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First published online September 7, 2007; 10.1104/pp.107.105023 Plant Physiology 145:1018-1030 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
Transcriptional Regulation of the Respiratory Genes in the Cyanobacterium Synechocystis sp. PCC 6803 during the Early Response to Glucose Feeding1,[C],[W],[OA]Laboratory of Plant Genomics, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305–806, Korea (S.L., J.-H.J.); Department of Biology, Chungnam National University, Daejeon 305–764, Korea (J.-Y.R., S.Y.K., J.Y.S., H.-T.C., Y.-I.P.); Department of Biological Sciences, Myongji University, Yongin, Kyunggi-do 449–728, Korea (S.-B.C.); Department of Plant Science, Seoul National University, Seoul 151–742, Korea (D.C.); and Institut Pasteur, Unité des Cyanobactéries (CNRS URA 2172) 28, F–75724 Paris cedex 15, France (N.T.d.M.)
The coordinated expression of the genes involved in respiration in the photosynthetic cyanobacterium Synechocystis sp. PCC 6803 during the early period of glucose (Glc) treatment is poorly understood. When photoautotrophically grown cells were supplemented with 10 mM Glc in the light or after a dark adaptation period of 14 h, significant increases in the respiratory activity, as determined by NAD(P)H turnover, respiratory O2 uptake rate, and cytosolic alkalization, were observed. At the same time, the transcript levels of 18 genes coding for enzymes associated with respiration increased with differential induction kinetics; these genes were classified into three groups based on their half-rising times. Transcript levels of the four genes gpi, zwf, pdhB, and atpB started to increase along with a net increase in NAD(P)H, while the onset of net NAD(P)H consumption coincided with an increase in those of the genes tktA, ppc, pdhD, icd, ndhD2, ndbA, ctaD1, cydA, and atpE. In contrast, the expression of the atpI/G/D/A/C genes coding for ATP synthase subunits was the slowest among respiratory genes and their expression started to accumulate only after the establishment of cytosolic alkalization. These differential effects of Glc on the transcript levels of respiratory genes were not observed by inactivation of the genes encoding the Glc transporter or glucokinase. In addition, several Glc analogs could not mimic the effects of Glc. Our findings suggest that genes encoding some enzymes involved in central carbon metabolism and oxidative phosphorylation are coordinately regulated at the transcriptional level during the switch of nutritional mode.
Respiration releases the energy stored in carbon compounds and also provides many carbon precursors for the biosynthesis of a wide variety of plant biomolecules, including amino acids, lipids, isoprenoids, and porphyrins. D-Glc (hereafter Glc) is an immediate substrate for the four major respiration processes: glycolysis, the oxidative pentose phosphate (OPP) pathway, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation. In plants, respiration is predominantly controlled by the key metabolites ADP and inorganic phosphate, and the ratio of NADPH to NADP+. The cellular concentration of ADP initially controls the rates of electron transfer and ATP synthesis, which in turn affect TCA cycle activity, finally resulting in the regulation of glycolytic reactions. The ratio of NADPH to NADP+ regulates the OPP pathway by directly influencing the activity of Glc-6-P dehydrogenase (G6PDH), the enzyme that catalyzes the initial reaction (for review, see Plaxton and Podesta, 2006  ).
