene (Eaton-Rye and Vermaas 1991) which has a phenotype otherwise indistinguishable from that of the wild type. Strains lacking PsbV and/or CyanoQ were produced as described in Summerfield et al. (2005). For DNA micorarray experiments, cultures were grown in BG-11 at pH 10.0 until approximately 8 × 107 cells ml−1 and transferred to BG-11 medium buffered at pH 7.5. Cells were harvested at 0, 1 and 2 h following transfer to pH 7.5, and cells from the 0 and 2 h time points were used for microarray experiments. To measure the impact of ROS on growth, either rose bengal or methyl violgen was added at 0 h. 77 K Fluorescence emission spectra Samples were assayed for fluorescence emission at 77 K with a modified Perkin-Elmer MPF-3L fluorescence spectrophotometer equipped with a custom-built silver Dewar. Samples for analysis were prepared from mixotrophic cultures grown to mid-logarithmic phase (OD730 nm ∼0.6) in BG-11 at the appropriate pH. Cells were washed three times in their respective media to remove glucose, resuspended to a Chl a concentration of 2 µg Chl ml−1 and left to stabilize for 30 min at 30°C at a light intensity of 50 µmol photons m−2 s−1. A 1 ml aliquot of cells was loaded into electron paramagnetic resonance tubes, quickly frozen in liquid nitrogen and kept in liquid nitrogen until emission spectra were collected. 77 K fluorescence emission spectra were measured using two different excitation energy wavelengths for each sample: 440 nm to excite specifically Chl a and 580 nm to excite specifically the PBS. The excitation slit wavelength was set at 12 and 10 nm for 440 and 580 nm excitation, respectively; the emission slit wavelength was set at 4 nm. Spectra were normalized to the emission peak at 725 nm arising from PSI. pH transition time course for 77 K fluorescence emission spectra The wild type and the ΔPsbO:ΔPsbU mutant of Synechocystis sp. PCC 6803 were grown in BG-11 buffered at pH 7.5 or pH 10.0, in the presence of 5 mM glucose and appropriate antibiotics. Cells were harvested at mid-logarithmic phase (OD730 nm ∼0.8) and re-suspended at pH 7.5 or pH 10.0 in BG-11 at 2 µg Chl ml−1. After an initial measurement, cultures were washed once with unbuffered BG-11 and resuspended in BG-11 pH 10.0 (for the cultures previously incubated at pH 7.5) or pH 7.5 (for the cultures previously incubated at pH 10.0) and incubated at 30°C at a light intensity of 50 µmol photons m−2 s−1. Samples were taken at 2, 6 and 12 h after the pH transition. Variable Chl a fluorescence yield measurements Cells were grown to an OD730 nm of ∼0.6–0.8 and washed as described for 77 K fluorescence emission spectra measurements but were resuspended at 6 µg Chl ml−1. After 30 min at 30°C and a light intensity of 50 µmol photons m−2 s−1, cells were diluted to 3 µg Chl ml−1 in dark flasks, and dark adapted for 15 min at 30°C with gentle shaking. Chl a fluorescence induction measurements were made using an FL-3500 fluorometer (Photon Systems Instruments) in 4 ml quartz cuvettes with a 1 cm optical path containing a 2 ml sample volume. Fluorescence induction was induced by a 455 nm constant actinic light applied at 2,800 µmol photons m−2 s−1 for 5 s. Fluorescence was measured using weak 455 nm probing flashes. RNA extraction Total RNA was extracted and purified using phenol–chloroform extraction and CsCl gradient purification as previously described (Reddy et al. 1990, Singh and Sherman 2002). Microarray design The DNA microarray platform and construction were as described in Postier et al. (2003), and the cDNA labeling, pre-hybridization and hybridization protocols are described in detail in Singh et al. (2003). The microarray experiment involved a loop design that compared the wild type and the ΔPsbO:ΔPsbU strain grown in BG-11 at pH 10.0 with the cells after 2 h following a transition to BG-11 at pH 7.5 by using an analysis of variance (ANOVA) model (Singh et al. 2003, Li et al. 2004). Data acquisition included the ANOVA model approach to test the null hypothesis that a particular gene’s expression level did not differ between the treatments and to calculate a P-value (Singh et al. 2003). This experiment contained two genotypes (wild type and the ΔPsbO:ΔPsbU strain) and two stimuli (growth at pH 10.0 and transition to medium at pH 7.5) for a total of four treatment combinations. The effects of the absence of PsbO and PsbU and the transition from pH 10.0 to pH 7.5 were examined in the ANOVA design essentially as described in Kerr and Churchill (2001a), Kerr and Churchill (2001b), Singh et al. (2003) and Li et al. (2004). We used the FDR of 5% to control the proportion of significant results that are Type I errors (false rejection of the null hypothesis) as described in Summerfield and Sherman (2007). Genes with an FDR = 0.05 (corresponding to 5% expected false positives) and that exhibited a change of at least 1.5-fold were considered interesting and retained for further analysis. The P-value of these genes ranged from 2.7 × 10−2 to 6.3 × 10−17. Semi-quantitative RT–PCR DNase I treatment and reverse transcription were performed as described in Summerfield et al. (2008). PCR was carried out using 94°C for 1 min, followed by 20–30 cycles of: 94°C for 30 s, 52°C for 30 s and 68°C for 30–120 s (depending on amplicon size), to amplify regions of the genes listed below. Semi-quantitative RT–PCR was used to examine mRNA levels in the wild type and the ΔPsbV:ΔCyanoQ strain. The genes amplified, the primers used along with the PCR product size and the number of PCR cycles were: sll0306 (F 5′-gagtacctcagtctgtcg, R 5′-ctgatgttgagatgctgg, 260 bp, 25 cycles); sll0789 (F 5′-tacgtagttgattgggtgc, R 5′-gcatctaaaccctcaacc, 212 bp, 25 cycles); sll1514 (F 5′-gataatttccagcagcag, R 5′- gtcaaagttaggataccg, 350 bp, 25 cycles); sll1577 (F 5′-ttcccaagctgatgctcg, R 5′-agcactgcaatcaccacg, 439 bp, 20 cycles), and rnpB (F 5′-tgtcacagggaatctgagg, R 5′-gagagagttagtcgtaagc, 405 bp, 25 cycles). The rnpB gene was included as this transcript is frequently used as a constitutively expressed control for gene expression. Amplification products were separated on a 2% agarose Tris-acetate-EDTA or TAE gel. Funding This work was supported by the New Zealand Marsden Fund [Grant 09-UOO-118 to T.C.S.]; US Department of Energy [Grant DE-FG02–99ER20342 to L.A.S.]. Acknowledgment We thank our anonymous reviewers for their helpful comments. 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Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected] http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant and Cell Physiology Oxford University Press

Environmental pH Affects Photoautotrophic Growth of Synechocystis sp. PCC 6803 Strains Carrying Mutations in the Lumenal Proteins of PSII

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Oxford University Press
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© The Author 2013. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected]
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0032-0781
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1471-9053
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10.1093/pcp/pct036
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23444302
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Abstract

Abstract Synechocystis sp. strain PCC 6803 grows photoautotrophically across a broad pH range, but wild-type cultures reach a higher density at elevated pH; however, photoheterotrophic growth is similar at high and neutral pH. A number of PSII mutants each lacking at least one lumenal extrinsic protein, and carrying a second PSII lumenal mutation, are able to grow photoautotrophically in BG-11 medium at pH 10.0, but not pH 7.5. We investigated the basis of this pH effect and observed no pH-specific change in variable fluorescence yield from PSII centers of the wild type or the pH-dependent ΔPsbO:ΔPsbU and ΔPsbV:ΔCyanoQ strains; however, 77 K fluorescence emission spectra indicated increased coupling of the phycobilisome (PBS) antenna at pH 10.0 in all mutants. DNA microarray data showed a cell-wide response to transfer from pH 10.0 to pH 7.5, including decreased mRNA levels of a number of oxidative stress-responsive transcripts. We hypothesize that this transcriptional response led to increased tolerance against reactive oxygen species and in particular singlet oxygen. This response enabled photoautotrophic growth of the PSII mutants at pH 10.0. This hypothesis was supported by increased resistance of all strains to rose bengal at pH 10.0 compared with pH 7.5. Introduction Cyanobacteria are known to be alkaliphilic microbes (Pikuta et al. 2007), although many strains are able to grow over a wide pH range. Nonetheless, a large body of research with model organisms, such as Synechocystis sp. strain PCC 6803 (hereafter Synechocystis 6803), has been conducted in buffered growth media typically around pH 7.5, which might not represent the most natural condition. Unexpectedly, several PSII-specific mutations introduced into Synechocystis 6803 were found to create strains unable to grow at pH 7.5, whereas photoautotrophic growth could be restored in these mutants at pH 10.0 (Eaton-Rye et al. 2003, Summerfield et al. 2005). The pH-sensitive strains all carried mutations targeting the lumenal proteins of PSII; these include two double mutants, ΔPsbO:ΔPsbU and ΔPsbV:ΔCyanoQ, and a third strain lacking PsbV and carrying a short segment deletion between Arg384 and Val392 in CP47 (Eaton-Rye et al. 2003, Summerfield et al. 2005). Additionally, mutation of the CP47 residue Glu364 to glutamine combined with the removal of PsbV created a pH-sensitive strain (Eaton-Rye et al. 2003). PSII is the light-driven water-plastoquinone