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First published online November 26, 2003; 10.1104/pp.103.029728 Plant Physiology 133:2069-2080 (2003) © 2003 American Society of Plant Biologists Inorganic Carbon Limitation Induces Transcripts Encoding Components of the CO2-Concentrating Mechanism in Synechococcus sp. PCC7942 through a Redox-Independent PathwayMolecular Plant Physiology Group, Research School of Biological Sciences, Australian National University, P.O. Box 475, Canberra, Australian Capital Territory 0200, Australia
The cyanobacterial CO2-concentrating mechanism (CCM) allows photosynthesis to proceed in CO2-limited aquatic environments, and its activity is modulated in response to inorganic carbon (Ci) availability. Real-time reverse transcriptase-PCR analysis was used to examine the transcriptional regulation of more than 30 CCM-related genes in Synechococcus sp. strain PCC7942 with an emphasis on genes encoding high-affinity Ci transporters and carboxysome-associated proteins. This approach was also used to test hypotheses about sensing of Ci limitation in cyanobacteria. The transcriptional response of Synechococcus sp. to severe Ci limitation occurs rapidly, being maximal within 30 to 60 min, and three distinct temporal responses were detected: (a) a rapid, transient induction for genes encoding carboxysome-associated proteins (ccmKLMNO, rbcLS, and icfA) and the transcriptional regulator, cmpR; (b) a slow sustained induction of psbAII; and (c) a rapid sustained induction of genes encoding the inducible Ci transporters cmpABCD, sbtA, and ndhF3-D3-chpY. The Ci-responsive transcripts investigated had half-lives of 15 min or less and were equally stable at high and low Ci. Through the use of a range of physiological conditions (light and Ci levels) and inhibitors such as 3-(3,4-dichlorophenyl)-1,1dimethylurea, glycolaldehyde, dithiothreitol, and ethoxyzolamide, we found that no strict correlation exists between expression of genes known to be induced under redox stress, such as psbAII, and the expression of the Ci-responsive CCM genes. We argue that redox stress, such as that which occurs under high-light stress, is unlikely to be a primary signal for sensing of Ci limitation in cyanobacteria. We discuss the data in relation to current theories of CO2 sensing in cyanobacteria.
In response to restrictions on the availability of CO2 in aquatic environments, cyanobacteria have evolved a unique ability to actively acquire inorganic carbon (Ci) and elevate CO2 up to 1,000-fold around the active site of Rubisco. In the unicellular, freshwater strains Synechococcus sp. PCC7942 and Synechocystis sp. PCC6803, this CO2-concentrating mechanism (CCM) consists of at least four Ci separate transport activities for the active uptake of both CO2 and HCO3 (for review, see Price et al., 2002  ; Shibata et al., 2002; Badger and Price, 2003). Ci is accumulated as HCO3- inside the cell but is converted to CO2 by a specifically localized carbonic anhydrase (CA) inside a microcompartment known as the carboxysome, which houses cellular Rubisco. The activity of the CCM is modulated according to the external availability of Ci and can occupy two extreme states—a low-affinity, constitutive state and a high-affinity, induced state—in addition to intermediate states (for review, see Kaplan and Reinhold, 1999; Price et al., 2002; Badger and Price, 2003).
Many of the genes encoding components of the cyanobacterial CCM have been identified in Synechococcus sp. PCC7942 and Synechocystis sp. PCC6803 (referred to as Synechococcus and Synechocystis hereafter), and a full genome sequence is available for Synechocystis. Two high-affinity, inducible HCO3- transport activities have been identified in these strains—an ABC-transporter, BCT1, encoded by the cmp operon (Omata et al., 1999
The signaling pathways that lead to up-regulation of the CCM in response to Ci limitation remain to be defined. It is unclear what sort of signal is actually perceived by cells subject to this stress. A number of competing theories have been proposed (for review, see Kaplan and Reinhold, 1999
The nature of the low-Ci signal remains elusive, and an integrated picture of the transcriptional regulation and physiological expression of the CCM is not yet available in Synechococcus, which has been favored for physiological and genetic studies of the CCM (Price et al., 2002
Early Transcriptional Response of Synechococcus to Severe Ci Limitation The responsiveness of several potential housekeeping genes to inorganic Ci limitation, including gap1 and ppc, encoding glyceraldehyde-3-phosphate dehydrogenase and phosphoenolpyruvate carboxylase, respectively, was determined. Exponentially growing Synechococcus high-Ci cells (bubbled with 1.7% CO2 in air) were harvested and immediately transferred to CO2-free air equilibrated buffer and aerated with CO2-free air for 3 h. First-strand cDNA was generated from normalized total RNA extracts from treated cells and quantitative real-time reverse transcriptase (RT)-PCR assays, using SYBR Green I to monitor product formation, were performed using ppc-specific primers (Table I). The gap1 transcript was found to vary considerably (results not shown), but the abundance of ppc was found to be relatively unresponsive to Ci limitation, varying by less than 35% in two independent experiments (Fig. 1.) Accordingly, ppc was used to normalize for small variations in starting template concentration and the efficiency of cDNA synthesis in all further experiments. To provide a "snapshot" of the early transcriptional response in Synechococcus, cells were subjected to a severe Ci limitation (as above) for 30 min. As a control, a culture was harvested, resuspended in high-Ci equilibrated medium, and returned to aeration with 1.7% CO2. The abundance of 35 transcripts encoding membrane transporters, carboxysome-associated genes, electron transport components, and regulatory factors in low-Ci-induced cells relative to high-Ci-grown cells was determined by real-time PCR (Fig. 2) using the gene-specific primers listed in Table I.