In cyanobacteria such as Synechocystis sp. PCC 6803 (hereafter Synechocystis), Glc derived from glycogen or supplied exogenously is catabolized via the OPP pathway, the lower energy-conserving phase of glycolysis, and an incomplete TCA cycle (Stal and Moezelaar, 1997
As in higher plants, the increased respiration of Synechocystis cells that had been adapted to steady-state growth could be attributed to the posttranslational regulation of enzymes involved in Glc catabolism and oxidative phosphorylation. The activities of the enzymes involved in the glycolytic and OPP pathways, such as G6PDH, Fru-1,6-bisphosphatase, phosphofructokinase (PFK), Fru-1,6-bisphosphate aldolase (FBA), and pyruvate kinase (PK)-A and -F, are influenced by changes in metabolite concentrations (Stal and Moezelaar, 1997
Several recent studies indicate the possibility of the presence of transcriptional regulation of respiratory genes in Synechocystis cells. Indeed, when Synechocystis cells that had been preadapted in the dark for 2 d were exposed to Glc with or without light, transcriptional regulation of several glycolytic genes, including glk, pfkA (sll1196), fbaA (sll0018), gpmB (slr1124), and pk, was observed (Tabei et al., 2007
In this study, we report that respiratory genes in Synechocystis are coregulated at the transcriptional level in response to Glc and that this regulation correlates with subsequent increases in respiratory activity. Previous studies have analyzed gene expression profiles (Yang et al., 2002
Effects of Glc Feeding on the Expression of Respiratory Genes
The putative genes involved in carbohydrate metabolism were identified during the sequencing of the Synechocystis genome (Yang et al., 2002
Analysis of Transcript Levels by RT-PCR Nine Glc-inducible genes that were identified by the DNA microarray analysis (Table I) were chosen as representatives for the central metabolic pathway, the TCA cycle, respiratory electron transport, and ATP synthesis, and their expression levels were examined in wild-type, glucokinase (glk)-defective, or Glc transporter (glcP)-defective mutant cells. Transcript levels were examined by semiquantitative RT-PCR in the presence or absence of the transcription inhibitor rifampicin (Rif). Consistent with the DNA microarray data, transcripts of the nine genes (Fig. 2A ) and their relative expression (Fig. 2B) sharply increased during the first 15 min of incubation in the presence of 10 mM Glc in wild-type Synechocystis under photoautotrophic growth conditions. This effect was not observed in glk- and glcP-defective mutants (data not shown). In addition, Rif treatment completely inhibited the effects of Glc, indicating that the Glc-induced accumulation of transcripts was due to transcriptional activation.
To determine whether the gene induction that took place in the light also occurred in the dark, wild-type cells were preadapted to the dark for 14 h. This period of darkness was enough to fully repress the transcript accumulation of the 18 genes that were examined (Fig. 3A ). The Glc induction that was observed under light conditions in wild-type cells (Fig. 2) was also observed in the dark but not in the presence of Rif, nor in glk- and glcP-defective mutants (Fig. 3B). To determine whether gene induction was mediated by Glc per se or by its phosphorylation or further metabolism, dark-adapted wild-type cells were incubated with the Glc analogs L-Glc (LGlc), 3-O-methyl-Glc (OMG), 2-deoxy-Glc (2dGlc), and Man in the dark for 1 h as reported previously for Synechocystis (Ryu et al., 2004 ; Lee et al., 2005). LGlc, the enantiomer of Glc, is very poorly transported into cells, whereas OMG is a Glc analog that is transported into the cell but not phosphorylated by hexokinase. Man and 2dGlc are sugars that are phosphorylated by hexokinases but inhibit the production of Glc-6-P, NADPH, and ATP. The Glc-induced induction of the 18 genes was not observed in response to any of the Glc analogs used in this experiment (Fig. 3B), suggesting that downstream glucokinase-mediated phosphorylation is implicated in the transcriptional induction.
We next determined the transcript levels of 18 genes from the early time regime to the time required for Glc induction in the dark. Wild-type Synechocystis cells that had been preadapted to the dark for 14 h were incubated in the presence of 10 mM Glc for 1, 2, 5, 10, 15, 20, and 60 min under continuous dark conditions with or without the translation inhibitor chloramphenicol (Cm; 250 µg mL–1). After Glc treatment for given periods of time, Rif (300 µg mL–1) was immediately added to inhibit any further transcriptional activity that might occur during the cell-harvesting procedure. As shown in Figure 4A , the transcripts of the 18 Glc-inducible genes were rapidly induced, although their induction kinetics were not the same. Transcript levels of gpi, zwf, pdhB, and atpB immediately reached a peak or maximum level within 1 to 3 min after Glc treatment, while the atpI, G, D, A, and C transcript levels started to increase after a lag period that was longer than 5 min, showing slow half-rise time ranges varying from 11.9 to 13.5 min (Fig. 4B). Transcripts of the remaining nine genes, tktA, ppc, icd, pdhD, ndhD2, ndbA, ctaD1, cydA, and atpE, accumulated with moderate half-rise times in the range of 1.7 to 6.3 min. Three groups of genes were therefore identified based on their induction kinetics: the early, medium, and late responsive genes, termed groups I, II, and III, respectively.
