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First published online September 10, 2004; 10.1104/pp.104.045047 Plant Physiology 136:3290-3300 (2004) © 2004 American Society of Plant Biologists Gene Expression Profiling Reflects Physiological Processes in Salt Acclimation of Synechocystis sp. Strain PCC 68031Universität Rostock, FB Biowissenschaften, Pflanzenphysiologie, 18051 Rostock, Germany (K.M., M.H.); Department of Regulation Biology, National Institute for Basic Biology, Myodaiji, Okazaki 444–8585, Japan (Y.K., N.M., I.S.); and Institute of Plant Physiology, 127276 Moscow, Russia (D.A.L.)
The kinetics of genome-wide responses of gene expression during the acclimation of cells of Synechocystis sp. PCC 6803 to salt stress were followed by DNA-microarray technique and compared to changes in main physiological parameters. During the first 30 min of salt stress, about 240 genes became induced higher than 3-fold, while about 140 genes were repressed. However, most changes in gene expression were only transient and observed among genes for hypothetical proteins. At 24 h after onset of salt stress conditions, the expression of only 39 genes remained significantly enhanced. Among them, many genes that encode proteins essential for salt acclimation were detected, while only a small number of genes for hypothetical proteins remained activated. Following the expression of genes for main functions of the cyanobacterial cell, i.e. PSI, PSII, phycobilisomes, and synthesis of compatible solutes, such as ion homeostasis, distinct kinetic patterns were found. While most of the genes for basal physiological functions were transiently repressed during the 1st h after the onset of salt stress, genes for proteins specifically related to salt acclimation were activated. This gene expression pattern reflects well the changes in main physiological processes in salt-stressed cells, i.e. transient inhibition of photosynthesis and pigment synthesis as well as immediate activation of synthesis of compatible solutes. The results clearly document that following the kinetics of genome-wide expression, profiling can be used to envisage physiological changes in the cyanobacterial cell after certain changes in growth conditions.
Microorganisms have often been used as model organisms to study basic physiological processes in living cells. Acclimation to high-salt concentrations is of high importance for basic as well as applied research, since a high percentage of irrigated land suffers from increasing levels of salts and can no longer be used to grow crop plants, which have generally a rather low halotolerance. The best-characterized photoautotrophic microorganism in relation to salt and osmotic stress response is the moderately halotolerant cyanobacterium Synechocystis sp. strain PCC 6803 (henceforth referred to as Synechocystis). Studies using this cyanobacterium are strongly promoted by the knowledge of its complete genome sequence (Kaneko et al., 1996  ). On this basis, DNA microarrays have been developed (CyanoCHIP from TaKaRa in Japan; Postier et al., 2003), which allow us to characterize transcriptional changes during environmental stress on the whole-genome level.
The basic response of Synechocystis cells to salt stress is well understood (Hagemann et al., 1999
Recently, the complete set of salt- and osmo-regulated genes after exposure of Synechocystis cells to salt stress for a short term (30 min) has been identified by DNA-microarray technique. In this study, 28 genes were found to be more than 3-fold induced specifically by salt, 11 genes by osmotic, and 34 genes by both stress treatments, respectively (Kanesaki et al., 2002 In this study, we intended to evaluate the kinetics of acclimation to salt stress by following changes in salt-regulated expression of genes by DNA-microarray analysis. Since this technique measures relative changes in mRNA levels and biological functions are fulfilled by proteins, it is a matter of discussion whether or not microarray data can shed light on physiological processes. Therefore, in parallel changes in main physiological parameters during salt acclimation were analyzed to evaluate the relationship between transcriptional and physiological changes. In general, surprisingly good correlations between kinetics of gene expression and of activities of corresponding processes have been found. However, even after comparisons of gene expression and physiological processes, it remains difficult to assign functions to the increasingly expressed genes, which encode proteins of unknown function during salt acclimation of Synechocystis cells.