oxidoreductase of oxygenic photosynthesis (Wydrzynski and Satoh 2005, Renger 2012). The photosystem includes a core complex of the PsbA (D1) and PsbD (D2) polypeptides that bind the P680 reaction center Chls, the Mn4CaO5 oxygen-evolving complex (OEC) and the majority of the remaining redox cofactors (Nixon et al. 2005, Kawakami et al. 2011, Müh et al. 2012). The PSII core is flanked by PsbB (CP47) and PsbC (CP43), which are Chl-binding proteins that serve as a proximal antenna transferring excitation energy from the peripheral phycobilisome (PBS) antenna to P680 (Eaton-Rye and Putnam-Evans 2005, Mullineaux 2008). Hydrophilic domains of CP47 and CP43, together with the extrinsic proteins, provide a cap over the OEC (Bricker et al. 2012). In cyanobacteria, additional lipoproteins are associated with the lumenal face of PSII; however, these proteins are not present in the available PSII crystal structures from either Thermosynechococcus elongatus or T. vulcanus (Fagerlund and Eaton-Rye 2011). The lumenal PSII extrinsic proteins play a role in protecting the OEC from bulk reductants and contribute to putative channels associated with the access of substrate H2O and the egress of O2 and H+ (Gabdulkhakov et al. 2009, Umena et al. 2011). The X-ray crystallographic structures from T. elongatus and T. vulcanus have confirmed the presence of PsbO, PsbU and PsbV as hydrophilic lumenal extrinsic proteins (Ferreria et al. 2004, Loll et al. 2005, Umena et al. 2011). In addition, the lipoprotein CyanoQ has been shown to be associated with highly active PSII preparations from Synechocystis 6803 (Roose et al. 2007). In cyanobacteria, the PsbO protein contributes to the stability of the PSII dimeric supercomplex through interactions with loop A and loop E of CP47 (De Las Rivas and Barber 2004, Sakurai et al. 2007, Bentley and Eaton-Rye 2008). Removal of PsbO increases the sensitivity of PSII to photoinactivation and results in a strict dependence on Ca2+ and Cl− for photoautotrophic growth (Bockholt et al. 1991, Burnap and Sherman 1991, Mayes et al. 1991, Philbrick et al. 1991, Burnap et al. 1996). A similar light sensitivity and a dependence on Ca2+ and Cl− for photoautotrophy has been observed when the psbV gene is deleted (Shen et al. 1995, Morgan et al. 1998, Shen et al. 1998). Susceptibility to photoinactivation is also observed when the PsbU subunit is removed (Clarke and Eaton-Rye 1999, Inoue-Kashino et al. 2005, Abasova et al. 2011). Removal of PsbU results in slower photoautotrophic growth in the absence of Ca2+ and Cl− (Shen et al. 1997); and, in the absence of both ions, the ΔPsbU mutant is not photoautotrophic (Inoue-Kashino et al. 2005, Summerfield et al. 2005). In Synechocystis 6803, the absence of PsbU affected both energy transfer and electron transfer in the PBS–PSII complex (Veerman et al. 2005). In addition, in the Synechococcus sp. PCC 7942 mutant lacking PsbU, cells exhibited an elevated resistance to oxidative stress (Balint et al. 2006, Abasova et al. 2011). Furthermore, the PsbO, PsbU and PsbV proteins are required for the thermal stability of PSII and for the development of cellular thermotolerance (Nishiyama et al. 1999, Kimura et al. 2002). It is therefore apparent that the extrinsic proteins enable cyanobacteria to adjust to a number of variable environmental parameters. Transfer of wild-type Synechocystis 6803 from pH 7.5 to pH 10.0 increased mRNA levels of genes encoding several low molecular weight intrinsic subunits as well as extrinsic proteins of PSII, including psbO and psbU (Summerfield and Sherman 2008). This was part of a cell-wide response that included mechanisms to maintain pH homeostasis in the cytosol such as the presence of monovalent cation/proton antiporters (Padan et al. 2005). Cyanobacteria exhibit additional complexity, compared with non-photosynthetic organisms, in maintaining a pH in the thylakoid lumen that is approximately 2 pH units more acid than the cytosol. Moreover, cyanobacterial cytoplasmic and lumen pHs are altered by the environmental pH, with an increase of 2 pH units in the external pH resulting in an internal increase of approximately 0.2 pH units (Belkin and Packer 1988, Ritchie, 1991). It appears that the external pH is able to influence the growth of mutants with an altered complement of PSII extrinsic proteins, even though these proteins are located in the acidic thylakoid lumen. To investigate how the different combinations of the extrinsic proteins influence the ability of Synechocystis 6803 cells to adapt to different pHs, we have investigated PSII activity in vivo using non-invasive measurements of variable Chl fluorescence yield. We have combined these PSII activity measurements with pH-dependent changes in global gene expression in the wild type and the pH-sensitive ΔPsbO:ΔPsbU mutant to investigate how modification of PSII activity by the presence or absence of specific lumenal extrinsic proteins can influence gene expression and facilitate photoautotrophic growth. Results Photoautotrophic but not photoheterotrophic growth is increased in growth medium buffered at pH 10.0 compared with pH 7.5 It has previously been observed that wild-type cultures grown at pH 10.0 and pH 7.5 have similar initial doubling times, oxygen evolution rates and number of assembled PSII centers (Eaton-Rye et al. 2003). However, following 100 h of photoautotrophic growth, wild-type cultures reached an OD730 nm of ∼3 in pH 10.0 medium, compared with an OD730 nm of <2 in medium buffered at pH 7.5 (Fig. 1A). In contrast, photoheterotrophic growth (in the presence of 20 µM atrazine and 5 mM glucose) of the wild type was similar in BG-11 buffered at either pH 10.0 or pH 7.5 (Fig. 1A). Furthermore, a strain lacking both the PsbO and PsbU proteins grew photoautotrophically at pH 10.0 but not at pH 7.5 (Eaton-Rye et al. 2003), whereas photoheterotrophic growth was similar at pH 7.5 and pH 10.0 (Fig. 1B). These growth characteristics indicate that the pH of the growth medium has a PSII-specific effect; this is supported by observations of additional PSII mutants that exhibit photoautotrophic growth at pH 10.0 but not pH 7.5 (Eaton-Rye et al. 2003, Summerfield et al. 2005). To investigate the impact of extracellular pH on PSII activity and assembly, variable Chl a fluorescence yield and 77 K fluorescence emission spectra were measured. Fig. 1 View largeDownload slide Growth of Synechocystis sp. PCC 6803 strains in BG-11 medium as measured by the optical density at 730 nm. (A) Wild type: photoautotrophic growth in media buffered at pH 7.5 (open circles) and in media buffered at pH 10.0 (filled circles); photoheterotrophic growth in the presence of 20 µM atrazine and 5 mM glucose in media buffered at pH 7.5 (open squares) and media buffered at pH 10.0 (filled squares). (B) ΔPsbO:ΔPsbU mutant: photoautotrophic growth in media buffered at pH 7.5 (open triangles) and in media buffered at pH 10.0 (filled triangles); photoheterotrophic growth in the presence of 20 µM atrazine and 5 mM glucose in media buffered at pH 7.5 (open diamonds) and media buffered at pH 10.0 (filled diamonds). In A and B, the data are the average ± SE of three independent experiments. Error bars not visible are smaller than the symbols. Fig. 1 View largeDownload slide Growth of Synechocystis sp. PCC 6803 strains in BG-11 medium as measured by the optical density at 730 nm. (A) Wild type: photoautotrophic growth in media buffered at pH 7.5 (open circles) and in media buffered at pH 10.0 (filled circles); photoheterotrophic growth in the presence of 20 µM atrazine and 5 mM glucose in media buffered at pH 7.5 (open squares) and media buffered at pH 10.0 (filled squares). (B) ΔPsbO:ΔPsbU mutant: photoautotrophic growth in media buffered at pH 7.5 (open triangles) and in media buffered at pH 10.0 (filled triangles); photoheterotrophic growth in the presence of 20 µM atrazine and 5 mM glucose in media buffered at pH 7.5 (open diamonds) and media buffered at pH 10.0 (filled diamonds). In A and B, the data are the average ± SE of three independent experiments. Error bars not visible are smaller than the symbols. Absence of extrinsic proteins of PSII alters Chl a variable fluorescence from PSII in strains grown at both pH 7.5 and pH 10.0 Chl a variable fluorescence arises from PSII and can be induced by actinic light applied to dark-adapted cells (Papageorgiou et al. 2007, Stamatakis et al. 2007, Kaňa et al. 2012). Fluorescence induction for the wild type and mutants lacking extrinsic proteins grown at pH 7.5 and pH 10.0 is presented in Fig. 2. The control strain exhibited a fluorescence induction curve typical of Synechocystis 6803 cells, with a pronounced O to J rise reflecting the photochemical reduction of QA to form QA−, followed by a slow J to I thermal phase on a millisecond time scale before undergoing a large I to P rise at around 0.5 to 1 s (Kaňa et al. 2012) (Fig. 2A). Removal of PsbU resulted in a reduction of the O to J rise, but a substantial I to P rise remained; in contrast, removal of PsbO resulted in a reduced J level and prevented any I to P rise (Fig. 2A). The ΔPsbO:ΔPsbU strain and a ΔPsbO:ΔPsbU pseudorevertant strain, that grew photoautotrophically at pH 7.5 (Summerfield et al. 2007), exhibited fluorescence induction curves similar to that observed with ΔPsbO cells (Fig. 2C). For each of the five strains, the fluorescence induction curves of cells grown at pH 10.0 and pH 7.5 were similar, except for a slight increase in the O to J rise at pH 10.0 (Fig. 2A–D). Fig. 2 View largeDownload slide Fluorescence induction kinetics of Synechocystis sp. PCC 6803 strains grown in BG-11 medium buffered at pH 7.5 (open symbols) or pH 10.0 (filled