When cells were treated with Glc in the presence of Cm, little or no change was detected in the induction kinetics of the genes investigated, with the exception of the atpD/A/C genes from group III (Supplemental Fig. S2). This indicates that the Glc-induced expression of respiratory genes in groups I and II is not dependent on de novo protein synthesis, but instead relies on the activation or inactivation of the regulatory proteins that were present before Glc treatment.
Glc treatment induces changes in cytosolic and membrane redox states, cellular energy charge (ATP to ADP ratio), and cytosolic pH, which affect metabolism and hormone signaling, and mediates Glc repression of yeast genes (Rolland et al., 2002
NAD(P)H produced by Glc metabolism is oxidized by NDH-1 (Mi et al., 1992 ; Cooley and Vermaas, 2001) in both photosynthetic and cytoplasmic membranes, resulting in increase in the redox state of the plastoquinone (PQ) pool (Kujat and Owttrim, 2000). The reduced PQ is oxidized by CtaI and quinol oxidase (Cyd; Cooley and Vermaas, 2001). Thus, the redox status of the PQ pool can be modulated using inhibitors such as Hg2+ and CN– since they inhibit electron inputs into the PQ pool via NDH-1 and outputs from the CtaI to the molecular oxygen, respectively. In other words, the PQ pool would tend to be oxidized by Hg2+ treatment and reduced by CN– treatment. When cells were treated with Glc for 2 min in the dark with various concentrations of Hg2+ or CN–, the Glc induction of group I genes was not inhibited, whereas the levels of group II genes were markedly decreased. These differential effects suggest that reduced NAD(P)H is required for group I gene expression, whereas either an oxidized cellular redox state or a reduced membrane redox status through consumption of NADPH via NDH-1 is responsible for the expression of group II genes. Indeed, after 8 min of Glc treatment, which resulted in the full expression of group II genes (Fig. 4), both inhibitors significantly inhibited the expression of group II genes. Despite their opposite effects on the PQ pool redox status, the similar inhibitory effects of both inhibitors on the expression of group II genes favor the view that a decrease in cytosolic redox rather than membrane redox modulation is involved in the expression of group II genes.
In cyanobacteria as well as in other organisms, respiratory electron transport is coupled to proton translocation (Schmetterer, 1994
To investigate whether the Glc-induced accumulation of respiratory gene transcripts correlates with an increase in respiration activity, NAD(P)H turnover, O2 uptake rates, PQ reduction, and the extent of cytosolic alkalization were measured in the presence of Glc under light and dark conditions. Synechocystis cells were grown in the light or adapted in the dark for 14 h, and then incubated with 10 mM Glc for up to 20 min. Upon the addition of Glc, the concentration of NAD(P)H increased to a peak after a lag of about 30 s, followed by a slow decline to levels below that initially observed in the light (40 µmol m–2 s–1) and especially in the dark (Fig. 6A ). This suggests that Glc treatment induces a rapid turnover of NAD(P)H that is particularly evident in the dark.