As has been observed in Synechocystis, we found that expression of transcripts encoding the known inducible Ci transport activities was induced by Ci limitation in Synechococcus. Previously, only expression of the cmp operon had been confirmed as being low-Ci responsive in this strain (Omata et al., 1999
Carboxysomes are known to increase in number in Synechococcus cells in response to Ci limitation (McKay et al., 1993
Severe Ci limitation is somewhat analogous to a high-light stress because depression of CO2 fixation would cause over-reduction of the plastoquinone pool. A preliminary report of the low-Ci inducibility of psbAII has emerged from another group (T. Omata, personal communication). We also found low-Ci-responsive expression of transcripts encoding other components of the photosynthetic electron transport chain. The psbAI/II/III and psbDI/II gene families, encoding alternative D1 and D2 proteins, responded to Ci limitation as they do to high-light stress (Schaefer and Golden, 1989
As with other Lys-R-type transcriptional regulators, expression of ndhR, a controller of the ndhD3/F3/chpY operon, and the cmpA-D operon regulator, cmpR, is stress responsive, and transcript abundance for these genes is induced in response to Ci limitation (Figge et al., 2001
To date, there has been no report of the kinetics of low-Ci-inducible gene expression using quantitative gene expression assays. High-Ci-grown Synechococcus cells were transferred to low Ci as described above, and cultures were sampled at 15, 30, 60, and 180 min after the switch. Changes in the abundance of a representative subset of transcripts from the full set previously assayed, compared with time zero, were quantified as before using real-time PCR assays. The low-Ci-inducible transcripts exhibited kinetic patterns of three types. First, the abundance of one transcript, psbAII, increased steadily over the first 60 min and was sustained at this level for 3 h (Fig. 3A). A second group of transcripts, encoding carboxysome-associated proteins and cmpR, was rapidly induced but, in the case of ccmM, rbcL, and icfA, returned to uninduced levels approximately 60 min after induction and in the case of cmpR, by 3 h (Fig. 3B). It is possible that carboxysome gene expression may also be strongly but transiently Ci responsive in Synechocystis and that there is no significant difference in the regulation of these genes between the two strains. A third group of transcripts, encoding the inducible Ci transport activities, was rapidly induced to maximum amounts by 30 to 60 min (Fig. 3C) and was maintained at relatively high amounts over the 3-h time course. A slight transient reduction in the amount of rbcR transcript was detected at 15 min, whereas the amount of transcript for psaE slowly declined over the 3 h (Fig. 3A).