symbols). Chl a fluorescence was induced with a 455 nm, 2,800 µmol photons m−2 s−1 actinic light and probed using a non-actinic measuring light of the same wavelength. (A–D) Control (squares); ΔPsbO (circles); ΔPsbU (triangles); ΔPsbO:ΔPsbU (diamonds); ΔPsbO:ΔPsbU pseudorevertant (inverted triangles). (E and F) Wild type (squares); ΔCyanoQ (triangles); ΔPsbV (circles); ΔCyanoQ:ΔPsbV (diamonds). Traces were normalized to (F – Fo)/Fo. Fig. 2 View largeDownload slide Fluorescence induction kinetics of Synechocystis sp. PCC 6803 strains grown in BG-11 medium buffered at pH 7.5 (open symbols) or pH 10.0 (filled symbols). Chl a fluorescence was induced with a 455 nm, 2,800 µmol photons m−2 s−1 actinic light and probed using a non-actinic measuring light of the same wavelength. (A–D) Control (squares); ΔPsbO (circles); ΔPsbU (triangles); ΔPsbO:ΔPsbU (diamonds); ΔPsbO:ΔPsbU pseudorevertant (inverted triangles). (E and F) Wild type (squares); ΔCyanoQ (triangles); ΔPsbV (circles); ΔCyanoQ:ΔPsbV (diamonds). Traces were normalized to (F – Fo)/Fo. Fluorescence induction was investigated in the pH-sensitive ΔPsbV:ΔCyanoQ mutant (Fig. 2E, F) as this strain exhibited a similar phenotype to theΔPsbO:ΔPsbU strain (Summerfield et al. 2005). The wild type and the ΔCyanoQ strain possessed a similar O–J–I–P transient, whereas the ΔPsbV and ΔPsbV:ΔCyanoQ strains exhibited suppressed fluorescence induction. The results in Fig. 2 indicate that the absence of an O–J–I–P transient does not preclude photoautotrophic growth, and the ability of the ΔPsbO:ΔPsbU or ΔPsbV:ΔCyanoQ strains to grow at pH 10.0 was not accompanied by restoration of an I to P rise. Since in all strains the O to J rise was slightly enhanced at pH 10.0, and this might correlate with an increased absorption cross-section for energy transfer to PSII from the peripheral antenna or PBS, energy transfer was investigated using 77 K fluorescence emission spectra. Growth medium pH alters 77 K fluorescence emission spectra of PSII mutants We measured 77 K fluorescence emission using 580 nm excitation of the PBS (Fig. 3). Compared with the control strain, the ΔPsbU and ΔPsbO strains had increased emission at 685 nm, suggesting an enhanced emission from the terminal emitter of the PBS in cultures grown at pH 7.5 (Fig. 3A). Increased fluorescence yield was previously reported for the ΔPsbU strain grown in unbuffered BG-11 (Veerman et al. 2005). Emission at 695 nm from the CP47 Chl a core antenna was not increased in either the ΔPsbU or ΔPsbO strains, supporting the interpretation that the increased fluorescence results from the terminal emitters of the PBS and not the CP43 Chl a core antenna which also emits at 685 nm. The ΔPsbO:ΔPsbU strain had an emission spectrum similar to that of the ΔPsbO strain, whereas the ΔPsbO:ΔPsbU pseudorevertant had decreased emission at ∼685 nm compared with the ΔPsbO and ΔPsbO:ΔPsbU strains, but this was still increased compared with the wild type (Fig. 3C). Notably, the fluorescence emission at 685 nm was markedly decreased in strains lacking PsbO and PsbU when grown at pH 10.0 (Fig. 3B, D). Fig. 3 View largeDownload slide 77 K fluorescence emission spectra of Synechocystis sp. PCC 6803 strains grown in BG-11 medium buffered at pH 7.5 (A, C) or pH 10.0 (B, D). Spectra were collected using excitation at 580 nm and normalized to a PSI emission peak at 725 nm. (A and B) Control (black); ΔPsbO (red); ΔPsbU (green). (C and D) ΔPsbO:ΔPsbU (blue); ΔPsbO:ΔPsbU pseudorevertant (green). (E and F) Time course following transition from BG-11 medium at pH 7.5 to pH 10.0 (E) and from BG-11 medium at pH 10.0 to pH 7.5 (F) in a ΔPsbO:ΔPsbU strain, pre-transfer (black); 2 h after transfer (blue); 6 h after transfer (green); and 12 h after transfer (red). The pre-transfer (black line) and 2 h after transfer (blue line) values are almost overlapping in E and F. Fig. 3 View largeDownload slide 77 K fluorescence emission spectra of Synechocystis sp. PCC 6803 strains grown in BG-11 medium buffered at pH 7.5 (A, C) or pH 10.0 (B, D). Spectra were collected using excitation at 580 nm and normalized to a PSI emission peak at 725 nm. (A and B) Control (black); ΔPsbO (red); ΔPsbU (green). (C and D) ΔPsbO:ΔPsbU (blue); ΔPsbO:ΔPsbU pseudorevertant (green). (E and F) Time course following transition from BG-11 medium at pH 7.5 to pH 10.0 (E) and from BG-11 medium at pH 10.0 to pH 7.5 (F) in a ΔPsbO:ΔPsbU strain, pre-transfer (black); 2 h after transfer (blue); 6 h after transfer (green); and 12 h after transfer (red). The pre-transfer (black line) and 2 h after transfer (blue line) values are almost overlapping in E and F. At pH 7.5, compared with the wild type, the ΔCyanoQ strain showed slightly increased emission at 648, 665 and 685 nm corresponding to phycocyanin, allophycocyanin and the terminal emitter of the PBS, respectively (Supplementary Fig. S1A). The ΔPsbV and ΔPsbV:ΔCyanoQ strains exhibited an increased emission at 685 nm when grown at pH 7.5 (Supplementary Fig. S1A), and this was reversed when these strains were grown at pH 10.0 (Supplementary Fig. S1B), similar to the changes observed in the 77 K fluorescence emission spectra of the ΔPsbU, ΔPsbO and ΔPsbO:ΔPsbU strains. Confirmation that the elevated fluorescence resulted from excitation of the PBS and not the core antenna pigments was obtained by measuring the Fo fluorescence level when probed with a red light (625 nm) compared with blue light (455 nm) at pH 7.5. The Fo fluorescence emission was elevated following excitation with red light which excited the PBS compared with when blue light (455 nm) was used to excite the core antenna pigments directly (Supplementary Fig. S1C). At pH 10.0, the Fo fluorescence levels probed with red light or blue light were similar (Supplementary Fig. S1D), consistent with quenching of fluorescence from the PBS at pH 10.0. In Synechocystis 6803, non-photochemical quenching (NPQ) involves interaction of the orange carotenoid protein (OCP) with the PBS to increase energy dissipation, therefore decreasing the amount of energy arriving at PSII (Wilson et al. 2006). To assess whether the decreased fluorescence at pH 10.0 was due to increased NPQ, we induced NPQ in the wild type and ΔPsbO:ΔPsbU strains grown at both pH 7.5 and pH 10.0. Induction of NPQ using blue light resulted in a small decrease in fluorescence at 648, 665 and 685 nm, but did not substantially reduce the elevated fluorescence at 685 nm in the ΔPsbO:ΔPsbU strain grown at pH 7.5 (Supplementary Fig. S2A, B). This demonstrates that NPQ is not the cause of fluorescence quenching in the PSII mutant strains at pH 10.0 and is consistent with increased coupling of the PBS to PSII at pH 10.0. Our measurements of 77 K fluorescence emission, using excitation at 580 nm, in ΔPsbO:ΔPsbU cells transferred from pH 7.5 to pH 10.0 also showed that decreased fluorescence did not occur until several hours after transfer to elevated pH (Fig. 3E). In addition, cells grown at pH 10.0 and transferred to pH 7.5 showed no increase in fluorescence at 685 nm even 12 h after transfer to the lower pH (Fig. 3F). The kinetics of these changes in fluorescence emission suggest that the altered coupling of the PBS to PSII was not a primary response to the change in pH of the growth medium. Furthermore, the pH-dependent coupling of the PBS by itself does not explain the photoautotrophic growth of the ΔPsbO:ΔPsbU and ΔPsbV:ΔPsbQ strains at pH 10.0, but not pH 7.5, as the ΔPsbO, ΔPsbU and ΔPsbV strains showed similar increased fluorescence emission at pH 7.5 and grew photoautotrophically at this pH (Supplementary Fig S1A). To identify mechanisms involved in recovery of photoautotrophic growth of pH-sensitive mutants at pH 10.0, we investigated the cell-wide response to altered growth medium pH by examining the transcriptional response following the transition from pH 10.0 to pH 7.5 in the wild type and the ΔPsbO:ΔPsbU strain. Global transcriptional response to transition from pH 10.0 to pH 7.5 in the wild type and ΔPsbO:ΔPsbU strain Synechocystis 6803 cultures were grown in continuous light in BG-11 medium buffered at pH 10.0. Samples were taken for RNA isolation at 0 h (t0), and 2 h (t2) following transfer from pH 10.0 to pH 7.5. As indicated in the Materials and Methods, we considered genes to be differentially regulated if they showed a fold change of ≥1.5 with a false discovery rate (FDR) = 0.05. A similar number of genes met these criteria in the two strains; 467 and 413 genes in the wild type and ΔPsbO:ΔPsbU strain, respectively. Increased transcript abundance at pH 7.5 was observed for 209 and 242 genes for the wild type and ΔPsbO:ΔPsbU strain, respectively. Genes were divided into functional categories according to the Cyanobase designation (http://genome.microbedb.jp/cyanobase), and the number of differentially expressed genes in each category is shown in Table 1. Changes in transcript level, across a range of different functional categories, indicated a cell-wide response to external pH; categories containing large numbers of genes exhibiting differential transcript abundance included: photosynthesis and respiration (20 and 42 genes in the wild type and the ΔPsbO:ΔPsbU strain, respectively); transport and binding proteins (36 and 28 genes in the wild type and the ΔPsbO:ΔPsbU cells, respectively); regulatory functions (20 genes in both strains); and cellular processes (14 and 17 genes in the wild type and the ΔPsbO:ΔPsbU strain, respectively). Table 1 Functional categories of pH-responsive genesa in wild-type and ΔPsbO:ΔPsbU Synechocystis sp. PCC 6803 strains Gene category  No. of genesb  Differentially regulated genes       WT (Up)c  OU (Up)c  OU/WT pH 7.5d  OU/WT pH 10.0d  Amino