In the light, the increase in the turnover rate of NAD(P)H should reduce the O2 concentration as a result of respiration, leading to a decrease in the net O2 evolution during steady-state photosynthesis. Indeed, it was previously shown that photosynthetic O2 evolution decreased significantly when cells were adapted to photomixotrophic (Lee et al., 2005 ) or heterotrophic (Kurian et al., 2006) conditions compared to those in photoautotrophic conditions. As expected, even during the early phase of the shift from photoautotrophic to photomixotrophic growth, the addition of Glc reduced the O2 evolution rate by 10% (Fig. 6B) due to the increased consumption of O2 during illumination, as indicated by the apparent 2.4-fold increase in postillumination O2 uptake (Fig. 6B). At the same time, under dark conditions Glc treatment increased respiratory O2 uptake by 3.2-fold (from 6.2 ± 0.4 to 20.1 ± 0.5; Fig. 6B).
To detect a Glc-induced increase in respiratory electron flow, we indirectly measured the redox state of the PQ pool in thylakoids by monitoring the chlorophyll (Chl) a fluorescence yield in darkness (Cooley et al., 2000
A significant increase in the respiratory electron flow from NADPH to O2 should result in greater alkalization of the cytosol due to coupling of electron flow to H+ translocation. We therefore measured the extent of cytosolic alkalization by means of AY fluorescence, which has been used as a cytosolic pH indicator (Teuber et al., 2001 The Glc-induced changes in O2 evolution, and NADPH, PSII, and AY fluorescence were observed only in wild-type cells; they were not detected in glk- and glcP-defective cells or in response to other Glc analogs, such as LGlc, OMG, 2dGlc, and Man (data not shown).
Respiratory activity observed in Synechocystis cells grown under photomixotrophic conditions was higher than those grown under photoautotrophic conditions (Lee et al., 2005 ; Kurian et al., 2006). This difference can be ascribed to the modulation of the activity of enzymes associated with the glycolytic and the OPP pathways, the TCA cycle, and oxidative phosphorylation at the posttranslational level (Yang et al., 2002; Knowles and Plaxton, 2003; Kahlon et al., 2006). In addition to posttranslational regulation, the transcriptional regulation of respiratory genes was also indicated in Synechocystis during the light-to-dark transition based on the global analysis of circadian expression (Kucho et al., 2005). In this study, we demonstrated the presence of transcriptional regulation of respiratory activity during the early phase of photomixotrophic growth or heterotrophic growth. The transcript levels of a number of respiratory genes in the light (Fig. 2) or dark (Figs. 3 and 4) were significantly up-regulated along with an enhanced respiratory activity as estimated by NADPH content, the PQ pool redox state, cytosolic alkalization, and O2 consumption (Fig. 6). This transcriptional regulation is not likely to be unique to Synechocystis, but instead may be general phenomenon presumably found in all aerobes, including B. subtilis (Blencke et al., 2003) and E. coli (Vemuri et al., 2006), and higher plants (Plaxton and Podesta, 2006). The presence of transcriptional regulation of respiratory genes in Synechocystis cells undergoing a change in nutrition mode along with the well-known posttranslational regulation observed in cells acclimated to photoautotrophy, mixotrophy, or heterotrophy (Yang et al., 2002; Knowles and Plaxton, 2003; Singh and Sherman, 2005) indicates the presence of differential regulatory mechanisms that operate specifically during nutrient transition periods and steady-state growth.
