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First published online November 23, 2005; 10.1104/pp.105.069146

Plant Physiology 139:1959-1969 (2005)
© 2005 American Society of Plant Biologists

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BIOENERGETICS AND PHOTOSYNTHESIS

Sensing of Inorganic Carbon Limitation in Synechococcus PCC7942 Is Correlated with the Size of the Internal Inorganic Carbon Pool and Involves Oxygen

Fiona J. Woodger, Murray R. Badger and G. Dean Price*

Molecular Plant Physiology Group, Research School of Biological Sciences, Australian National University, Canberra, Australian Capital Territory 0200, Australia (F.J.W., M.R.B., G.D.P.); and Australian Research Council Centre of Excellence Australian Research Council in Plant Energy Biology, Research School of Biological Sciences, Australian National University, Canberra, Australian Capital Territory 0200, Australia (M.R.B.)


   ABSTRACT
TOP
 ABSTRACT
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 LITERATURE CITED
 
Freshwater cyanobacteria are subjected to large seasonal fluctuations in the availability of nutrients, including inorganic carbon (Ci). We are interested in the regulation of the CO2-concentrating mechanism (CCM) in the model freshwater cyanobacterium Synechococcus sp. strain PCC7942 in response to Ci limitation; however, the nature of Ci sensing is poorly understood. We monitored the expression of high-affinity Ci-transporter genes and the corresponding induction of a high-affinity CCM in Ci-limited wild-type cells and a number of CCM mutants. These genotypes were subjected to a variety of physiological and pharmacological treatments to assess whether Ci sensing might involve monitoring of fluctuations in the size of the internal Ci pool or, alternatively, the activity of the photorespiratory pathway. These modes of Ci sensing are congruent with previous results. We found that induction of a high-affinity CCM correlates most closely with a depletion of the internal Ci pool, but that full induction of this mechanism also requires some unresolved oxygen-dependent process.

Cyanobacterial CO2-concentrating mechanisms (CCMs) feature multiple inorganic carbon (Ci) transport systems for the active uptake of both CO2 and HCO3 (Shibata et al., 2002Go; for review, see Price et al., 2002Go; Badger and Price, 2003Go). Ci is accumulated inside the cell as HCO3– and converted to CO2 inside a microcompartment known as the carboxysome, which contains the CO2-fixing enzyme Rubisco. In the unicellular, freshwater strains Synechococcus sp. strain PCC7942 and Synechocystis sp. strain PCC6803 (hereafter referred to as Synechococcus PCC7942 and Synechocystis PCC6803, respectively) two high-affinity, inducible HCO3– transport activities have been characterized. These are an ABC transporter, BCT1, encoded by the cmp operon (Omata et al., 1999Go), and a Na+-dependent HCO3– uptake activity encoded by the sbtA gene (Shibata et al., 2002Go). A third HCO3– transport activity of medium affinity, termed BicA, has recently been identified in Synechocystis PCC6803 and a number of marine cyanobacteria but is not found in Synechococcus PCC7942 (Price et al., 2004Go). The active transport of CO2 is associated with specialized NADH dehydrogenase complexes (Type 1; NDH-1) that are proposed to convert CO2 to HCO3– within the cell—the ndhF4/ndhD4/chpX (cupB) genes are required for a low-affinity constitutive CO2 transport activity termed NDH-14, whereas an inducible, high-affinity CO2 transporter, NDH-13, requires expression of the ndhD3/ndhF3/chpY (cupA) genes (Klughammer et al., 1999Go; Ohkawa et al., 2000Goa, 2000Gob; Shibata et al., 2001Go; Maeda et al., 2002Go).

Freshwater cyanobacteria are subjected to large seasonal fluctuations in the availability of nutrients, including Ci (Talling, 1985Go), and the activity of the cyanobacterial CCM is modulated in response to Ci. The apparent photosynthetic affinity for Ci, as measured by K0.5(Ci), ranges between a low-affinity, constitutive state (approximately 200 µM) and a high-affinity, induced state (10–15 µM; for review, see Kaplan and Reinhold, 1999Go; Price et al., 2002Go; Badger and Price, 2003Go). The transition between these states is characterized by dynamic and large changes in the abundance of transcripts encoding the BCT-1, SbtA, and NDH-13 transporters (Ohkawa et al., 1998Go; Omata et al., 1999Go, 2001Go; Shibata et al., 2002Go; McGinn et al., 2003Go; Woodger et al., 2003Go; Wang et al., 2004Go). By contrast, carboxysome-associated genes are expressed at relatively high constitutive levels at nonlimiting Ci and are weakly induced after the transition to limiting Ci (Woodger et al., 2003Go; Wang et al., 2004Go; F. Woodger, unpublished data).