The physiological response of Synechococcus cells to Ci limitation was also monitored for 3 h after the transition from bubbling with 1.7% CO2 to bubbling with CO2-free air. The maximum photosynthetic rate of cells during this period was largely constant, indicating that cells were not photo-inhibited (Fig. 3D). The relative affinity for Ci, as determined by net K0.5 (Ci), started to increase soon after transfer to Ci limitation (Fig. 3D). The maximal increase in affinity was achieved between 1 and 1.5 h (Fig. 3D), similar to what has been reported recently in Synechocystis (McGinn et al., 2003
Relatively little data exists about the relaxation of high-affinity CCMs and consequently the rate of turnover of CCM components. To address this question, exponentially growing high-Ci Synechococcus cells were transferred to CO2-free equilibrated growth medium and bubbled for 2 h with CO2-free air. Cells were then swapped to bubbling with 1.7% CO2 (designated time zero) and supplemented with NaHCO3 to a final concentration of 5 mM. This amount of supplementary Ci equals the total concentration of Ci found in actively growing cultures bubbled with 1.7% CO2 under steady-state conditions (data not shown). Cultures were sampled over the next 48 h to determine the relative affinity for Ci, as K0.5 (Ci) (Fig. 4). The K0.5 (Ci) decreased from 163 to 15 µM during the initial CO2-free air induction. For 25 h after the return to high-CO2 conditions, the K0.5 (Ci) rose steadily to 133 µM, about 80% of the initial high-CO2 value, but cells required a further 24 h growth at high CO2 to return to the initial low-affinity state. Previous work on the relaxation of the high-affinity CCM in Synechocystis found that it took considerably longer (over 40 h) for cells to approach a low-affinity state (Benschop et al., 2003
To examine the turnover and stability of CCM-related transcripts in cells subject to either Ci limitation or Ci sufficiency, an exponentially growing high-Ci Synechococcus cell culture was transferred to CO2-free equilibrated growth medium and bubbled for 30 min with CO2-free air. This period is sufficient to strongly induce Ci-responsive CCM transcripts but brief enough to capture the transient increases observed for transcripts such as ccmM and cmpR (Fig. 3, A-C). Subsequently, the culture was split four ways. Two cultures were supplemented with 5 mM NaHCO3 (designated time zero) and swapped to bubbling with 1.7% CO2 in the presence or absence of 200 µg mL-1 of the transcriptional inhibitor rifampicin for a further 2 h. The remaining two cultures were supplemented with 5 mM NaCl, to balance sodium, and bubbled with CO2-free air with or without rifampicin as above. The four cultures were sampled for transcript analysis at various times after the treatments. Changes in the abundance of cmpA, sbtA, chpY, cmpR, ccmM, and psbAII compared with time zero, were quantified as before using real-time PCR assays (Fig. 5). The cmpA transcript had a half-life of approximately 15 min at both high and low Ci. However, it was even more rapidly degraded in high-Ci cells in the absence of rifampicin, being two-thirds less abundant at 15 min than in high-Ci cells treated with rifampicin. This result suggests that the synthesis of a factor is required for rapid degradation of the cmpA transcript. The sbtA and chpY transcripts responded similarly to cmpA (results not shown), however, both sbtA and chpY exhibited a transient insensitivity to rifampicin treatment followed by rapid degradation. This type of response has been observed in Escherichia coli and is thought possibly to result from different sigma factors influencing the interaction of rifampicin with the RNA polymerase holoenzyme (Selinger et al., 2003
To assess whether high-light stress is sufficient to induce CCM-related gene expression, as has been reported in Synechocystis (Hihara et al., 2001
To examine some competing theories about the nature of the low-Ci signal that is sensed by Synechococcus cells, high-Ci-grown cells were treated for 30 min with 20 µM 3-(3,4-dichlorophenyl)-1,1dimethylurea (DCMU) or 20 µM 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone (DBMIB)—agents that increase the reduction state of the electron carriers between photosystems II and I. Cells were also treated with 5 mM dithiothreitol (DTT)red, 200 µM CA of the inhibitor ethoxyzolamide (EZ), and 10 mM of the carbon fixation inhibitor glycolaldehyde (GLY). The concentration of GLY used has only a minor effect on CO2 transport (Salon et al., 1998
We also tested the effect of GLY on low-Ci induction of CCM-related gene expression. Equal volumes of cells from an exponentially growing high-Ci Syn- echococcus culture were harvested and resuspended in medium containing approximately 100 µM total Ci. Cells were then bubbled slowly with air, using a standard pipette (approximately 0.15 L min-1) for 30 min to create a moderate level of Ci limitation. At the swap over, one of the cultures was treated with 10 mM GLY, and cells were incubated in the dark for 5 min. As a control, an equivalent number of high-Ci cells were resuspended in high-Ci-equilibrated medium and returned to bubbling with 1.7% CO2. Treatment of cells with GLY reduced the low-Ci induction of chpY and cmpR by more than 80% and that of cmpA and sbtA by more than 99% (Fig. 7C). By contrast, GLY treatment resulted in a 10-fold greater induction in psbAII expression than that observed in the untreated air-bubbled culture.
