Short-Term Temporal Metabolic Behavior in Halophilic Cyanobacterium Synechococcus sp. Strain PCC 7002 after Salt Shock

In response to salt stress, cyanobacteria increases the gene expression of Na+/H+ antiporter and K+ uptake system proteins and subsequently accumulate compatible solutes. However, alterations in the concentrations of metabolic intermediates functionally related to the early stage of the salt stress response have not been investigated. The halophilic cyanobacterium Synechococcus sp. PCC 7002 was subjected to salt shock with 0.5 and 1 M NaCl, then we performed metabolomics analysis by capillary electrophoresis/mass spectrometry (CE/MS) and gas chromatography/mass spectrometry (GC/MS) after cultivation for 1, 3, 10, and 24 h. Gene expression profiling using a microarray after 1 h of salt shock was also conducted. We observed suppression of the Calvin cycle and activation of glycolysis at both NaCl concentrations. However, there were several differences in the metabolic changes after salt shock following exposure to 0.5 M and 1 M NaCl: (i): the main compatible solute, glucosylglycerol, accumulated quickly at 0.5 M NaCl after 1 h but increased gradually for 10 h at 1 M NaCl; (ii) the oxidative pentose phosphate pathway and the tricarboxylic acid cycle were activated at 0.5 M NaCl; and (iii) the multi-functional compound spermidine greatly accumulated at 1 M NaCl. Our results show that Synechococcus sp. PCC 7002 acclimated to different levels of salt through a salt stress response involving the activation of different metabolic pathways.


Introduction
Cyanobacteria are oxygenic photosynthetic prokaryotes that hold promise for producing sustainable fuels and chemicals because of their fast growth compared to higher plants. In addition, some halophilic cyanobacteria can grow under high salinity conditions that are incompatible with plant growth [1,2]. Advances in our understanding of the salt stress response in halophilic cyanobacteria capillary electrophoresis/mass spectrometry (CE/MS) and gas chromatography/mass spectrometry (GC/MS). We also analyzed the expression levels of multiple genes after 1 h of salt shock using a microarray to support our metabolomic analysis data.

Results and Discussion
2.1. Cell Growth after Transfer to 0. 5

and 1 M NaCl Conditions
Cell growth curves after transfer to the different media are shown in Figure 1. Cell growth in 0.5 M NaCl was slightly slower than in the control, with the cell density of the control and 0.5 M NaCl cultures being 0.76 and 0.65 g dry-cell weight (DW) L −1 at 10 h, respectively. The cell growth stopped until 10 h upon suddenly increasing the salt concentration to 1 M from 0 M NaCl (Pre-cultured condition), and then they grew again from 10 h (Figure 1). The cells have probably acclimated to 1 M NaCl condition after 10 h.

Gene Expression of Na + /H + Antiporters and K + Transporters
Synechococcus sp. PCC 7002 possesses six different genes annotated as Na + /H + antiporters, identified from the genome on the National Center for Biotechnology Information website (https: //www.ncbi.nlm.nih.gov). The gene expression level of nhaS3 (A0577) increased 2.4-fold under the 1 M NaCl condition compared with the control (Table 1). Marin et al. reported the transient induction of several Na + /H + antiporter genes (nhaS1, nhaS3, and nhaS6) in Synechocystis sp. PCC 6803 after the addition of 684 mM NaCl [14]. Since NhaS3 is mainly localized in the thylakoid membrane in Synechocystis sp. PCC 6803 [10], NhaS1 and NhaS6 are predicted to play a role in the exocytic release of Na + from the cytoplasm. However, we did not observe two-fold higher induction of other Na + /H + antiporter genes (nhaS1, nhaS2, nhaS4, nhaS5, and nhaS6) under both NaCl conditions. Another candidate Na + transport system in the cytoplasmic membrane of Synechococcus sp. PCC 7002 is the F 1 F 0 -type Na + -ATPase homologous operon (atpA-II, atpB-II, atpC-II, atpD(A0749), atpF-II, atpG-II, and atpH-II), which may be absent in the Synechocystis sp. PCC 6803 genome [21]. The F 1 F 0 -type Na + -ATPase has the broad ion specificity for Na + , H + , or Li + in an anaerobic bacterium Propionigenium modestum [22,23]. The expression of these genes was enhanced in Synechococcus sp. PCC 7002 after 2 h of salt shock at 0.5 M and 1 M NaCl (Table 1). However, such gene expression changes have not observed after acclimating to hyper saline condition in Synechococcus sp. PCC 7002 [24]. Accordingly, F 1 F 0 -type Na + -ATPase are likely induced for short-term after salt shock in Synechococcus sp. PCC 7002. F 1 F 0 -type Na + -ATPase in cyanobacteria is generally believed to be an ATP synthase that uses the ∆Na + between the inside and the outside of the cell [21]. However, when driven in reverse, Na + -ATPase performs as an efficient sodium pump in P. modestum [25]. Na + -ATPase may play two roles in Synechococcus sp. PCC 7002: as an ATP synthase, and as a sodium pump in the salt stress response. Table 1. Gene expression of Na + /H + antiporters, F 1 F 0 -type Na + -ATPase subunits, and K + transporters with more than two-fold change at either 0.5 M or 1 M NaCl.