Salt-Induced Gene Expression Is a Dynamic Process
During the experiments, cells were harvested at various time points (0 h or control before NaCl addition, 0.25, 0.5, 2, 6, and 24 h, respectively) after addition of 684 mM NaCl that corresponds to 4% (w/v). Since gene expression and physiological data of cyanobacterial cells depend critically on growth parameters, all experiments were performed at exactly the same light, CO2, and nutrient conditions. From a subset of samples, total RNA was isolated and used for genome-wide transcriptional analyses using a DNA-microarray technique. In a control experiment, total RNA samples from two independent control cultures were used for cDNA synthesis with Cy5 and Cy3, respectively. In the subsequent DNA-microarray experiment with these two cDNAs from control cells, for all genes induction values around 1 were found, which were always inside the range of 0.5 to 2.0 (data not shown). Since for the time course one representative experiment was chosen, a gene was regarded as induced when the induction factor was higher than 3.0 and regarded as repressed when the induction factor was lower than 0.33. In long-term salt-acclimated cells, genes were regarded as induced or repressed when exceeding the factors 2.0 and 0.5 which were found to represent significant changes (Kanesaki et al., 2002 The salt stress conditions used here induced the expression of approximately 360 genes and repressed the expression of approximately 200 genes, which revealed different kinetics. According to the time of maximum induction or repression, the salt-stress-regulated genes were categorized into four groups: Group 1 included all genes that revealed a maximum of induction or repression at 0.25 h (64/19 genes), group 2 included all genes with a maximum of induction or repression at 0.5 h (155/77 genes), group 3 included genes with a maximum of induction or repression at 2 h (123/106 genes), and group 4 included genes with a maximum of induction or repression at 6 h (15/11 genes) after salt shock, respectively. In Figure 1, the induction or repression of genes from the different groups was followed for each time point during salt stress acclimation. The top 10 genes, which showed the highest induction factor in each group, are listed in Table I.
The genes of group 1 were only shortly induced, and after 2 h most of them returned to the original level of expression, while at 24 h the expression of none of these genes was still regarded to be enhanced (Fig. 1A). Similar kinetics were found for the 155 genes of group 2, which showed their maximal induction at 30 min. At the first time point (15 min), 78 genes of group 2 were already regarded as induced, whereas the other 77 genes were newly induced at 30 min. Two hours after salt addition, only 43 genes of group 2 were still regarded as induced and the number decreased further, so that at 24 h the expression of no gene from this group was still enhanced (Fig. 1B). In the same manner, the induction of gene expression was followed for groups 3 and 4. In contrast to groups 1 and 2, the expression of 12 genes of group 3 and 1 gene of group 4 remained higher than 3-fold induced after salt treatment for 24 h (Fig. 1, C and D).
Comparable kinetics of the salt-regulated expression of genes regarded as repressed during salt stress was observed among the groups 1 to 4 (Fig. 1, E–H). However, the number of genes observed as repressed was lower than the number of induced genes, in particular in group 1. In the case of induction and repression, a large number of genes were transiently induced and only a small number of genes were still differently expressed 24 h after salt addition (Fig. 1). As was found previously (Kanesaki et al., 2002