acid biosynthesis  97  17 (10)  11 (6)  11 (4)d  5 (1)d  Biosynthesis of cofactors, prosthetic groups and carriers  124  13 (5)  15 (7)  13 (6)  8 (4)  Cell envelope  67  7 (6)  19 (19)  15 (15)  2 (1)  Cellular processes  76  14 (2)  17 (10)  11 (8)  9 (9)  Central intermediary metabolism  31  4 (3)  4 (3)  1 (1)  1 (0)  DNA replication, restriction, recombination and repair  60  5 (2)  6 (4)  4 (1)  3 (3)  Energy metabolism  132  19 (14)  17 (12)  12 (7)  7 (4)  Hypothetical  1076  169 (53)  133 (64)  92 (51)  70 (52)  Other categories  306  38 (22)  21 (9)  16 (7)  8 (3)  Photosynthesis and respiration  141  20 (13)  42 (37)  18 (14)  12 (11)  Purines, pyrimidines, nucleosides and nucleotides  41  5 (2)  3 (1)  2 (1)  1 (1)  Regulatory functions  146  20 (8)  20 (11)  9 (4)  6 (4)  Transcription  30  8 (6)  6 (3)  2 (0)  2 (2)  Translation  168  23 (15)  17 (12)  8 (3)  4 (3)  Transport and binding proteins  196  36 (27)  28 (13)  28 (9)  16 (4)  Unknown  474  69 (21)  54 (31)  37 (31)  34 (22)  Total number  3165b  467 (209)  413 (242)  279 (162)  188 (124)  Gene category  No. of genesb  Differentially regulated genes       WT (Up)c  OU (Up)c  OU/WT pH 7.5d  OU/WT pH 10.0d  Amino acid biosynthesis  97  17 (10)  11 (6)  11 (4)d  5 (1)d  Biosynthesis of cofactors, prosthetic groups and carriers  124  13 (5)  15 (7)  13 (6)  8 (4)  Cell envelope  67  7 (6)  19 (19)  15 (15)  2 (1)  Cellular processes  76  14 (2)  17 (10)  11 (8)  9 (9)  Central intermediary metabolism  31  4 (3)  4 (3)  1 (1)  1 (0)  DNA replication, restriction, recombination and repair  60  5 (2)  6 (4)  4 (1)  3 (3)  Energy metabolism  132  19 (14)  17 (12)  12 (7)  7 (4)  Hypothetical  1076  169 (53)  133 (64)  92 (51)  70 (52)  Other categories  306  38 (22)  21 (9)  16 (7)  8 (3)  Photosynthesis and respiration  141  20 (13)  42 (37)  18 (14)  12 (11)  Purines, pyrimidines, nucleosides and nucleotides  41  5 (2)  3 (1)  2 (1)  1 (1)  Regulatory functions  146  20 (8)  20 (11)  9 (4)  6 (4)  Transcription  30  8 (6)  6 (3)  2 (0)  2 (2)  Translation  168  23 (15)  17 (12)  8 (3)  4 (3)  Transport and binding proteins  196  36 (27)  28 (13)  28 (9)  16 (4)  Unknown  474  69 (21)  54 (31)  37 (31)  34 (22)  Total number  3165b  467 (209)  413 (242)  279 (162)  188 (124)  WT, wild type; OU, ΔPsbO:ΔPsbU mutant. a Genes were considered differentially regulated when fold change was >1.5 fold. b Total number of genes based on Kazusa annotation prior to May, 2002. c Number of genes with increased mRNA levels at pH 7.5 compared with pH 10.0 in a functional category. d Number of genes with increased mRNA levels in the mutant compared with the wild type in a functional category. View Large There were 198 genes up-regulated both on transfer from pH 7.5 to pH 10.0 and on transfer from pH 10.0 to pH 7.5: these had previously been identified and designated pH independent (Summerfield and Sherman 2008), and these genes are shown in Supplementary Table S1. The majority of these genes (157/198) had increased mRNA levels following transfer and represented functional categories including: translation (51 genes, 43 encoding ribosomal proteins); respiration and photosynthesis (23 genes, including genes encoding inducible bicarbonate transporters, carboxysome components and ATP synthase); biosynthesis of cofactors, prosthetic groups and carriers (11 genes); energy metabolism (10 genes); and hypothetical (22 genes). The majority of genes with decreased transcript abundance following transfer (34/41 genes) belonged to categories hypothetical, unknown or other. All except four of these genes were similarly regulated or unchanged in the ΔPsbO:ΔPsbU strain on transition from pH 10.0 to pH 7.5. Differences in the transcript level response to transfer from pH 10.0 to pH 7.5 in the wild type and the ΔPsbO:ΔPsbU strain Large numbers of genes involved in photosynthesis and respiration were increased in the ΔPsbO:ΔPsbU strain but not in the wild type at pH 7.5. These included genes encoding Cyt c oxidase (slr1136–slr1138); this is the major terminal oxidase in Synechocystis 6803 with a role in both the thylakoid and plasma membranes (Howitt and Vermaas 1998). This up-regulation probably reflects increased respiration in this strain due to the inability of the mutant to grow photoautotrophically at pH 7.5. In addition, genes encoding several components of the electron transport chain had increased transcript abundance in the ΔPsbO:ΔPsbU mutant at pH 7.5 compared with pH 10.0, but were unchanged in the wild type. This included transcripts encoding: PSI components (PsaL, PsaK1 and PsaE); core subunits of NADH dehydrogenase (NdhB and NdhD1); the Cyt b559 subunits together with other low molecular weight PSII proteins from the same operon (PsbEFLJ); and three genes (ssl0020, sll1584 and slr1828) encoding ferredoxin or ferredoxin-like proteins, including the most highly expressed ferredoxin gene, ssl0020, that is essential for viability (Poncelet et al. 1998) (Table 2). Ferredoxin is the final electron acceptor of the photosynthetic electron transport chain, interacting with both regulatory and metabolic polypeptides (Hanke et al. 2011). Table 2 Selected genesa showing altered transcript abundance on transition from pH 10.0 to pH 7.5 in Synechocystis sp. PCC 6803 wild type and a ΔPsbO:ΔPsbU strain No.  Gene Name  H2O2b  WTc  OUc  WT/OUd             pH 10.0  pH 7.5  Biosysthesis of cofactors, prosthetic groups      slr0233  trxQ  I  −2.3  −1.6  1.2  −1.2      slr0600  ntr  –  −1.5  −1.6  −1.3  −1.3      slr1562  grxX1  –  −1.5  −1.4  1.0  1.1  Cellular processes      sll0170  dnaK2  I  −2.1  −1.4  1.3  −1.1      sll0430  htpG  I  −2.3  −2.2  1.0  1.0      sll1514  hspA  I  −6.0  −1.7  1.6  −2.2      sll1933  dnaJ  –  −1.6  −1.1  1.4  1.1      sll0093  dnaJ  I  −1.7  −1.2  1.0  −1.3      sll0755  tpx  I  −1.3  −1.5  −1.2  1.0  Hypothetical      sll0939  hypo  I  −4.0  −2.9  1.0  −1.4      slr1128  hypo  –  −2.2  −1.5  1.0  −1.4      slr1963  ocp  I  −1.7  −1.1  1.8  1.2  Other      sll0550  flv3  I  −1.7  −1.3  1.2  −1.1      sll1621  ahpC  I  −1.1  −1.9  −1.4  −1.3      ssl2542  hliA  I  −1.4  −1.5  −1.6  −1.5      ssr2595  hliB  I  −1.7  −4.1  −2.6  −1.1  Photosynthesis and respiration   Respiration      slr1136  ctaD1  –  1.0  2.1  1.0  −2.0      slr1137  ctaC1  –  −1.1  1.5  1.2  −1.5      slr1138  ctaE1  –  1.0  1.6  1.0  −1.6   NADH dehydrogenase      sll0026  ndhF4  R  1.8  2.0  1.1  1.0      sll0027  ndhD4  R  2.1  1.8  1.2  1.4      sll0223  ndhB  –  −1.3  1.6  1.6  −1.3      slr0331  ndhD1  R  −1.5  2.9  2.4  −1.8      PSI                  slr1655  psaL  R  1.0  1.9  1.8  −1.1      ssr0390  psaK1  −  1.1  1.9  1.7  1.0      ssr2381  psaE  R  1.0  1.5  1.0  −1.6   PSII      sll0258  psbV  R  1.7  1.6  −1.1  1.0      sll1398  psb28  −  1.6  1.2  −1.2  1.1      slr1739  psb28-2  I  −3.2  −2.4  1.1  −1.2      Phycobilisomes                  sll1577  cpcB  –  1.2  1.7  1.4  1.0      sll1579  cpcC2  R  1.8  2.6  1.2  −1.1      sll1580  cpcC1  R  1.4  1.7  1.2  1.0      slr0335  apcE  R  1.0  2.1  2.0  −1.1      ssr3383  apcC  R  1.8  3.2  1.6  −1.2      slr1687  nblB  I  −2.3  −2.0  −1.4  −1.6      ssl0452  nblA1  I  −1.2  −1.5  −1.4  −1.1      ssl0453  nblA2  I  −2.1  −2.4  −1.1  1.0      Soluble electron carriers                  sll1584  fdl  –  1.3  1.7  1.1  −1.3      slr1828  petF  R  1.3  1.7  −1.4  −1.8      ssl0020  petF  –  1.1  2.1  1.6  −1.2  Regulatory functions      sll0797  rppA  –  1.5  1.5  −1.1  −1.1      sll0798  rppB  –  1.6  2.2  1.1  −1.2      sll1392  pfsR  –  −3.9  −3.3  1.0  −1.1      slr0311  hik29  –  1.5  2.0  1.0  −1.3      slr0533  hik10  –  1.5  2.2  1.0  −1.3      slr0947  rpaB  I  −1.7  −2.3  −1.2  −1.1      slr1285  hik34  I  −2.8  −1.7  1.2  −1.3      slr1529  ntrX  –  1.5  1.9  −1.1  −1.4  Transcription      sll0184  sigC  I  −1.9  −1.2  1.1  −1.4      sll0306  sigB  I  −2.5  −1.7  1.0  −1.5      sll1818  rpoA  –  2.9  1.4  −2.1  1.0      slr1129  rne  –  2.7  1.5  −1.4  1.3  Translation      sll0020  clpC  I  −2.0  −1.2  1.1  −1.5      slr0257  ctpB  –  −1.7  −1.2  1.3  −1.1      slr1641  clpB1  I  −1.6  −1.2  1.1  −1.2      slr1751  ctpC  –  −3.1  −1.3  1.9  −1.2  Gene clusters      sll0788  hypo  I  −4.0  −5.8  1.1  1.5      sll0789  rre34  I  −3.5  −11.5  −2.1  1.6      sll0790  hik31  R  −1.3  −12.5  −6.2  1.5      slr0074  ycf24  I  −2.0  −2.3  −1.1  1.0      slr0075  ycf16  I  −1.5  −2.1  1.0  1.4  No.  