Gene Accession No. Function
Induction Factor 0.5 M NaCl 1 M NaCl The gene expression level of ktrB, a Ktr system subunit, is enhanced three-fold within 1 h of NaCl salt shock in Synechocystis sp. PCC 6803, while that of kdpA, a Kdp system subunit, does not change [7]. The Kdp system was previously reported to be of minor importance in the cyanobacterial salt stress response [2,20]. However, the gene expression levels of the Kdp system subunits were enhanced by salt shock in Synechococcus sp. PCC 7002 (Table 1), while those of the Ktr system subunits did not change. The expression levels of kdpA, kdpB, kdpC, kdpD, and kdpF was increased 32.0-, 33.0-, 13.9-, 20.2-, and 20.6-fold at 0.5 M NaCl, respectively (Table 1), although the gene expression of Ktr system was less than two-fold changes. Further investigations are needed to clarify the role of the Kdp system in the salt stress response of cyanobacteria. However, to our knowledge, this is the first report of the Kdp system responding to NaCl stress in cyanobacteria. Synechococcus sp. PCC 7002 is a good model strain for studying the cyanobacterial Kdp system.

Compatible Solute Synthesis
The moderately halotolerant cyanobacterium Synechococcus sp. PCC 7002 accumulates glucosylglycerol as the major compatible solute and sucrose as a secondary compatible solute [26]. Figure 2 shows the changes under different NaCl conditions in metabolite concentrations in glucosylglycerol synthesis, glycogen metabolism, glycolysis/gluconeogenesis, the Entner-Doudoroff (ED) pathway, the Calvin cycle, the OPP pathway, the TCA cycle, the urea cycle, and polyamine synthesis. Glucosylglycerol accumulated much faster at 0.5 M than 1 M NaCl. The glucosylglycerol level at 0.5 M NaCl was 70 µmol g −1 DW after 1 h and 84 µmol g −1 DW after 10 h. In contrast, the glucosylglycerol level at 1 M NaCl was just 3 µmol g −1 DW after 1 h, gradually increasing to 74 µmol g −1 DW after 10 h. Glucosylglycerol is synthesized from ADP-glucose and glycerol 3-phosphate via glucosylglycerol-phosphate by the cooperation of glycerol-3-phosphate dehydrogenase (GlpD), glycerol-phosphate synthase (GgpS) and glucosylglycerol-phosphate phosphatase ( Figure 2). The GlpD and GgpS genes were highly induced at both NaCl conditions (Table 2). However, glucosylglycerol accumulation was much slower at 1 M than 0.5 M NaCl. Glycerol 3-phosphate, the precursor metabolite of glucosylglycerol, accumulated after 3 h only at 1 M NaCl (50 µmol g −1 DW), which is more than 20-fold of that observed at 0.5 M NaCl. In addition, the concentration of ADP-glucose was up to 1 µmol g −1 DW at 1 M NaCl after 1-24 h, which was much lower than that obtained at 0.5 M NaCl. These results show that glucosylglycerol synthesis might be inhibited by a deficiency of ADP-glucose at 1 M NaCl.

Gene Accession No. Function
Induction Factor

M NaCl 1 M NaCl
Each value indicates the ratio of the level expression in stressed cells to that in control cells. Values shown are means (±SD) of three independent experiments.