Signals responsible for the Glc-induced changes in respiratory gene expression presented in this study might be generated by metabolism-related events downstream of Glc phosphorylation as the effects of Glc were not mimicked by several Glc analogs or by the functional loss of a Glc transporter or major glucokinase (Fig. 3). These results also rule out the possibility that the Glc transporter (or homologs) and the glucokinase have regulatory roles as they do in higher plants and yeast (Rolland et al., 2002
Consistent with these views, expression of all respiratory genes investigated in this study was inhibited by the G6PDH inhibitor in a concentration-dependent manner (Fig. 5). However, the nature of the signals that induced the expression of each group was different; increased or decreased cytosolic redox was required for group I or group II expression, respectively, whereas increased cytosolic pH was required for group III expression. Expression of group I genes was inhibited by 6-AN, but not by electron transport inhibitors Hg2+ and CN–. The G6PDH inhibitor would inhibit NADPH production by G6PDH in the presence of Glc and hence would maintain a lower intracellular NADPH to NADP+ ratio, whereas the direct (by Hg2+) or indirect (by CN–) NADPH oxidation inhibitors would keep NADPH in a reduced state, which would maintain a higher intracellular NADPH to NADP+ ratio (Woodmansee and Imlay, 2002 To act as regulatory signals, it is necessary for these signals to be induced prior to the Glc-induced accumulation of respiratory genes. Thus, we compared the mRNA induction kinetics of 18 genes to those of NADPH turnover and cytosolic alkalization (Fig. 7 ). We could distinguish approximately three groups of respiratory genes, whose grouping was consistent with the results of the half-rising times (Fig. 4). Genes encoding enzymes of the glycolytic and OPP pathways and ATP synthesis, including gpi, zwf, pdhB, and atpB, were induced earlier than the onset of the net decrease in NADPH content. Transcripts of genes in group II that were involved in the TCA cycle and respiratory electron transport chains as well as ATP synthesis (tktA, ppc, icd, pdhD, ndhD2, ndbA, ctaD1, cydA, and atpE) started to accumulate along with the onset of net NADPH consumption. After the establishment of cytosolic alkalization, transcript levels of atp1 operon genes (atpI, G, D, A, and C) started to increase (Fig. 7). Thus, it is most likely that Glc induction of group I genes of the glycolytic and OPP pathways is mediated by increases in cytosolic redox potential, whereas the expression of group II genes involved in the TCA cycle and respiratory electron transport chains is tightly correlated with a decrease in cellular redox potential. It seems that an increased cytosolic pH would be responsible for the Glc-induced expression of group III genes.
Molecular sensing for Glc-induced changes in the expression of respiratory genes has not been reported; however, regulatory proteins that participate in Glc signaling should be present in the cell prior to Glc feeding as the Glc-induced expression of genes in groups I and II was not inhibited in the presence of the translation inhibitor Cm (Supplemental Fig. S2). In contrast, Cm treatment inhibited the accumulation of atpD/A/C gene transcripts, indicating the requirement for regulatory proteins that are newly synthesized in response to Glc feeding (Supplemental Fig. S2). In Synechocystis, the atpI/G genes form the atp1 operon together with the atpD/A/C genes (Lill and Nelson, 1991 ); however, accumulation of these transcripts was differentially affected by Cm treatment (Supplemental Fig. S2), indicating the possibility of either the selective regulation of transcription or differences in mRNA stability in response to Glc feeding. Indeed, this is the case for the atp2 operon genes atpB and atpE, which have been classified into groups I and II, respectively. This Glc-induced differential regulation of the atp2 operon genes is not unique as heat stress has been shown to selectively up-regulate the atpF gene over other atp1 operon genes (Suzuki et al., 2005).
Recently, several sensors have been identified in Synechocystis that are responsible for the Glc-induced signaling of several glycolytic and OPP pathway genes during steady-state growth under photoautotrophic, mixotrophic, and heterotrophic conditions. One example of these sensors is His kinase 8 (Hik8), a homolog of SasA from Synechococcus that interacts with KaiC, a protein that is involved in circadian control (Iwasaki et al., 2000
In addition to two-component signaling regulatory proteins, sigma factors are also important regulators that determine the profile of transcripts via a direct interaction with the promoter sequence. SigE is one of the nine sigma factors present in Synechocystis and has been shown to positively regulate the expression of sugar catabolic genes. The transcript level of sigE peaks at the end of the day; therefore, it was previously proposed that sigE was under the control of Hik8 in order to regulate the expression of sugar catabolic genes, including the three glycolytic genes pfkA, gap1, and pyk1 and the four OPP pathway genes zwf, opcA, gnd, and tal (Osanai et al., 2005
Glc sensors that are specific for Glc feeding could be activated by a signaling molecule such as
The exogenous supplementation of Synechocystis cells with Glc could mimic the physiological state of cells under the light-to-dark transition. Glycogen synthesized during active photosynthesis is subsequently catabolized via the OPP pathway during dark periods (Stal and Moezelaar, 1997 Genes with similar induction kinetics, for example, those belonging to the same group, should contain a common cis-element(s) in their putative promoter regions. Indeed, we carried out promoter analysis of four genes in group I using the LuxAB reporter system and found a putative 12-bp-long consensus sequence (data not shown). This sequence was not found in genes in groups II and III. Base substitution experiments for putative group I common cis-elements as well as experiments to investigate group II- and III-specific cis-elements are currently in progress.