Gene Name  H2O2b  WTc  OUc  WT/OUd             pH 10.0  pH 7.5  Biosysthesis of cofactors, prosthetic groups      slr0233  trxQ  I  −2.3  −1.6  1.2  −1.2      slr0600  ntr  –  −1.5  −1.6  −1.3  −1.3      slr1562  grxX1  –  −1.5  −1.4  1.0  1.1  Cellular processes      sll0170  dnaK2  I  −2.1  −1.4  1.3  −1.1      sll0430  htpG  I  −2.3  −2.2  1.0  1.0      sll1514  hspA  I  −6.0  −1.7  1.6  −2.2      sll1933  dnaJ  –  −1.6  −1.1  1.4  1.1      sll0093  dnaJ  I  −1.7  −1.2  1.0  −1.3      sll0755  tpx  I  −1.3  −1.5  −1.2  1.0  Hypothetical      sll0939  hypo  I  −4.0  −2.9  1.0  −1.4      slr1128  hypo  –  −2.2  −1.5  1.0  −1.4      slr1963  ocp  I  −1.7  −1.1  1.8  1.2  Other      sll0550  flv3  I  −1.7  −1.3  1.2  −1.1      sll1621  ahpC  I  −1.1  −1.9  −1.4  −1.3      ssl2542  hliA  I  −1.4  −1.5  −1.6  −1.5      ssr2595  hliB  I  −1.7  −4.1  −2.6  −1.1  Photosynthesis and respiration   Respiration      slr1136  ctaD1  –  1.0  2.1  1.0  −2.0      slr1137  ctaC1  –  −1.1  1.5  1.2  −1.5      slr1138  ctaE1  –  1.0  1.6  1.0  −1.6   NADH dehydrogenase      sll0026  ndhF4  R  1.8  2.0  1.1  1.0      sll0027  ndhD4  R  2.1  1.8  1.2  1.4      sll0223  ndhB  –  −1.3  1.6  1.6  −1.3      slr0331  ndhD1  R  −1.5  2.9  2.4  −1.8      PSI                  slr1655  psaL  R  1.0  1.9  1.8  −1.1      ssr0390  psaK1  −  1.1  1.9  1.7  1.0      ssr2381  psaE  R  1.0  1.5  1.0  −1.6   PSII      sll0258  psbV  R  1.7  1.6  −1.1  1.0      sll1398  psb28  −  1.6  1.2  −1.2  1.1      slr1739  psb28-2  I  −3.2  −2.4  1.1  −1.2      Phycobilisomes                  sll1577  cpcB  –  1.2  1.7  1.4  1.0      sll1579  cpcC2  R  1.8  2.6  1.2  −1.1      sll1580  cpcC1  R  1.4  1.7  1.2  1.0      slr0335  apcE  R  1.0  2.1  2.0  −1.1      ssr3383  apcC  R  1.8  3.2  1.6  −1.2      slr1687  nblB  I  −2.3  −2.0  −1.4  −1.6      ssl0452  nblA1  I  −1.2  −1.5  −1.4  −1.1      ssl0453  nblA2  I  −2.1  −2.4  −1.1  1.0      Soluble electron carriers                  sll1584  fdl  –  1.3  1.7  1.1  −1.3      slr1828  petF  R  1.3  1.7  −1.4  −1.8      ssl0020  petF  –  1.1  2.1  1.6  −1.2  Regulatory functions      sll0797  rppA  –  1.5  1.5  −1.1  −1.1      sll0798  rppB  –  1.6  2.2  1.1  −1.2      sll1392  pfsR  –  −3.9  −3.3  1.0  −1.1      slr0311  hik29  –  1.5  2.0  1.0  −1.3      slr0533  hik10  –  1.5  2.2  1.0  −1.3      slr0947  rpaB  I  −1.7  −2.3  −1.2  −1.1      slr1285  hik34  I  −2.8  −1.7  1.2  −1.3      slr1529  ntrX  –  1.5  1.9  −1.1  −1.4  Transcription      sll0184  sigC  I  −1.9  −1.2  1.1  −1.4      sll0306  sigB  I  −2.5  −1.7  1.0  −1.5      sll1818  rpoA  –  2.9  1.4  −2.1  1.0      slr1129  rne  –  2.7  1.5  −1.4  1.3  Translation      sll0020  clpC  I  −2.0  −1.2  1.1  −1.5      slr0257  ctpB  –  −1.7  −1.2  1.3  −1.1      slr1641  clpB1  I  −1.6  −1.2  1.1  −1.2      slr1751  ctpC  –  −3.1  −1.3  1.9  −1.2  Gene clusters      sll0788  hypo  I  −4.0  −5.8  1.1  1.5      sll0789  rre34  I  −3.5  −11.5  −2.1  1.6      sll0790  hik31  R  −1.3  −12.5  −6.2  1.5      slr0074  ycf24  I  −2.0  −2.3  −1.1  1.0      slr0075  ycf16  I  −1.5  −2.1  1.0  1.4  WT, wild type; OU,ΔPsbO:ΔPsbU mutant. a Genes were considered differentially regulated when fold change was >1.5 fold. b Genes previously shown to have altered mRNA levels following exposure to H2O2 (Li et al. 2004, Kanesaki et al. 2007). R, repressed; I, induced. c Genes with altered mRNA levels at pH 7.5 vs. pH 10.0. d Genes with altered mRNA levels in the WT vs. OU. View Large Oxidative stress-responsive genes exhibit decreased transcript abundance in the wild type and the ΔPsbO:ΔPsbU strain on transfer to pH 7.5 Several genes with increased mRNA levels following exposure to hydrogen peroxide had decreased mRNA levels at pH 7.5; conversely, genes with decreased mRNA levels following hydrogen peroxide treatment were increased at pH 7.5 (Table 2). Genes that exhibited decreased transcript abundance in the wild type included dnaK2, dnaJ, hspA and htpG (Table 2) (cf. Li et al. 2004, Kanesaki et al. 2007). Two of these genes (htpG and hspA) had decreased mRNA levels in the mutant strain at pH 7.5. Increased transcript levels of these genes form part of a global response to numerous environmental factors, including oxidative, heat, UV light, high light and osmotic stresses (Hihara et al. 2001, Li et al. 2004, Singh et al. 2006, Rupprecht et al. 2007). Genes with roles in scavenging reactive oxygen species (ROS) exhibited decreased transcript levels at pH 7.5 compared with pH 10.0 in both strains. This included trxQ (slr0233) encoding one of the four thioredoxins in Synechocystis 6803 that is up-regulated in response to hydrogen peroxide (Perez-Perez et al. 2009a), and ntr encoding a putative NADP+ thioredoxin reductase (NTR); it has been suggested that NTR donates electrons to TrxQ (Perez-Perez et al. 2009a). Deletion of either trxQ or ntr increased sensitivity to oxidative stress (Hishiya et al. 2008, Perez-Perez et al. 2009a). One of the two genes encoding glutaredoxin (grx1, slr1562) showed decreased transcript abundance at pH 7.5 in the wild type: the Grx1 protein has been shown to accept electrons from NTR (Marteyn et al. 2009). In the ΔPsbO:ΔPsbU strain, the detoxification gene tpx (sll0755) encoding a peroxiredoxin shown to accept electrons from thioredoxin showed decreased mRNA levels at pH 7.5; this gene is also up-regulated under high light and heat exposure (Perez-Perez et al. 2009b). Similarly, the gene ahpC (sll1621) exhibited decreased mRNA levels at pH 7.5 in the ΔPsbO:ΔPsbU strain; this gene encodes a protein with sequence identity to type 2 peroxiredoxin-like hypothetical proteins, and may play a critical role in adapting to photooxidative stress (Kobayashi et al. 2004). Oxidative stress-responsive genes with roles in maintaining photosynthetic performance under stress conditions had decreased mRNA levels in both strains at pH 7.5. These included the hli genes encoding high-light-inducible polypeptides (HLIPs), also known as small CAB (Chl a/b-binding)-like proteins or SCPs (Dolganov et al. 1995, Funk et al. 1999), that are up-regulated under various stress conditions, and are thought to maintain photosynthetic performance by absorbing excess excitation energy or enabling the cells to cope with elevated ROS (He et al. 2001). Two of the four hli genes, hliA and hliB, had decreased mRNA levels at pH 7.5 and were up-regulated following hydrogen peroxide treatment. The HLIPs interact with Slr1128, a protein of unknown function (Wang et al. 2008): slr1128 transcript levels are decreased at pH 7.5 in both strains. The gene encoding OCP is induced following hydrogen peroxide treatment and has decreased mRNA levels in the wild type at pH 7.5. Several genes involved in photosynthesis that exhibited altered transcript levels following exposure to hydrogen peroxide were inversely affected by transfer to pH 7.5 (Table 2). In the wild type, decreased mRNA levels were observed for sll0550, encoding a flavoprotein (Flv3) involved in the Mehler reaction (Helman et al. 2003). The genes encoding NADH dehydrogenase subunits NdhD4 and NdhF4 had increased mRNA levels in both strains at pH 7.5; these were decreased following hydrogen peroxide treatment. These subunits are involved in CO2 uptake (Ogawa et al. 2000) and more recently they have been shown to be involved in PSI-mediated cyclic electron flow (Bernat et al. 2011). The gene encoding the PsbV PSII extrinsic protein had increased mRNA levels at pH 7.5 and decreased mRNA levels following hydrogen peroxide treatment, and the psb28-2 gene, which has similarity to psb28, had decreased mRNA levels at pH 7.5 and was up-regulated under stress including exposure to hydrogen peroxide, UV light and osmotic stress (see Table 2; Huang et al. 2002, Li et al. 2004, Paithoonrangsarid et al. 2004). Increased transcript levels of PBS structural genes (apcABC and cpcBA), and decreased transcription of genes nblA1, nblA2 and nblB, that encode proteins involved in PBS degradation, were observed at pH 7.5 in the wild type and to a greater extent in the ΔPsbO:ΔPsbU strain (Table 2). These PBS structural genes are repressed by hydrogen peroxide treatment while the nblA and nblB genes are induced. Response regulator RppA (Sll0797) is involved in regulation of the PBS structural genes and the nblA genes (Li and Sherman 2000). At pH 7.5, genes encoding this response regulator and neighboring histidine kinase (sll0798, rppB) exhibited increased transcript levels. Genes involved in transcriptional regulation that were induced by hydrogen peroxide treatment exhibited decreased mRNA levels in both strains at pH 7.5: hik34 encoding a histidine kinase and sigB encoding a sigma factor. In addition, the sigma factor-encoding gene, sigC, had decreased mRNA levels in the wild type. The gene cluster sll0788–sll0790 encodes a hypothetical protein, a response regulator (Rre34) and a histidine kinase (Hik31), respectively. Two of these genes, sll0789 and sll0788, were repressed by hydrogen peroxide treatment and exhibited decreased transcript levels at pH 7.5 in both strains. The gene encoding Hik31 was decreased in the mutant but not in the wild type at pH 7.5, and this gene was repressed by hydrogen peroxide treatment (Li et al. 2004, Kanesaki et al. 2007; see Table 2). Hik31 is involved in glucose metabolism (Kahlon et al. 2006) and has been shown to be involved in acclimation to low oxygen conditions, where it plays a role in down-regulating transcripts involved in photosynthesis (including the PBS components), chaperones and ribosomal proteins (Summerfield et al. 2011). Regulatory gene sll1392 (pfsR), which is involved in photosynthesis and an Fe-homeostasis stress response, had decreased mRNA levels at pH 7.5. In addition, transcriptional regulator slr0947 exhibited a decreased mRNA level at pH 7.5. Down-regulation of this gene has been shown to decrease energy transfer from the PBS to PSII (Ashby and Mullineaux, 1999); this is consistent with the decoupling of the PBS observed at pH 7.5 (Fig. 3). Under normal light conditions, Slr0947 (RpaB) binds upstream of the stress-responsive hliB gene and this binding is weaker under high light conditions (Kappell and Van Waasbergen 2007). Under low light conditions, RpaB acts a repressor of stress-responsive genes in Synechococcus elongatus strain PCC 7942, and these include hliA, although immunoprecipitation experiments indicate that additional regulatory mechanisms may be involved in this response (Seki et al., 2007, Hanaoka and Tanaka 2008). The down-regulation of rpaB at pH 7.5 is consistent with previous reports of altered energy transfer from the PBS to PSII, but is not consistent with the decreased levels of hliB under these conditions. Overall, transcriptional changes following transition from pH 10.0 to pH 7.5 indicated that growth at pH 10.0 stimulated a response with numerous similarities to the oxidative stress response in both strains. Expression of stress-responsive genes is decreased in