Glycogen Metabolism and ADP-Glucose Synthesis
Cyanobacteria accumulate glycogen as a carbon sink during photosynthesis and consume glycogen as a carbon source to control intracellular metabolic homeostasis. The glycogen content fluctuates because of the changes in metabolic balance in response to cultivation conditions such as light intensity, CO 2 concentration, and nutrient concentration [28,29]. Glycogen is likely interconverted to glucosylglycerol by salt stress in some cyanobacteria [18,20], including Synechococcus sp. PCC 7002 [19]. A decrease in glycogen at 0.5 M and 1 M NaCl was inversely correlated with an increase in glucosylglycerol in this study ( Figure 2). The glycogen content decreased to 14.2% of DW after 1 h at 0.5 M NaCl compared to the control, and decreased to 12.7% of DW after 10 h at 1 M NaCl. There was no significant difference in glycogen metabolism gene expression between 0.5 M and 1 M NaCl. The gene expressions levels of alpha-glucan phosphorylase (GlgP) and 4-alpha-glucanotransferase (MalQ), which are related to glycogen degradation, were enhanced around two-fold at both conditions. The gene expression of glycerol-3-phosphate dehydrogenase (GlgC), a regulatory enzyme for glycogen production [30], and 1,4-alpha-glucan branching enzyme (A2819), were suppressed by around half at both conditions ( Table 2). Glycogen degradation was probably enhanced by salt stress at both NaCl conditions. ADP-glucose synthesis by GlgC enzyme is dependent on the ATP concentration. The oxygen consumption rate, oxygen evolution rate, and net photosynthetic activity related to ATP production under the control, 0.5 M and 1 M NaCl conditions are shown in Figure 3, in addition to the ATP concentrations. The oxygen consumption rate, oxygen evolution rate, and net photosynthetic activity after 2 h at 0.5 M NaCl were similar to the control. However, the oxygen consumption rate and oxygen evolution rate decreased to one-third and one-fourth after 2 h at 1 M NaCl. Net photosynthetic activity, calculated from the sum of the oxygen consumption rate and the oxygen evolution rate, decreased to one-third after 2 h at 1 M NaCl. The ATP concentration was much lower (around half) after 1 h at 1 M NaCl than at 0.5 M NaCl ( Figure 3). ADP-glucose synthesis might be suppressed at 1 M NaCl because of a decrease in photosynthetic ATP-production, resulting in the delay of glucosylglycerol synthesis at 1 M NaCl as shown in Figure 2.