In conclusion, we have shown that in Synechocystis respiratory activity is regulated at the transcriptional level upon Glc feeding either in the light or the dark in a coordinated manner. It is highly likely that some of the respiratory genes possess a common cis-element(s) that permits the convergence and integration of Glc catabolism-related signals generated downstream of Glc phosphorylation. These signals would be detected by unknown regulatory proteins that are probably subject to modulation by cytosolic redox states and cytosolic pH. Furthermore, it remains to be determined whether PmgA or
Strains and Culture Conditions
Synechocystis wild type and Glc transporter (glcP; gene locus sll0771)- and glucokinase (glk; gene locus sll0593)-defective strains were grown in BG-11 inorganic medium buffered with 5 mM TES-NaOH, pH 8.0 (Ryu et al., 2004
Measurements of NAD(P)H (Ryu et al., 2003
For the analysis of transcript levels, total RNA was isolated using Trizol reagent (Life Technologies) and treated with RNase-free DNaseI (Promega). RT-PCR analyses were carried out using total RNA and gene-specific primers as employed in the PCR cloning for the DNA microarray construction (Supplemental Table S1). One microgram of total RNA was used for each reverse transcription in a 20-µL reaction volume, and 0.8 µL of the reaction mixture was subjected to PCR amplification under the following conditions: initial denaturation at 94°C for 5 min, followed by varying numbers of cycles of 94°C for 30 s, 55°C (60°C for the rRNA gene) for 20 s, and 72°C for 30 to 50 s, and a final extension step at 72°C for 7 min. These cDNA concentrations were within the linear response range of the PCR amplification (data not shown). The 16S rRNA gene (rrn16Sa) was included as a control. To detect possible DNA contamination, control reactions were performed in the absence of reverse transcriptase but in the presence of Taq DNA polymerase. The PCR products were routinely viewed with a GELDOC2000 densitometer and a computer-aided image analysis system (Bio-Rad). The identities of all RT-PCR products were confirmed by DNA sequencing. The relative expression of respiratory genes at each incubation time was calculated after quantifying the intensity of each band divided by that of rRNA. Four to five independent experiments were performed.
The partial-genome microarray contained 351 genes consisting of 11 functional KEGG categories (www.genome.ad.jp/kegg/), including photosynthesis, light harvesting, CO2 fixation, glycolysis, TCA cycle, the OPP pathway, and oxidative phosphorylation (Supplemental Table S1). Primers (Supplemental Table S1) were designed to produce approximately 0.5-kb DNA fragments with a Tm of 60°C at 50 mM NaCl and 20 nucleotides in length. Full or partial open reading frames were PCR amplified under the following conditions: initial denaturation at 95°C for 5 min, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 min, and a final extension at 72°C for 5 min. The PCR-amplified products were purified using Sephadex G-50 columns, dried, resuspended in 50% (v/v) dimethyl sulfoxide solution, and then spotted using an OmniGrid Microarrayer (Gene Machines) onto silanized glass slides (CMT-GAPS; Corning). Human gap1 (positive control), rrn16, and rnpB (negative controls) were included. Each slide was cross-linked with 300 mJ of short-wave UV irradiation (Stratalinker; Stratagene) and stored in a desiccator until use.