the ΔPsbV:ΔCyanoQ strain at pH 7.5 To determine whether decreased mRNA levels of the stress responsive genes were shared by other pH-sensitive mutants, semi-quantitative reverse transcription–PCR (RT–PCR) was performed on RNA extracted from the ΔPsbV:ΔCyanoQ strain. The mRNA levels of a subset of genes that showed differential transcript abundance in the wild type and the ΔPsbO:ΔPsbU strain were examined. Genes encoding the heat shock protein HspA and sigma factor SigB are up-regulated following peroxide stress, and these genes showed decreased mRNA levels in the ΔPsbV:ΔCyanoQ strain on transfer from pH 10.0 to pH 7.5 (Fig. 4). The gene encoding Rre34 that is in an operon with the gene encoding the histidine kinase Hik31 had decreased transcript levels at pH 7.5, whereas sll1577 encoding a PBS subunit was unchanged. This is similar to the response for the wild type and the ΔPsbO:ΔPsbU strain. Hence, the wild type and the ΔPsbO:ΔPsbU and ΔPsbV:ΔCyanoQ mutants showed decreased mRNA levels of stress-responsive genes following transfer to pH 7.5. Based on our observations, we hypothesized that increased transcript abundance of oxidative stress-responsive genes at pH 10.0 improves resistance to oxidative stress. Fig. 4 View largeDownload slide Semi-quantitative RT–PCR of Synechocystis sp. PCC 6803 wild type and a ΔPsbV:ΔCyanoQ strain grown photoautotrophically at pH 10.0 and transferred to pH 7.5. Wild-type and ΔPsbV:ΔCyanoQ cells were grown in BG-11 at pH 10.0 and samples were harvested at t0; the remaining cells were transferred to pH 7.5 and samples were harvested at 1 and 2 h following transfer, as indicated above the lane. Equal amounts of RNA were used for each time point. Transcripts amplified were: sll0306, sigB; sll0789, rre34; sll1514, hspA; sll1577, cpcB; and rnpB. Fig. 4 View largeDownload slide Semi-quantitative RT–PCR of Synechocystis sp. PCC 6803 wild type and a ΔPsbV:ΔCyanoQ strain grown photoautotrophically at pH 10.0 and transferred to pH 7.5. Wild-type and ΔPsbV:ΔCyanoQ cells were grown in BG-11 at pH 10.0 and samples were harvested at t0; the remaining cells were transferred to pH 7.5 and samples were harvested at 1 and 2 h following transfer, as indicated above the lane. Equal amounts of RNA were used for each time point. Transcripts amplified were: sll0306, sigB; sll0789, rre34; sll1514, hspA; sll1577, cpcB; and rnpB. The wild type is more sensitive to rose bengal when grown in BG-11 medium at pH 7.5 compared with pH 10.0 To test whether the cells have increased resistance to oxidative stress at pH 10.0 compared with pH 7.5, we exposed wild-type cultures grown at pH 10.0 and at pH 7.5 to the singlet oxygen generator rose bengal. Photoautotrophic wild-type cultures at pH 7.5 exhibited increased sensitivity to rose bengal compared with cultures grown at pH 10.0 (Fig. 5A). The addition of either 5 or 2.5 µM rose bengal to BG-11 medium at pH 7.5 prevented photoautotrophic growth of the wild type (Fig. 5A). However, addition of 2.5 µM rose bengal to the wild type at pH 10.0 had no impact on doubling time, and the presence of 5 µM rose bengal only slightly decreased the growth rate (Fig. 5A). Absorbance at 543 nm was used to demonstrate the stability of rose bengal; this was similar in BG-11 at pH 7.5 and pH 10.0 (Fig. 5B). Photomixotrophic growth of the wild type in BG-11 medium buffered at pH 7.5 or pH 10.0 in the presence of 5 mM glucose was similar for the first 50 h, but by 100 h the pH 10.0 culture had reached a much higher OD730 nm than the pH 7.5 culture (almost 5 compared with ∼2.7), indicating a pH effect similar to that observed in photoautotrophically grown cultures (cf. Figs. 1A and 6A). Photomixotrophically grown cultures also showed pH-dependent sensitivity to rose bengal, with 2.5 µM rose bengal preventing photomixotrophic growth of the wild type at pH 7.5 but not pH 10.0 (Fig. 6A). Fig. 5 View largeDownload slide (A) Photoautotrophic growth of the wild-type Synechocystis sp. PCC 6803 strains in BG-11 medium as measured by the optical density at 730 nm. Growth of the wild-type strain plus 5 µM rose bengal (squares) and 2.5 µM rose bengal (circles), in BG-11 medium buffered at pH 7.5 (open symbols) or pH 10.0 (filled symbols). (B) Absorbance at 543 nm of 5 µM rose bengal in BG-11 medium buffered at pH 7.5 (open symbols) or pH 10.0 (filled symbols). Fig. 5 View largeDownload slide (A) Photoautotrophic growth of the wild-type Synechocystis sp. PCC 6803 strains in BG-11 medium as measured by the optical density at 730 nm. Growth of the wild-type strain plus 5 µM rose bengal (squares) and 2.5 µM rose bengal (circles), in BG-11 medium buffered at pH 7.5 (open symbols) or pH 10.0 (filled symbols). (B) Absorbance at 543 nm of 5 µM rose bengal in BG-11 medium buffered at pH 7.5 (open symbols) or pH 10.0 (filled symbols). Fig. 6 View largeDownload slide Photomixotrophic growth of the Synechocystis sp. PCC 6803 strains in BG-11 medium in the presence of 5 mM glucose as measured by the optical density at 730 nm. Cells were incubated in BG-11 medium buffered at pH 7.5 (open symbols) or pH 10.0 (filled symbols). (A) Growth of the wild-type strain (diamonds), plus 2.5 µM rose bengal (circles). (B) Growth of theΔPsbO:ΔPsbU strain (diamonds) and with the addition of 1 µM rose bengal (triangles). (C) Growth of the ΔPsbV:ΔCyanoQ strain (diamonds) and with the addition of 1 µM rose bengal (triangles). (D) Growth of the ΔPsbO:ΔPsbU pseudorevertant strain (diamonds) and with the addition of 1 µM rose bengal (triangles). (E) Growth of the wild-type strain plus 0.5 µM (diamonds), 1 µM (squares) or 2 µM methyl viologen (circles). (F) Growth of the ΔPsbO:ΔPsbU strain (diamonds) and with the addition of 0.1 µM (triangles), 0.5 µM (diamonds) or 2 µM methyl viologen (circles). Fig. 6 View largeDownload slide Photomixotrophic growth of the Synechocystis sp. PCC 6803 strains in BG-11 medium in the presence of 5 mM glucose as measured by the optical density at 730 nm. Cells were incubated in BG-11 medium buffered at pH 7.5 (open symbols) or pH 10.0 (filled symbols). (A) Growth of the wild-type strain (diamonds), plus 2.5 µM rose bengal (circles). (B) Growth of theΔPsbO:ΔPsbU strain (diamonds) and with the addition of 1 µM rose bengal (triangles). (C) Growth of the ΔPsbV:ΔCyanoQ strain (diamonds) and with the addition of 1 µM rose bengal (triangles). (D) Growth of the ΔPsbO:ΔPsbU pseudorevertant strain (diamonds) and with the addition of 1 µM rose bengal (triangles). (E) Growth of the wild-type strain plus 0.5 µM (diamonds), 1 µM (squares) or 2 µM methyl viologen (circles). (F) Growth of the ΔPsbO:ΔPsbU strain (diamonds) and with the addition of 0.1 µM (triangles), 0.5 µM (diamonds) or 2 µM methyl viologen (circles). The ΔPsbO:ΔPsbU and ΔPsbV:ΔCyanoQ strains are more sensitive to rose bengal than the wild type The ΔPsbO:ΔPsbU strain grew photomixotrophically at both pH 10.0 and pH 7.5, although cultures grew faster at pH 10.0 (Fig. 6B). The mutant was more sensitive than the wild type to rose bengal, with the mutant showing no growth in the presence of 2.5 µM rose bengal at pH 10.0 or pH 7.5 (data not shown). The presence of 1 µM rose bengal prevented growth of the ΔPsbO:ΔPsbU strain at pH 7.5 but not pH 10.0, although growth was reduced at pH 10.0 (Fig. 6B). Addition of 1 µM rose bengal had a similar impact on photomixotrophic growth of the ΔPsbV:ΔCyanoQ strain, with no growth of this strain at pH 7.5 and decreased growth at pH 10.0 (Fig. 6C). This strain appeared more sensitive than the ΔPsbO:ΔPsbU strain to the presence of rose bengal at pH 10.0 (Fig. 6B, C). The ΔPsbO:ΔPsbU pseudorevertant grew photomixotrophically at both pH 10.0 and pH 7.5, with a similar doubling time for the first ∼50 h, similar to the wild type (Fig. 6D). However, this strain was unable to grow photomixotrophically in the presence of 1 µM rose bengal at pH 7.5 (Fig. 6D). Growth was retarded under photomixotrophic conditions at pH 10.0 in the presence of 1 µM rose bengal to a similar extent to the ΔPsbO:ΔPsbU strain (Fig. 6B, D). The pseudorevertant’s increased sensitivity to rose bengal at pH 7.5 compared with pH 10.0 indicates that the mechanism enabling photoautotrophic growth of the pseudorevertant at pH 7.5 is not sufficient to confer resistance to rose bengal and is not the same mechanism enabling growth of the mutants at pH 10.0. To examine whether the increased sensitivity at pH 7.5 was observed in the presence of other ROS generators, methyl viologen was added to growth media as this generates superoxide. In both the wild type and ΔPsbO:ΔPsbU strain, sensitivity to methyl viologen was similar at pH 7.5 and pH 10.0 (Fig. 6E, F). At pH 7.5 and pH 10.0, growth of the ΔPsbO:ΔPsbU strain and the wild type was slightly retarded by the addition of 0.5 and 1 µM methyl viologen, respectively and neither strain grew in the presence of 2 µM methyl viologen. These data indicate that the observed pH sensitivity is not induced by all ROS generators. Discussion Altered Chl a variable fluorescence from PSII in pH-sensitive PSII mutants grown at pH 10.0 is not sufficient to explain recovery of photoautotrophic growth At elevated pH, we observed improved growth of the wild-type strain and growth of PSII mutants under photoautotrophic conditions, but there was no pH effect on photoheterotrophic growth, indicating a PSII-specific pH effect. Fluorescence induction measurements showed that the PSII mutants differed from the wild type, but growth under elevated pH did not restore a typical O–J–I–P transient as observed with wild-type cells. The 77 K fluorescence showed an increased emission at ∼685 nm in cells grown at pH 7.5; this may reflect partial decoupling of the PBS resulting in