Carbon Dioxide Fixation, Glycolysis, the ED Pathway and the OPP Pathway
Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) is inactivated in the presence of a high concentration of NaCl in both halophytes and glycophytes [1,31]. In this study, RuBP accumulated 3-4-fold higher at both NaCl conditions than in the control after 1 h (Figure 2), indicating that Rubisco in Synechococcus sp. PCC 7002 is inhibited by a sudden increase in NaCl concentration, similar to higher plants. Transcriptomic analysis showed that expression of the Rubisco large subunit gene (rbcL), Rubisco small subunit gene (rbcS), and Rubisco chaperone gene (rbcX) were greatly decreased at both NaCl conditions ( Table 3). The genes encoding NADP(H)-dependent glyceraldehyde-3-phosphate dehydrogenase (Gapdh2) and phosphoribulokinase (Prk), which are believed to regulate the Calvin cycle by altering the ratio of NADP(H)/NAD(H) [32,33], were downregulated at both NaCl conditions. In addition, the fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase gene (glpX) and the CO 2 concentrating mechanism (CCM) genes ccmK, ccmL, and ccmM were also downregulated at both conditions (Table 3). These results show that Synechococcus sp. PCC 7002 may suppress gene expression levels related to carbon dioxide fixation, along with inactivation of the Rubisco enzyme. CcmO is a predominant protein of β-carboxysome shell, and the CCM activity of the ccmO deletion mutant is significantly decreased [34].The gene expression level of ccmO in Synechococcus sp. PCC 7002 does not increase after acclimating to 1.5 M NaCl condition [24]. However, CcmO gene was up-regulated 2.4-fold after 0.5 M NaCl salt shock, while that was not changed at 1 M NaCl (Table 3). CcmO might play a role for maintaining β-carboxysome activity under moderately salt stress condition, although further investigations are needed to clarify the mechanism. Table 3. Gene expression related to the Calvin cycle, the CCM, glycolysis, the ED pathway, and the OPP pathway with more than two-fold change at either 0.5 M or 1 M NaCl.
Each value indicates the ratio of the level expression in stressed cells to that in control cells. Values shown are means (±standard deviations) of three independent experiments.
The concentration of FBP, DHAP, and GAP increased further after 3 h at 1 M NaCl, but decreased to the same level as the control at 0.5 M NaCl (Figure 2). Glycolysis must remain active even after 3 and 10 h at 1 M NaCl because the glucosylglycerol concentration was insufficient for establishing adequate intracellular pressure.
The ED pathway is also important for sugar catabolism of cyanobacteria in addition to glycolysis and the OPP pathway [36]. The gene expression of 6-phosphogluconate dehydratase was suppressed at both NaCl conditions (Table 3). In contrast, 2-Keto-3-deoxygluconate-6-phosphate aldolase gene was upregulated 3.5-fold at both NaCl conditions. The ED pathway might be suppressed after salt shock of 0.5 M and 1 M NaCl by the downregulation of 6-phosphogluconate dehydratase gene.
The OPP pathway metabolite 6-phosphogluconate (6PG) greatly accumulated after 1-10 h at 0.5 M NaCl compared to the control (Figure 2). The genes encoding the OPP pathway enzymes glucose-6-phosphate 1-dehydrogenase and fructose-1,6-bisphosphatase (Fbp) were upregulated at 0.5 M NaCl ( Table 3). The reactive oxygen species are more generated by salt-stress in cyanobacteria, which are detoxified via NADPH-dependent enzymes such as catalase, ascorbate peroxidase, and glutathione reductase [37]. The OPP pathway generate NADPH and are also involved in the production of a precursor metabolite for glucosylglycerol synthesis. The OPP pathway is probably activated for producing NADPH and glucosylglycerol under salt-stressed condition. However, the OPP pathway was less activated at 1 M NaCl than at 0.5 M NaCl. The 6PG concentration at 1 M NaCl was lower than at 0.5 M NaCl after 1-10 h (Figure 2). The gene expression levels of zwf and fbp were lower at 1 M than 0.5 M NaCl (Table 3). Glycolysis may be activated in preference to NADPH production via the OPP pathway at 1 M NaCl.