The expression of 39 genes was enhanced more than 2-fold after salt treatment for 24 h. Most of them remained enhanced in fully salt-acclimated cells (Table II). The increased expression of these genes was confirmed in three independent experiments encompassing culture of cells, RNA isolation, cDNA synthesis, and DNA-microarray hybridization and evaluation. Most of the stably up-regulated genes belonged to group 3 with the maximum induction around 2 h during salt stress. Many of these genes encode proteins for salt acclimation, namely the ggpS, stpA, glpK, and glpD genes for enzymes involved in GG biosynthesis; the ggtABC genes for the ABC-type translocator for compatible solutes; the gene nhaS6 (sll0556) for one of the six Na+/H+ antiporters in Synechocystis; and the gene slr0765 for one of the nine mechano-sensitive channel-like proteins, such as the gene slr1894 for a probable DNA-binding stress protein. Some other stress-related genes also revealed an enhanced expression 24 h after the salt acclimation but returned to lower values during further cultivation in high salt medium. They include ssr2595 and ssl2542 for high light-induced proteins, sll0430 for the heat shock protein HtpG, and slr0193 for the RNA-binding protein Rbp3 (Table II). Three genes for proteins acting in basic carbohydrate metabolism (pfkA for phosphofructokinase, cbbA for Fru-1,6-biphosphate aldolase, and cfxE for pentose-5-phoshate-3-epimerase) were also found to be up-regulated after salt acclimation for 24 h, but they showed an unchanged expression after long-term salt acclimation. In addition, the genes sll1898 and sll1899 for proteins involved in cytochrome oxidase folding or assembly were detected. The enhanced cytochrome oxidase activity is characteristic for salt-stressed Synechocystis cells (Jeanjean et al., 1993
In order to evaluate the salt-dependent expression of genes, RNA was isolated from cells completely acclimated to salt concentrations of 1%, 2%, 3%, and 4% NaCl (w/v), respectively, and analyzed by DNA-microarray technique. Thereby the expressions of 13 genes shown in Table II were stepwise enhanced in correlation to external salt concentrations. In contrast to the most transiently induced genes of groups 1 and 2, only 8 genes existed among the up-regulated genes at 24 h, which encode proteins of unknown function (Table II).
In order to evaluate whether or not DNA-microarray experiments gave a reliable reflection of gene expression pattern in salt-shocked Synechocystis cells, the expression of several genes was followed by northern-blotting experiments (Fig. 2). In all cases, a good correspondence of both methods was found: genes whose expression was induced or repressed by microarray experiments showed the same behavior in northern-blotting assays (compare Fig. 2 and Figs. 3, 4, or 5 for selected genes). For genes encoding enzymes involved in osmoregulation (ggpS, stpA, and ggtB—all group 3 genes) and the stress-related protease subunit ClpC, an induction of the mRNA content was observed, while the mRNA contents of genes for subunits of the PSI, PSII, or phycobiliosmes (psaB, psbCD, and cpcABCD, respectively, all group 3 genes) were drastically repressed. The gene nhaS2 was selected as an example which shows almost no significant changes in the mRNA level in northern-blotting analysis as in DNA-microarray experiments. Furthermore, the comparison of both methods clearly indicates that changes found in DNA-microarray experiments do not only reflect relatively higher or lower levels of mRNA, but also reveal real induction and repression, respectively, of genes.
Synthesis and Uptake of Compatible Solutes During acclimation to salt stress, very high values of induction were observed for genes, which encode for proteins involved in GG synthesis (ggpS) and in the metabolism of glycerol-3-phosphate (glpD, glpK), a precursor of GG (Fig. 3A). The enhanced level of ggpS-mRNA corresponded to the increased level of GgpS protein as demonstrated by immunoblotting analysis and was also accompanied by an immediate increase in the GG content (Fig. 3B). The transient accumulation of low levels of Suc during salt stress was reflected by the transient induction of the sps gene for the key enzyme in Suc synthesis. Besides the synthesis, the uptake system Ggt of compatible solutes was activated at the level of transcription (Fig. 3A). After 24 h of salt stress, a steady-state level of GG is adjusted; the expression of all genes involved in GG synthesis or GG uptake was still found to be enhanced (Table II), whereby the level of sps transcript returned to the original level and no Suc was further detected.