increased fluorescence from the terminal phycobilin emitters in the PSII mutants and to a lesser extent in the wild type. At pH 10.0, the increased fluorescence emission is reduced; this was not due to NPQ but is probably due to improved energy transfer from the PBS to PSII, and this may account in part for the pH-induced recovery of photoautotrophy in these strains. However, increased coupling of the PBS to PSII at pH 10.0 is not sufficient to explain fully the restoration of photoautotrophic growth in the PSII mutants as the ΔPsbO and ΔPsbV mutants exhibited increased fluorescence emission at pH 7.5, but grew photoautotrophically at this pH. Furthermore, the time course experiment (Fig. 3E, F) indicated that the decoupling of the PBS at pH 7.5 was a secondary effect. Transcript level changes on transition from pH 10.0 to pH 7.5 indicate maintenance of cellular homeostasis in the wild type and ΔPsbO:ΔPsbU strain Cell-wide transcriptional changes were observed in both the wild type and the ΔPsbO:ΔPsbU strain on transfer from growth medium buffered at pH 10.0 to pH 7.5. These changes were consistent with the results of a previous pH 7.5 to pH 10.0 transition experiment with the wild type (Summerfield and Sherman 2008), reflecting maintenance of cellular homeostasis, as genes with altered mRNA levels were involved in osmotic, pH and ion homeostasis. However, the kinetics of transcript level changes differ markedly depending on whether the transition is from pH 7.5 to pH 10.0 or from pH 10.0 to pH 7.5. The pH-independent differential regulation of 198 genes following both transfer from pH 7.5 to pH 10.0 and transfer from pH 10.0 to pH 7.5 identified further genes involved in maintenance of cellular homeostasis including adjusting to changes such as an altered bicarbonate/carbon dioxide ratio (Summerfield and Sherman 2008). In the ΔPsbO:ΔPsbU mutant, increased transcript levels of genes involved in photosynthesis may result from perturbation of the photosynthetic electron transport chain A major difference in the response of the two strains was increased transcript abundance of photosynthesis genes in the ΔPsbO:ΔPsbU mutant; these changes may result from perturbation of the photosynthetic electron transport chain due to the absence of PsbO and PsbU. The impaired PSII centers in the ΔPsbO:ΔPsbU and ΔPsbV:ΔCyanoQ mutants may increase ROS generation preventing photoautotrophic growth at pH 7.5. Balint et al. (2006) observed up-regulation of antioxidative mechanisms in their ΔpsbU mutant compared with wild-type Synechococcus sp. PCC 7942 and attributed this to an increased production of ROS in their ΔpsbU cells. Furthermore, the removal of PsbU in Synechocystis 6803 was shown to increase PBS fluorescence and to alter primary photochemistry of PSII (Veerman et al. 2005); here we have demonstrated a similar increase in PBS fluorescence in the absence of PsbO and PsbV but not CyanoQ. These findings are suggestive of a role for the extrinsic proteins in moderating light entering PSII and are consistent with the decreased psbO and psbU transcript levels observed by Tucker et al. (2001) in the unicellular diazotrophic cyanobacterium Cyanothece sp. ATCC 51142 in the dark, and the hypothesis that alterations on the oxidizing side of PSII mediate PSII activity as Cyanothece sp. ATCC 51142 proceeds through a 12 h light−12 h dark diurnal cycle (Meunier et al. 1998). Increased abundance of transcripts encoding oxidative stress-responsive genes at pH 10.0 may facilitate photoautotrophic growth of the pH-sensitive PSII mutants A proposed mechanism for the recovery of the pH-sensitive ΔPsbO:ΔPsbU and ΔPsbV:ΔCyanoQ mutants at pH 10.0 involves increased abundance of transcripts encoding oxidative stress-responsive genes at pH 10.0 (Fig. 7). In the mutant strains, we suggest increased mRNA levels of stress-responsive genes protect the impaired PSII centers, as well as the rest of the cell, from excessive ROS damage and facilitate photoautotrophic growth. Many genes exhibiting increased transcript abundance under oxidative stress also showed increased mRNA levels in the wild type following transfer from pH 7.5 to pH 10.0 (Summerfield and Sherman 2008). In addition, increased inorganic carbon availability at elevated pH may act to mitigate oxidative damage. However, there was no evidence to support inorganic carbon limitation at pH 7.5 compared with pH 10.0, as wild-type cells grown and measured at pH 7.5 and pH 10.0 without the addition of electron donors or acceptors had similar oxygen evolution rates (data not shown). Furthermore, comparison of DNA microarray data from our study with those of Wang et al. (2004) showed that none of the genes that exhibited increased transcript levels following a shift to lower carbon dioxide (up to 1–3 h) was increased on transfer to pH 7.5, and only three of the 23 genes with decreased mRNA levels following a shift to increased carbon dioxide were decreased on transfer to pH 7.5. Increased inorganic carbon availability may increase carbon fixation and reduce damage to PSII, contributing to the recovery of the strains at pH 10.0, but this does not appear sufficient to explain fully the recovery of the PSII mutants at elevated pH. Fig. 7 View largeDownload slide Model of the response to transition from pH 10.0 to pH 7.5 of Synechocystis sp. PCC 6803 wild type (WT) and a ΔPsbO:ΔPsbU mutant that is unable to grow photoautotrophically at pH 7.5, and comparison of a ΔPsbO:ΔPsbU mutant with a ΔPsbO:ΔPsbU pseudorevertant strain that is able to grow photoautotrophically at pH 7.5. Selected differentially abundant genes (≥1.5-fold change) at pH 10.0 compared with pH 7.5 in the wild type and the ΔPsbO:ΔPsbU strain are listed. The results are presented in the form of a Venn diagram that highlights the overlap among oxidative stress-induced genes and genes involved in scavenging ROS that exhibit increased mRNA levels at pH 10.0. In addition, genes with increased transcript levels in the pseudorevertant compared with the ΔPsbO:ΔPsbU strain at pH 7.5 are shown (Summerfield et al. 2007). Genes designated with a superscript 1 were reported previously to exhibit elevated transcripts following exposure to oxidative stress, and genes designated with a superscript 2 exhibited decreased transcripts following exposure to oxidative stress (Li et al. 2004, Kanesaki et al. 2007). We suggest that global stress-induced gene expression changes are sufficient to account for restoration of photoautotrophic growth in the ΔPsbO:ΔPsbU strain and the increased resistance to rose bengal (RB) observed in all strains at pH 10.0 and directly or indirectly led to changes in light harvesting. Fig. 7 View largeDownload slide Model of the response to transition from pH 10.0 to pH 7.5 of Synechocystis sp. PCC 6803 wild type (WT) and a ΔPsbO:ΔPsbU mutant that is unable to grow photoautotrophically at pH 7.5, and comparison of a ΔPsbO:ΔPsbU mutant with a ΔPsbO:ΔPsbU pseudorevertant strain that is able to grow photoautotrophically at pH 7.5. Selected differentially abundant genes (≥1.5-fold change) at pH 10.0 compared with pH 7.5 in the wild type and the ΔPsbO:ΔPsbU strain are listed. The results are presented in the form of a Venn diagram that highlights the overlap among oxidative stress-induced genes and genes involved in scavenging ROS that exhibit increased mRNA levels at pH 10.0. In addition, genes with increased transcript levels in the pseudorevertant compared with the ΔPsbO:ΔPsbU strain at pH 7.5 are shown (Summerfield et al. 2007). Genes designated with a superscript 1 were reported previously to exhibit elevated transcripts following exposure to oxidative stress, and genes designated with a superscript 2 exhibited decreased transcripts following exposure to oxidative stress (Li et al. 2004, Kanesaki et al. 2007). We suggest that global stress-induced gene expression changes are sufficient to account for restoration of photoautotrophic growth in the ΔPsbO:ΔPsbU strain and the increased resistance to rose bengal (RB) observed in all strains at pH 10.0 and directly or indirectly led to changes in light harvesting. The wild type and mutant strains are more resistant to singlet oxygen at pH 10.0 than at pH 7.5 All strains exhibited decreased sensitivity to rose bengal when grown at pH 10.0 compared with pH 7.5, thus demonstrating an increased resistance of these cells to oxidative stress at elevated pH. This pH 10.0 acclimation to rose bengal is similar to the acclimation response to low levels of singlet oxygen observed in Chlamydomonas reinhardtii (Ledford et al. 2007) where exposure to low levels of rose bengal resulted in increased transcript abundance of genes involved in the oxidative stress response and led to increased tolerance to exposure to higher levels of rose bengal. In our model, at pH 10.0, increased resistance to exogenous singlet oxygen from rose bengal arises due to changes in transcript levels of stress-responsive genes (Fig. 7). It is also noteworthy that singlet oxygen can give rise to other ROS such as hydrogen peroxide and superoxide through reactions with ascorbate (Miyake et al. 1991, Kramarenko et al. 2006). Moreover, since the primary site of singlet oxygen production in oxygenic photosynthesis is the antenna of PSII, our observed down-regulation of energy transfer from the PBS to the PSII reaction center in our mutants would be consistent with a mechanism to reduce singlet oxygen production at the non-permissive pH for photoautotrophic growth of these strains. The wild type and ΔPsbO:ΔPsbU strain did not show increased resistance to methyl viologen at pH 10.0. The different impact of the two ROS generators may be explained by their different