Tricarboxylic Acid Cycle, Urea Cycle, and Polyamine Synthesis
Isocitrate and malate, the substrates for reducing NAD cofactor in the TCA cycle, accumulated temporarily after 1 h at 0.5 M NaCl, then decreased after 3 h (Figure 2). The gene expression levels of 2-ketoglutarate decarboxylase (A2770), succinate-semialdehyde dehydrogenase (A2771), and succinate dehydrogenase subunit (SdhA) were upregulated 2-3-fold at 0.5 M NaCl ( Table 4). The TCA cycle is likely activated to produce more reducing agent for salt stress response, similar to the OPP pathway. In addition, succinate dehydrogenase may act as the main respiratory donor [38]. Energization of both the thylakoid and cytoplasmic membrane electron transfer chains is important under salt stress conditions in cyanobacteria [39], possibly to provide a proton gradient for Na + /H + transporter and ATP production to supplement photosynthetic activity. Synechococcus sp. PCC 7002 may obtain protons and electrons from succinate, in addition to the photosynthetic oxidation of water at 0.5 M NaCl.
Citrate and isocitrate levels were lower at 1 M NaCl than in the control after 1-10 h (Figure 2). The gene expression of pyruvate dehydrogenase (A0353, A0655, A0110) were decreased at both NaCl conditions (Table 4). However, since the pyruvate dehydrogenase is a salt sensitive enzyme in wheat plant Triticum aestivum [40], the pdh dehydrogenase activity at 1 M NaCl may be suppressed more than that at 0.5 M NaCl. In addition, the gene expression of methylisocitrate dehydratase (AcnB) was suppressed less at 1 M NaCl than at 0.5 M NaCl, and gene expression levels of A2771 were induced less at 1 M NaCl than at 0.5 M NaCl (Table 4). In contrast, metabolites related to the urea cycle, including arginine and aspartate, accumulated at 1 M NaCl after 1 and 3 h. Arginine and aspartate are the precursor metabolites of polyamines, and thus polyamine synthesis may be activated in preference to the production of reducing agents via the TCA cycle at 1 M NaCl.
Polyamines are polycationic aliphatic amines found in all living organisms and affect DNA folding, protein synthesis, membrane stability, photosystem II stability, and stress responses, although the underlying mechanisms are not completely understood [41]. Intracellular polyamine level increases because of salt stress in both halophytes and glycophytes [42,43]. Some polyamines, including putrescine, spermine, and spermidine, are found in cyanobacteria. An exogenous addition of spermidine enhanced the cell growth of Synechocystis sp. PCC 6803 in the presence of 0.5 M NaCl [44]. As shown in Figure 2, spermidine concentrations increased to 2.0 and 13.8 µmol g −1 DW at 0.5 M NaCl and 1 M NaCl after 1 h. The spermidine concentration remained high at 1 M NaCl (above 10 µmol g −1 DW) but decreased gradually at 0.5 M NaCl (Figure 2). The spermine concentration was much lower than that of spermidine but increased at both NaCl conditions after 1 h. The concentration of putrescine remained essentially constant under all conditions. Spermidine is the major polyamine accumulating in Synechococcus sp. PCC 7002 under salt stress conditions. Short-term increases in polyamines are observed in various higher plants such as a narrow-leafed ash Fraxinus angustifolia and a pepper Capsicum annuum [45,46], but such increases have not been investigated in cyanobacteria.
Since polyamines accumulate transiently also in the presence of 0.2 M mannitol in F. angustifolia [45], polyamine is likely also related to osmotic response in addition to ionic adjustment. We believe that polyamines aid short-term salt tolerance in cyanobacteria also.
The spermidine concentration increased at both NaCl conditions after 1 h. However, the expression levels of genes related to polyamine synthesis (speB, speE, metK, and speH) and the urea cycle (argG and argH) significantly decreased at both 0.5 M and 1 M NaCl after 1 h ( Table 4). The expression levels of genes related to polyamine synthesis might be enhanced in the very short term, then suppressed within several hours. Our results show that polyamines, especially spermidine, are probably important in the early stage of the salt stress response in Synechococcus sp. PCC 7002. Table 4. Gene expression related to the TCA cycle, the urea cycle, and polyamine synthesis with more than two-fold change at either 0.5 M or 1 M NaCl.

Gene Accession No. Function
Induction Factor 0.5 M NaCl 1 M NaCl

Gene Expression of Photosynthetic Components
Transcriptomic analysis revealed down-regulation of a large number of genes encoding subunits of photosystem I (PSI), subunits of photosystem II (PSII), phycobilisome (PBS), cytochrome b 6 f complex, F type H + -transporting ATP synthase, and electron carrier proteins (ferredoxin, ferredoxin-NADP reductase, and cytochrome c 6 ) at both 0.5 M and 1 M NaCl conditions. Several of the genes involved in the photosynthetic apparatus are listed in Table 5 and others are listed in Supplementary Materials  Table S1.
In contrast, the A2164 gene (which encodes PsbA) was up-regulated at both conditions, and the genes encoding phycocyanobilin lyase CpcE and CpcF were up-regulated at 1 M NaCl. Two main isoforms of protein PsbA, D1:1 and D1:2, are found in some cyanobacteria such as Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7942 [47]. The isoform D1:1 is more abundant in Synechococcus sp. PCC 7942 under optimal growth conditions, whereas D1:2 is upregulated when the cells are exposed to stress conditions such as high illumination or UVB levels [47]. The A0157 gene and the A2164 gene are upregulated, and the A1418 gene is downregulated in Synechococcus sp. PCC 7002 after acclimating to hyper saline condition [24]. We could presume that the D1:1 isoform is A0157 and A2164 and the D1:2 isoform is A1418. However, in contrast to long-term response, the A0157 gene was downregulated at short-term response (Table 5). Accordingly, there is likely also different function between A0157 and A2164 in Synechococcus sp. PCC 7002. Table 5. Gene expression of the photosynthesis reaction centers, phycobilisome, cytochrome b 6 f, electron carrier proteins, and ATP synthase with more than two-fold change at either 0.5 M or 1 M NaCl.