Sodium export, potassium uptake, and water flux across the membrane are involved in ion homeostasis during salt stress response in Synechocystis. A transient induction of the genes nhaS1 (slr1727; data not shown), nhaS3 (sll0689; data not shown), and nhaS6 (sll0556) was observed, whereby other genes of this multigene family like nhaS2 (sll0273) were not found to be salt-regulated (Fig. 4A) as also demonstrated by northern-blotting analysis (Fig. 2). By flame photometry, we could not detect the very fast influx of Na+ after the onset of salt treatment as reported for other cyanobacterial strains (Blumwald et al., 1984
After addition of NaCl, the photosynthetic evolution of oxygen and the maximal efficiency of PSII photochemistry (Fv/Fm) decreased immediately (Fig. 5D). On the level of gene expression, there is also a pronounced decrease in mRNA levels of numerous genes for subunits of PSI (Fig. 5A) and PSII (Fig. 5B). For all main components of PSI, a transient decrease of the RNA levels was observed during salt stress response. Only a few genes for small subunits, such as psaK, showed no significant changes in transcript level. However, 24 h after salt addition, levels of mRNA of all psa genes did not differ from control levels.
Most genes for main components of PSII showed a decreased level of mRNA with the only exception of the psbA genes (Fig. 5B). The psbA2 (slr1311) and psbA3 (sll1867) genes, which were found to be induced after high light stress (Hihara et al., 2001 ), showed a decreased mRNA content immediately after shock, while a transient increase up to 2.0 was observed at 2 h after salt shock (Fig. 5B). Contrarily, for the psbA1 gene (slr1181), which is not transcribed in Synechocystis (Bouyoub et al., 1993), no change in the signal ratio was found after the salt shock treatment (data not shown). The psbV and psbO genes for proteins associated with the oxygen-evolving complex at the lumenal site of PSII displayed a decrease in levels of mRNA. Compared to the lowered expression of genes for PSI subunits, the decrease in the levels of mRNA was less pronounced among genes for PSII subunits. However, 24 h after salt addition, all genes for PSII subunits showed levels of mRNA comparable to those of control cells (Fig. 5B) as observed for PSI genes.
Levels of light-harvesting pigments, chlorophyll a (data not shown), and phycocyanin (Fig. 5D) changed during salt acclimation of Synechocystis cells. Corresponding to the decrease in the phycocyanin level, a strong decrease in transcript level of genes for phycocyanin and allo-phycocyanin linker proteins (cpc, apc; Fig. 5C) was found. Furthermore, an induction of the nblA genes for polypeptides involved in phycobilisome degradation and an nblB homolog (slr1687) for phycocyanin
The analyses of salt-induced gene expression at various time points revealed that salt-regulated genes could be categorized into four groups regarding their times of maximal induction. While the early induced genes are mostly induced only transiently, the genes that belong to groups characterized by maximum expression at later time usually showed a longer enhanced expression, and some were also observed as induced in cells acclimated for 24 h or several days to elevated salt concentrations (Table II). Among them are genes that are recognized to be essential for salt acclimation, such as ggpS and stpA involved in GG synthesis (Hagemann et al., 1999 ) and cfxE (Karandashova et al., 2002).
How far some of the early salt-induced genes are involved in the induction of genes appearing later during salt acclimation should be analyzed in further experiments. Among the early salt-induced genes, two for group 2 sigma factors (SigB, SigD) have been found (Kanesaki et al., 2002
For the investigation of long-term physiological acclimation in relation to gene expression under our conditions, incubation for at least 24 h at higher concentrations of salt was necessary. Thereby a rather small number of genes were recognized that show changes in the gene expression. Among them are only a few genes with unknown function. To investigate their function for salt acclimation, most of the genes (sll1862/63, sll0528, slr1687, slr1670, slr1544, and slr1932), which encode for hypothetical proteins and were found to be increasingly expressed at 24 h after salt shock, were mutated. In each case, completely segregated mutants were obtained. However, none of them showed any significant reduction in salt resistance level compared to cells of the wild type (M. Hagemann and K. Marin, unpublished data). Therefore, even a stable and high up-regulation of genes for unknown proteins such as for the highly induced sll1862/1863 genes does not necessarily indicate that these proteins are essential for salt acclimation. Additional analyses are necessary to elucidate the possible function of these and other hypothetical proteins. In Saccharomyces cerevisiae, only 0.88% of the genes that exhibited a significant increase in mRNA expression after salt shock also showed after mutation a significant decrease in fitness under NaCl stress conditions (Giaever et al., 2002