Total RNA (30 µg) was labeled by the direct incorporation of either Cy3- or Cy5-conjugated dUTP (Amersham Pharmacia Biotech) following the protocol of Dr. Patrick Brown's laboratory (cmgm.stanford.edu/pbrown/protocols/4_yeast_RNA.html). The purified and labeled probe was concentrated to a final volume of 5 µL for hybridization and mixed with 10 µL of formamide and 5 µL of 4x hybridization solution (Amersham Pharmacia Biotech). The probe was denatured for 3 min at 95°C, applied to the microarray slide, and then covered with a coverslip. After incubation for 16 h at 42°C, the microarray was successively washed at room temperature with 2x SSC (3.0 M NaCl, 300 mM sodium citrate, pH 7.0) containing 0.03% (w/v) SDS, 2x SSC, 1x SSC and then 0.2x SSC for 2 min each at room temperature and spin-dried at 700g for 5 min. In total, five biological replicates were conducted for each of the five different time points examined. The microarray slide was scanned with an Axon GenePix 4000A scanner (Molecular Devices) to generate a TIFF image. The PMT voltage was adjusted to yield a Cy3/Cy5 signal intensity that was as close to 1.0 as possible. After image acquisition, the spot intensities were measured using the Axon GenePix Pro 4.0 (Molecular Devices) image analysis software. Global normalization of the microarray data was conducted using the calculated ratio of median factor. The software package Acuity 3.1 (Molecular Devices) was then used to analyze the microarray data. The expression ratios were presented as log-transformed base 2. To remove the unreliable spots, we eliminated all the spots flagged as "bad" or "not found" by the image analysis software. In addition, all the spots with a forward intensity weaker than the background intensity were removed. All the genes with at least four validated data (from five replicates) were selected. Replicate data were combined and filtered by coefficient of variation, the value generated by dividing the SD with the mean. In this study we assumed that data with a coefficient of variation value of less than 0.5 were reliable data and selected for further analysis. Finally, a 2-fold change (the value of log-transformed base 2 was 1.0) between the mean expression intensities of the genes was considered to be indicative of differentially expressed genes. To assess the reliability of the microarray data, we compared the expression levels of several genes that increased (crtP) or decreased (psbA2 and cph1) upon addition of Glc on the DNA microarray and RT-PCR analysis. Transcript levels estimated by RT-PCR correlated well with those obtained with the microarray (r = 0.93, Supplemental Fig. S1).
The following materials are available in the online version of this article.
We thank G.E. Edwards and A.G. Ivanov for their critical reading the manuscript. Received July 2, 2007; accepted September 4, 2007; published September 7, 2007.
1 This work was supported by grants from the Crop Functional Genomics Center (CG2122) and KOSEF (R01–2004–000010246–0) funded by the Ministry of Science and Technology, Korea. S.Y.K. is a recipient of a BK21 Fellowship from the Ministry of Education, Korea. 
2 These authors contributed equally to the article. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Youn-Il Park (yipark{at}cnu.ac.kr).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.105023 * Corresponding author; e-mail yipark{at}cnu.ac.kr.
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ABSTRACT
RESULTS
F is the increased AY fluorescence due to the presence of Glc in the light (Light) or dark (Dark). 
-tocopherol. Indeed, Synechocystis mutant cells that were impaired in the biosynthesis of 
0.6) were supplemented with 10 mM Glc and further incubated in the light for up to 3 h. To test Glc specificity, photoautotrophically grown cells were incubated in the dark for 14 h and then exposed to 10 mM Glc and its analogs for an additional hour in the dark with or without the translation inhibitor Cm (250 µg mL–1). When required, various concentrations of the OPP pathway (6-AN) and the respiratory electron transport inhibitors (HgCl2 and KCN), as well as an uncoupler (CCCP), were added to the cell cultures 3 min before Glc treatment in the dark. Before harvesting, the transcription inhibitor Rif (300 µg mL–1) was added to prevent further transcriptional activity. Cell density and Chl a content were determined using a UV-VIS spectrophotometer (Myers et al., 1980
factor sigE. J Biol Chem 280: 30653–30659