M NaCl 1 M NaCl
.6 (± 3.1) *1, the value is not described because the induction factor is less than 1.0.
Each value indicates the ratio of the level expression in stressed cells to that in control cells. Values shown are means (±standard deviations) of three independent experiments.

Concluding Remarks on Salt Stress
Response of Synechococcus sp. PCC 7002 at 0.5 M and 1 M NaCl Figure 4 shows activated and suppressed metabolic pathways at 0.5 M and 1 M NaCl after 1-3 h of salt shock. Glycogen degradation and glycolysis were activated at both NaCl conditions and the Calvin cycle was suppressed. However, there were also differences between cells exposed to 0.5 M and 1 M NaCl. The TCA cycle and the OPP pathway, accompanied by the production of reducing agents, were activated at 0.5 M NaCl, whereas high levels of the cationic polyamine spermidine accumulated at 1 M NaCl. These differences are probably due to the rate of glucosylglycerol synthesis. The level of glucosylglycerol at 1 M NaCl after 10 h was the same as at 0.5 M NaCl at 1 h ( Figure 2). The delay of glucosylglycerol synthesis at 1 M NaCl was likely due to a decrease in photosynthetic ATP-production ( Figure 3). Furthermore, the slow synthesis of glucosylglycerol at 1 M NaCl prolonged the inhibition of cell growth, photosynthetic activity, and Rubisco enzyme activity because of the high intracellular Na + level. Correspondingly, as the glucosylglycerol concentration increased at 1 M NaCl, photosynthesis activity and Rubisco enzyme activity recovered gradually, and cell growth resumed after 10 h (Figures 1-3). These results show that rapid glucosylglycerol accumulation is among the most important responses by Synechococcus sp. PCC 7002 for acclimating to higher salt conditions. However, even with high intracellular Na + levels, Synechococcus sp. PCC 7002 continued photosynthesis and produced glucosylglycerol slowly, indicating that it endures high salt stress by accumulating spermidine, thereby maintaining a suitable cation-anion balance and stabilizing membranes. Synechococcus sp. PCC 7002 thus overcomes metabolic inhibition because of salt stress by activating different metabolic pathways, depending on the salt stress level.

Strain and Culture Medium
Synechococcus sp. PCC 7002 was obtained from the Pasteur Culture Collection (Paris, France). Cells were pre-cultured in hexagonal flat flasks containing 600 mL fresh modified A medium without NaCl (AF medium) under continuous illumination at 400 µmol photons m −2 s −1 at 32 • C and bubbling air containing 2% (v/v) CO 2 at 100 mL min −1 . AF medium contained 1. . All reagents were purchased from Nacalai Tesque, Inc., (Kyoto, Japan). After pre-culturing in medium without NaCl for 24 h, the cells were inoculated into three type of medium without added NaCl (defined as control), containing 0.5 M NaCl, and containing 1 M NaCl at 0.32 g DW L −1 , and then cultivated at the same culturing conditions as the pre-culture for 48 h.

Cell Density
Cell growth was monitored turbidimetrically using the optical density at 750 nm (OD 750 ) measured using a UVmini-1240 (Shimadzu, Kyoto, Japan). Cell concentration was calculated as DW in medium from the linear correlation between DW and OD 750 . We determined OD 750 of 1 that corresponds to approximately 0.33 g DW L −1 .