stabilities. Methyl viologen is more stable than rose bengal, and methyl viologen stability and redox properties are not altered by pH (Bird and Kuhn, 1981). The stability of methyl viologen coupled with the fact that exposure to this herbicide stimulates a rapid transcriptional response in Synechocystis 6803 (Kobayashi et al., 2004) suggests that cultures grown at both pH 7.5 and pH 10.0 in the presence of methyl viologen may exhibit similar transcriptional profiles and this induces similar tolerance of ROS at both pH values. A subset of stress-responsive genes with elevated transcripts in the wild type or ΔPsbO:ΔPsbU strain at pH 10.0 compared with pH 7.5 also exhibited increased mRNA levels in an experiment comparing the ΔPsbO:ΔPsbU pseudorevertant with the ΔPsbO:ΔPsbU strain at pH 7.5 (Summerfield et al. 2007; Fig. 7), and we suggest that these transcripts may be involved in photoautotrophic growth of the pseudorevertant at pH 7.5. The fact that only a subset of the stress-responsive genes that had increased transcript levels in the wild type and ΔPsbO:ΔPsbU at pH 10.0 were increased in the pseudorevertant at pH 7.5 may account for the ability of this strain to grow photoautotrophically but still exhibit increased sensitivity to rose bengal at pH 7.5. Furthermore, additional stress-responsive genes such as tpx and sll1615 exhibited elevated transcript levels at pH 10.0 compared with pH 7.5 in the ΔPsbO:ΔPsbU mutant and these were not changed in the wild type and were not elevated in the pseudorevertant compared with the ΔPsbO:ΔPsbU strain at pH 7.5 (Fig. 7). Materials and Methods Cyanobacterial strains and growth conditions The glucose-tolerant variant of Synechocystis 6803 (Williams 1988) was used in this study. Cultures were maintained on BG-11 plates containing 5 mM glucose, 20 µM atrazine and appropriate antibiotics. In both solid and liquid media, chloramphenicol was present at a concentration of 15 µg ml−1, and erythromycin, kanamycin and spectinomycin were present at 25 µg ml−1. The BG-11 solid media were supplemented with 10 mM TES-NaOH (pH 8.2) and 0.3% sodium thiosulfate. Liquid cultures were bubbled with air and grown in BG-11 media containing either 25 mM HEPES (pH 7.5) or 25 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS; pH 10.0). Photomixotrophic and photoheterotrophic cultures contained 5 mM glucose and appropriate antibiotics; in addition, photoheterotrophic grown cultures contained 20 µM atrazine. Cultures were maintained at 30°C under constant illumination at 30 µmol photons m−2 s−1 in an MLR-350 growth cabinet (Sanyo Electric Biomedical Co. Ltd.). Growth curves were initiated from cultures harvested at log phase (OD730 nm between 0.4 and 0.8, measured with a Jasco V-550 UV/vis spectrophotometer; Jasco International) Mutants lacking PsbO and/or PsbU were produced as described in Eaton-Rye et al. (2003); these strains were constructed in the background of a control strain that has a kanamycin-resistance cassette located downstream of the psbB gene (Eaton-Rye and Vermaas 1991) which has a phenotype otherwise indistinguishable from that of the wild type. Strains lacking PsbV and/or CyanoQ were produced as described in Summerfield et al. (2005). For DNA micorarray experiments, cultures were grown in BG-11 at pH 10.0 until approximately 8 × 107 cells ml−1 and transferred to BG-11 medium buffered at pH 7.5. Cells were harvested at 0, 1 and 2 h following transfer to pH 7.5, and cells from the 0 and 2 h time points were used for microarray experiments. To measure the impact of ROS on growth, either rose bengal or methyl violgen was added at 0 h. 77 K Fluorescence emission spectra Samples were assayed for fluorescence emission at 77 K with a modified Perkin-Elmer MPF-3L fluorescence spectrophotometer equipped with a custom-built silver Dewar. Samples for analysis were prepared from mixotrophic cultures grown to mid-logarithmic phase (OD730 nm ∼0.6) in BG-11 at the appropriate pH. Cells were washed three times in their respective media to remove glucose, resuspended to a Chl a concentration of 2 µg Chl ml−1 and left to stabilize for 30 min at 30°C at a light intensity of 50 µmol photons m−2 s−1. A 1 ml aliquot of cells was loaded into electron paramagnetic resonance tubes, quickly frozen in liquid nitrogen and kept in liquid nitrogen until emission spectra were collected. 77 K fluorescence emission spectra were measured using two different excitation energy wavelengths for each sample: 440 nm to excite specifically Chl a and 580 nm to excite specifically the PBS. The excitation slit wavelength was set at 12 and 10 nm for 440 and 580 nm excitation, respectively; the emission slit wavelength was set at 4 nm. Spectra were normalized to the emission peak at 725 nm arising from PSI. pH transition time course for 77 K fluorescence emission spectra The wild type and the ΔPsbO:ΔPsbU mutant of Synechocystis sp. PCC 6803 were grown in BG-11 buffered at pH 7.5 or pH 10.0, in the presence of 5 mM glucose and appropriate antibiotics. Cells were harvested at mid-logarithmic phase (OD730 nm ∼0.8) and re-suspended at pH 7.5 or pH 10.0 in BG-11 at 2 µg Chl ml−1. After an initial measurement, cultures were washed once with unbuffered BG-11 and resuspended in BG-11 pH 10.0 (for the cultures previously incubated at pH 7.5) or pH 7.5 (for the cultures previously incubated at pH 10.0) and incubated at 30°C at a light intensity of 50 µmol photons m−2 s−1. Samples were taken at 2, 6 and 12 h after the pH transition. Variable Chl a fluorescence yield measurements Cells were grown to an OD730 nm of ∼0.6–0.8 and washed as described for 77 K fluorescence emission spectra measurements but were resuspended at 6 µg Chl ml−1. After 30 min at 30°C and a light intensity of 50 µmol photons m−2 s−1, cells were diluted to 3 µg Chl ml−1 in dark flasks, and dark adapted for 15 min at 30°C with gentle shaking. Chl a fluorescence induction measurements were made using an FL-3500 fluorometer (Photon Systems Instruments) in 4 ml quartz cuvettes with a 1 cm optical path containing a 2 ml sample volume. Fluorescence induction was induced by a 455 nm constant actinic light applied at 2,800 µmol photons m−2 s−1 for 5 s. Fluorescence was measured using weak 455 nm probing flashes. RNA extraction Total RNA was extracted and purified using phenol–chloroform extraction and CsCl gradient purification as previously described (Reddy et al. 1990, Singh and Sherman 2002). Microarray design The DNA microarray platform and construction were as described in Postier et al. (2003), and the cDNA labeling, pre-hybridization and hybridization protocols are described in detail in Singh et al. (2003). The microarray experiment involved a loop design that compared the wild type and the ΔPsbO:ΔPsbU strain grown in BG-11 at pH 10.0 with the cells after 2 h following a transition to BG-11 at pH 7.5 by using an analysis of variance (ANOVA) model (Singh et al. 2003, Li et al. 2004). Data acquisition included the ANOVA model approach to test the null hypothesis that a particular gene’s expression level did not differ between the treatments and to calculate a P-value (Singh et al. 2003). This experiment contained two genotypes (wild type and the ΔPsbO:ΔPsbU strain) and two stimuli (growth at pH 10.0 and transition to medium at pH 7.5) for a total of four treatment combinations. The effects of the absence of PsbO and PsbU and the transition from pH 10.0 to pH 7.5 were examined in the ANOVA design essentially as described in Kerr and Churchill (2001a), Kerr and Churchill (2001b), Singh et al. (2003) and Li et al. (2004). We used the FDR of 5% to control the proportion of significant results that are Type I errors (false rejection of the null hypothesis) as described in Summerfield and Sherman (2007). Genes with an FDR = 0.05 (corresponding to 5% expected false positives) and that exhibited a change of at least 1.5-fold were considered interesting and retained for further analysis. The P-value of these genes ranged from 2.7 × 10−2 to 6.3 × 10−17. Semi-quantitative RT–PCR DNase I treatment and reverse transcription were performed as described in Summerfield et al. (2008). PCR was carried out using 94°C for 1 min, followed by 20–30 cycles of: 94°C for 30 s, 52°C for 30 s and 68°C for 30–120 s (depending on amplicon size), to amplify regions of the genes listed below. Semi-quantitative RT–PCR was used to examine mRNA levels in the wild type and the ΔPsbV:ΔCyanoQ strain. The genes amplified, the primers used along with the PCR product size and the number of PCR cycles were: sll0306 (F 5′-gagtacctcagtctgtcg, R 5′-ctgatgttgagatgctgg, 260 bp, 25 cycles); sll0789 (F 5′-tacgtagttgattgggtgc, R 5′-gcatctaaaccctcaacc, 212 bp, 25 cycles); sll1514 (F 5′-gataatttccagcagcag, R 5′- gtcaaagttaggataccg, 350 bp, 25 cycles); sll1577 (F 5′-ttcccaagctgatgctcg, R 5′-agcactgcaatcaccacg, 439 bp, 20 cycles), and rnpB (F 5′-tgtcacagggaatctgagg, R 5′-gagagagttagtcgtaagc, 405 bp, 25 cycles). The rnpB gene was included as this transcript is frequently used as a constitutively expressed control for gene expression. Amplification products were separated on a 2% agarose Tris-acetate-EDTA or TAE gel. Funding This work was supported by the New Zealand Marsden Fund [Grant 09-UOO-118 to T.C.S.]; US Department of Energy [Grant DE-FG02–99ER20342 to L.A.S.]. Acknowledgment We thank our anonymous reviewers for their helpful comments. 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Journal

Plant and Cell Physiology – Oxford University Press

Published: Apr 24, 2013

Keywords: Alkaline pH Gene expression Oxidative stress Photosynthesis PSII extrinsic proteins Synechocystis sp. PCC 6803

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