As a reason for the inhibition of photosynthesis and growth after the sudden increase in salt concentration, the influx of high concentrations of sodium ions within seconds into the cytoplasm was discussed (Blumwald et al., 1984
Two hours after salt addition, the potassium concentration decreased whereby the content of the compatible solute GG increased in parallel. An exchange of high concentrations of potassium and chloride ions by GG was also demonstrated for Synechocystis sp. strain PCC 6714 (Reed et al., 1985
Besides genes for proteins of de novo synthesis of GG, also genes for the GG-uptake system were induced and showed an enhanced long-term expression. This observation is in agreement with the results of Mikkat and Hagemann (2000)
For a functional interpretation of our results, we compared physiological data obtained during salt stress response with the expression of genes for components involved in the acclimation process. During salt stress, a long known observation in cyanobacteria is the breakdown of photosynthetic oxygen evolution and a decrease in the efficiency of photochemistry (Fig. 5; Reed et al., 1985
During a decrease in the photosynthetic electron-transport rate, an unchanged absorption of excitation energy could lead to a harmful production of reactive oxygen species. The degradation of the light-harvesting antenna complex, the phycobilisomes, can minimize this problem and was first observed in cyanobacteria during starvation for sulfur or nitrogen (Collier and Grossman, 1992
The dynamic process of salt acclimation is multiphasic, and the physiological responses correlate well in many cases with changes in the expression pattern of corresponding genes. Thereby, many known and numerous unknown genes were regarded to be differently expressed. In a functional description, main processes of the cyanobacterial metabolism like light harvesting and photosynthetic electron transport are affected, which is at least accompanied by the repression of genes for main components of the PSI and PSII such as the phycobilisomes. Since in bacteria, transcription and translation are closely connected, one may assume that a significant increase or decrease in mRNA levels should induce similar changes in protein amount, as has been shown for GgpS. The stress response comprises the regulation of ion homeostasis by activation of sodium exporters and potassium-uptake systems such as the synthesis of the compatible solute GG. Most of the processes are activated on the gene expression level only transiently, whereas during long-term acclimation the changed expression of only a few mostly known genes is necessary. It cannot be excluded that among them exist some genes for proteins whose up-regulation does not necessarily mean that they exhibit a function during salt acclimation. Overall, the genome-wide gene expression data obtained by DNA-microarray technique seem to be a valuable tool to predict concerted regulatory and physiological alterations at least during acclimation of a cyanobacterium to an environmental stress.
Strains and Culture Conditions
A strain of Synechocystis sp. PCC 6803, which was selected for higher transformation efficiency, kindly provided by Dr. S. Shestakov (Moscow State University, Department of Genetics), served as the wild type. Axenic cells were cultivated in batch cultures at 30°C bubbling with CO2-enriched air (5% [v/v]) and permanent illumination (170 µmol m–2 s–1; Osram L58W/32 Lumilux deluxe; Munich) using BG11 buffered with [tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid to pH 8.0 that contained 20 mM Na+ as a standard medium (Rippka et al., 1979
Interposon mutagenesis was used to inactivate genes that showed an enhanced expression in salt-acclimated cells (for a detailed description, see Huckauf et al., 2000