Extraction of Intercellular Metabolites
Synechococcus sp. PCC 7002 cells, equivalent to 5 mg DW, were collected from the cultivation media and injected at a ratio of 4:1 (v/v) into a solution of 32.5% (v/v) methanol in water pre-chilled at −30 • C to rapidly terminate metabolism. The cells were harvested by centrifugation at 15,000× g for 5 min using a centrifuge and rotor precooled at −20 • C. The cells were resuspended in 2 mL methanol containing 190 nM (+)-10-camphorsulfonic acid, 31 µM L-methionine sulfone, and 31 µM piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES) as internal standards for mass analysis. Intracellular metabolites were extracted with a cold mixture of methanol-chloroform-water (10:3:1, by vol.), as described previously [48]. The mixture was shaken at 1200 rpm in a Bioshaker MBR-022UP (TAITEC, Saitama, Japan) for 30 min at 4 • C in the dark, then centrifuged at 22,000× g for 5 min at 4 • C. The supernatant cell extract (980 µL) was transferred to a clean tube, 440 µL MilliQ water was added, and the aqueous and organic layers were separated by centrifugation at 22,000× g for 5 min at 4 • C. Two aliquots (450 µL each) of the aqueous layer were transferred to clean tubes for analysis by CE/MS and GC/MS. After removing the solubilized proteins using a 5 kDa cut-off filter (Millipore, Bedford, MA, USA), the extracts were evaporated under reduced pressure using a Plus freeze dry system (FreeZone 2.5, Labconco, Kansas, MO, USA). The dried extracts were stored at -80 • C until used for metabolite analysis.

Metabolite Analysis Using CE/MS
The dried extracts were dissolved in 20 µL Milli-Q water and analyzed using an Agilent G7100 CE system, an Agilent G6224AA LC/MSD time-of-flight (TOF) system, and an Agilent 1200 series isocratic high-performance liquid chromatography (HPLC) pump equipped with a 1:100 splitter for delivery of the sheath liquid (Agilent Technologies, Palo Alto, CA, USA). Agilent Chem-Station software for CE/MS and MassHunter software for TOFMS were used as the system control and data acquisition software. The CE/MS analytical conditions for cationic and anionic metabolite analyses were as described previously [49].

Glycogen Quantification
Glycogen was extracted from cells as described previously [49]. Cells (10 mg DW) in 200 µL KOH (30%, w/v) were incubated in a heat block for 90 min at 95 • C and then placed on ice. The glycogen was precipitated by adding 600 µL ethanol pre-chilled to 4 • C to each cell extract and keeping the samples on ice for 1 h. The extracts were centrifuged at 3000× g for 5 min at 4 • C, then the pellets were washed twice with cold ethanol and dried for 10 min at 60 • C in a heat block. Each dried sample was reconstituted in 100 µL water, centrifuged at 10,000× g for 5 min at 4 • C, and the supernatant was subjected to HPLC analysis as described previously [51].

Measurement of Oxygen Consumption Rate and Oxygen Evolution Rate
The oxygen consumption and evolution rates were measured at 32 • C with a Clark-type oxygen electrode DW2/2 (Hansatech Instruments Ltd., King's Lynn, UK) controlled by a computerized oxygen monitoring system (OMS, Hansatech Instruments Ltd.). Oxygen consumption was monitored for 2 min under dark conditions, then oxygen evolution was monitored for 3 min by irradiation at 400 µmol photons m −2 s −1 using a halogen light source. The cell concentration in the reaction mixture was adjusted to 1.0 OD 750 for each cultivation medium. The net photosynthesis rate was calculated from the sum of the oxygen consumption rate and the oxygen evolution rate.

Profiles of Transcriptional Activity
Gene expression profiles of Synechococcus sp. PCC 7002 cultivated under different NaCl conditions were analyzed using a custom 8 × 15K microarray for 3187 Synechococcus sp. PCC 7002 genes (Agilent Technologies). After 1 h cultivation, the cells were harvested by centrifugation at 22,000× g for 5 min at 4 • C and immediately frozen in liquid nitrogen. Total RNA was extracted using a Total RNA Isolation Mini kit (Agilent Technologies) and complementary DNA labeled with cyanine 3 was generated using a Low Input Quick Amp Labeling kit (Agilent Technologies) according to the manufacture's protocol. After incubation at 65 • C for 17 h, the slides were scanned using an Agilent DNA Microarray scanner (Agilent Technologies). The scanning data were quantified and analyzed using Agilent feature extraction software and GeneSpring GX software (Agilent Technologies). We calculated fold-changes from the transcript level under 0.5 M and 1 M NaCl relative to control condition, and set fold-change threshold to more than 2.
Supplementary Materials: The following are available online at http://www.mdpi.com/2218-1989/9/12/297/s1. Table S1: Gene expression of the photosynthesis reaction center, phycobilisome, cytochrome b 6 f, and electron carrier proteins with less than two-fold induction at either 0.5 M or 1 M NaCl, except for the genes shown in Table 5.