We have profiled changes in the expression of genes involved in the CCM of Synechococcus when cells are transferred from Ci sufficiency to conditions of Ci limitation. We have also tested a number of ideas about the sensory mechanisms leading to induction of a high-affinity CCM. As has been observed in Synechocystis, the transcriptional response of this strain to severe Ci limitation occurs rapidly, reaching a maximum within 30 to 60 min (Fig. 3). One of our key findings includes the characterization of three distinct temporal responses of low-Ci-inducible transcripts. The temporal responses of these transcripts to Ci limitation align with functional groupings of the encoded proteins: (a) a slow, sustained induction for psbAII, (b) a rapid, transient induction for genes encoding the carboxysome-associated proteins (ccmKLMNO and rbcL) and cmpR, and (c) a rapid, sustained induction for genes encoding the inducible Ci uptake systems (sbtA, cmpABCD, and ndhF3-D3-chpY). In all cases, the transcriptional response of low-Ci-inducible CCM-related genes preceded the full physiological response by 30 to 60 min, however, the three distinct temporal responses are suggestive of the existence of alternative controls on expression of subsets of these genes. Furthermore, the pattern of decay of CCM-related transcripts at high- and low-Ci suggests that the abundance of these transcripts is primarily controlled at the level of transcription rather than at the level of mRNA stability (Fig. 5).
Previous work has shown that light is a prerequisite condition for induction of CCM-related gene expression (McGinn et al., 2003
We detected multiple conditions under which the regulation of a classic light-inducible gene, psbAII, and CCM-related genes is uncoupled in Synechococcus cells. First, a high-light treatment at high-Ci levels, although sufficient to induce psbAII expression, failed to significantly induce any of the CCM genes tested at high Ci (Fig. 6). The only effect of high light was found at low Ci, where cmpA transcript abundance was 4-fold higher in cells exposed to the higher irradiance. Our results indicate that any high-light responsiveness of CCM-related gene expression in Synechococcus is confined to conditions under which Ci is limiting, and this has also been confirmed in Synechocystis (McGinn et al., 2003
The preceding arguments point to just two of the proposed sensory mechanisms, namely that changes within the internal Ci pool are perceived directly and dictate the degree of induction of the CCM in Synechococcus or that cells sense altered photorespiratory activity. In support of the latter theory, it is already established that Anabaena variabilils cells grown at low O2 are slow to adapt to Ci limitation (Marcus et al., 1983
Cyanobacterial Strains and Culture Conditions
Cells of the cyanobacterium Synechococcus sp. strain PCC7942 were cultured in modified BG-11 medium (Price and Badger, 1989a
Isolation of total RNA from harvested cell pellets stored at -80°C was essentially as previously described (McGinn et al., 2003
Primers were designed using Jellyfish software (LABVELOCITY, v1.5) and, with the exception of the psbA primers, forward and reverse primers in every pair had melting temperatures that varied by less than 3°C and GC contents not greater than 55%. For specificity, it was necessary to anchor the psbAI/II/III forward primers in the 5'-untranslated region and to relax design constraints slightly. Primers for sbtA were designed after sequencing of a fragment of sbtA obtained with degenerate primers (GenBank accession no. AY365060). All other sequences were obtained from GenBank. The relative amount of specific cDNA templates between different samples was quantified using real-time PCR. HotstarTaq PCR kit components (Qiagen, Hilden, Germany) were used in 20-µL reactions containing 3 mM MgCl2, 200 µM each of dATP, dTTP, dCTP, and dGTP, 0.5 µM each of forward and reverse primer, 0.5 to 1 unit Taq, and cDNA template equivalent to 25 ng of total RNA. For every reaction, an RNA sample without reverse transcriptase was included to control for genomic DNA contamination. Product formation was monitored by the inclusion of SYBR Green I at a final dilution of 1:40,000 (Fisher Biotech, Springfield, NJ). Thermocycling was conducted in a Rotorgene 2000 Thermal Cycler (Corbett Research, Sydney, Australia) for 35 cycles consisting of denaturation for 30 s at 95°C, annealing at 54°C for 30 s, extension at 72°C for 30 s, and fluorescence acquisition at 84°C for 15 s. Cycling was preceded by a 15-min 95°C activation step. Specificity of amplification was confirmed through a melt curve analysis of final PCR products by ramping the rotor temperature from 55°C to 99°C at 0.2°C s-1 with fluorescence acquired after every 1°C increase.
The amplification efficiencies of primer pairs used in real-time PCR assays (Table I) varied by less than 10% compared with that of the normalizer ppc, which was 1.9. These values were determined by the slope of the curve generated by amplification of serially diluted cDNA over at least 3 orders of magnitude (r
Cells were prepared and analyzed in the mass spectrometer as previously described (Sültemeyer et al., 1995 Received July 3, 2003; returned for revision August 8, 2003; accepted September 1, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.029728. * Corresponding author; e-mail Price{at}rsbs.anu.edu.au; fax 61-2-61255075.
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ABSTRACT
RESULTS
0.965). Accordingly, all primer pairs were nominated as having an efficiency of 2 and -fold changes in transcript abundance were calculated using the 2-