Exponentially growing cells (OD750 approximately 1.0) were harvested from 10 mL of the culture by centrifugation at 4,000g for 5 min at 2°C and then immediately frozen in liquid nitrogen and stored at –80°C. RNA was extracted with hot phenol and chloroform and purified using the High Pure RNA Isolation kit (Roche Diagnostics, Mannheim, Germany). A Synechocystis DNA microarray (CyanoCHIP) was obtained from TaKaRa (Kyoto). This microarray covered 3,079 of the 3,168 ORFs of Synechocystis, excluding genes for transposases. As hybridization probes, Cy3 dye-labeled or Cy5 dye-labeled cDNAs were used, which were synthesized by reverse transcription of 20 µg of total RNA using an RNA Fluorescence Labeling kit (M-MLV) provided by TaKaRa. After incubation for 16 h, the microarrays were rinsed with 2x SSC (1x SSC is 150 mM NaCl and 15 mM Na-citrate) at room temperature. They were washed with 2x SSC at 60°C for 10 min and 0.2x SSC, 0.1% SDS at 60°C for 5 min. Finally, they were rinsed with distilled water at room temperature. Moisture was removed with an air sprayer before analysis with an array scanner (GMS418; Affymetrix, Santa Clara, CA). For quantification with the ImaGene version 4.0 program (BioDiscovery, El Segundo, CA), the local background of each spot was subtracted and the signal was normalized by transforming it to the ratio of the spot-specific intensity relative to the total intensity of signals from all genes with the exception of rDNA genes. Therefore, changes in the level of transcript of each gene relative to the total level of mRNAs were calculated. Each gene is spotted twice on the microarray, allowing signal evaluation and error exclusion. The time-dependent expression of genes such as the analysis of gene expression in cells acclimated to 1%, 2%, or 3% NaCl, respectively, was analyzed by one DNA microarray per sample. The gene expression in cells acclimated for 5 d to 4% NaCl was analyzed by three independent biological replications and three DNA-microarray experiments.
Cells for these RNA extractions were cultivated in exactly the same conditions that were used for cells in DNA-microarray experiments. However, cells were harvested from cultures independent from that used for RNA isolations in microarray experiments. RNA was extracted and purified as above. Five micrograms of total RNA was transferred onto a nylon membrane (Roti-Nylon plus; Carl Roth GmbH, Karlsruhe, Germany) by semidry blotting. Gene-specific DNA probes for the northern-blotting experiments were obtained by PCR amplification of sequences that encode the genes ggpS (sll1566), psbDC (sll0849/0851), ggtB (slr0529), psaB (slr1834), stpA (slr0746), nhaS2 (sll0273), clpC (sll0020), and cpcBAC2C1 (sll1577/1578/1579/1580). Primers (18-mers) corresponding to the start and stop of the encoding sequences of the corresponding genes were designed according to the known genome sequence of Synechocystis (Kaneko et al., 1996
Cells for physiological characterization were cultivated in exactly the same conditions that were used for cells in DNA-microarray experiments. Salt stress experiments were performed in batch cultures by addition of crystalline NaCl to obtain the final concentration of 684 mM (4% [w/v]). In acclimation experiments, cells were used after precultivation for 5 d at the desired NaCl concentration with daily medium exchange. The content of GG and Suc was analyzed by HPLC (Schoor et al., 1996 Received April 22, 2004; returned for revision July 12, 2004; accepted July 18, 2004.
1 This work was supported by the Deutsche Forschungsgemeinschaft (grant to M.H.), by the Russian Foundation for Basic Research (grant no. 03–04–48581), by the Russian Science Support Foundation (grant to D.L.), by Grants-in-Aid for Scientific Research (grant no. 13854002 to N.M. and I.S.) and for Exploratory Research (grant no. 14654169 to I.S.), by the Japan Society for the Promotion of Science, by Grants-in-Aid for Scientific Research on Priority Areas (grant nos. 14086207 to N.M. and 15013260 to I.S.), by the Ministry of Education, Science, Sports and Culture of Japan, and by the Salt Science Research Foundation (grant no. 03S1 to I.S.). 
2 Present address: Institute of Biochemistry, University Cologne, D–50674 Cologne, Germany. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.045047. * Corresponding author; e-mail martin.hagemann{at}biologie.uni-rostock.de; fax 49–(0)381–4986112.
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ABSTRACT
RESULTS
-subunit phycocyanobilin lyase was measured. However, 24 h after salt addition, all genes for subunits of phycobilisomes with the exception of nblB displayed unchanged transcript levels in comparison to control cells, whereby the phycocyanin content in salt-acclimated cells remained lower (