The signal-response pathways that regulate expression of genes encoding high-affinity Ci transporters in response to fluctuating Ci concentrations require definition. A variety of theories have been proposed to describe how cyanobacterial cells might sense Ci limitation (for review, see Kaplan and Reinhold, 1999Go). These include the direct sensing of external Ci or the internal Ci pool, the detection of changes in the concentration of photorespiratory or Calvin cycle intermediates, or changes in the redox potential of the photosynthetic electron transport chain. Under Ci limitation, the activity of the CCM is also responsive to ambient light (McGinn et al., 2003Go; Woodger et al., 2003Go; Takahashi et al., 2004Go) and, based on limited evidence in PII signaling mutants, possibly also nitrogen status (for review, see Forchhammer, 2004Go). The downstream events in the Ci signaling pathway are better characterized, and LysR-type transcriptional factors are known to be involved in the regulation of high-affinity Ci-transporter gene expression. NdhR (CcmR) and CmpR regulate ndhF3/D3/chpY (cupA), and cmpABCD and sbtA expression, respectively, in Synechocystis PCC6803 (Figge et al., 2001Go; Omata et al., 2001Go; Wang et al., 2004Go), while RbcR is thought to be involved in control of the rbc operon (Mori et al., 2002Go). Only rbcR and cmpR have been identified in Synechococcus PCC7942. A putative transcriptional regulator, lexA from Synechocystis PCC6803, has also been implicated in the control of high-affinity Ci-transporter genes (Domain et al., 2004Go).

Current evidence for the nature of primary Ci-sensing events suggests it is unlikely that cyanobacterial cells modulate the activity of their CCM through direct sensing of the external Ci concentration. The use of Calvin cycle and PSII inhibitors uncouples the link between external Ci concentration and the activity of the CCM in Synechococcus PCC7942 (Woodger et al., 2003Go; McGinn et al., 2004Go). Furthermore, Synechocystis PCC6803 and Synechococcus PCC7942 do not induce a high-affinity CCM upon Ci limitation in the dark (McGinn et al., 2003Go; F. Woodger, unpublished data). Alternatively, the redox potential of the photosynthetic electron transport chain could signal, indirectly, the Ci status of the cell. In Synechocystis PCC6803, Ferrodoxin1, a light-responsive gene encoding a mediator of electron transfer between PSI and FNR, may be negatively regulated by NdhR (CcmR) activity (Mazouni et al., 2003Go). Although light enhances expression of genes encoding high-affinity Ci transporters, including cmpABCD, sbtA, and ndhF3/D3/chpY (cupA) in Synechocystis PCC6803 and Synechococcus PCC7942 (Hihara et al., 2001Go; Huang et al., 2002Go; McGinn et al., 2003Go; Woodger et al., 2003Go; Takahashi et al., 2004Go), light stress is not sufficient to induce expression of high-affinity Ci-transporter genes in cells at high concentrations of Ci (McGinn et al., 2003Go; Woodger et al., 2003Go). These results suggest perception of Ci limitation is not simply the perception of an indirect light stress.

A variety of evidence is consistent with the idea that primary sensing of Ci limitation in cyanobacterial cells may involve sensing of alterations in photorespiratory activity. First, the Calvin-cycle inhibitor glycolaldehyde (GLY), which blocks regeneration of ribulose 1,5-bisphosphate (RuBP; Sicher, 1984Go) and consequently photorespiration, suppressed the induction of key CCM genes, including the high-affinity Ci-transporter genes in Synechococcus PCC7942 (Woodger et al., 2003Go). Second, in Anabaena variabilis, the physiological induction of a high-affinity CCM was impaired under conditions of low oxygen (Marcus et al., 1983Go). Third, analysis of a Synechocystis PCC6803 mutant lacking functional carboxysomes, and where the endogenous Rubisco enzyme had been replaced with the soluble Rhodospirillum rubrum enzyme, revealed that the mutant expressed a relatively high-affinity CCM (i.e. higher rates of Ci transport) compared with the wild type (Marcus et al., 1992Go). This mutant would be predicted to perform higher rates of photorespiration, suggesting a link between photorespiration and CCM induction.

Other results, from inhibitor studies, are instead consistent with the idea that Ci-limited Synechococcus PCC7942 cells sense a transient drop in the size of the internal Ci pool. First, increased induction of a high-affinity CCM was observed in the presence of ethoxyzolamide (EZ), which blocks CO2 uptake and reduces the internal Ci-pool size (Woodger et al., 2003Go). Second, CCM induction was inhibited in the presence of GLY, an inhibitor of CO2 fixation, which prevents depletion of the Ci pool (Woodger et al., 2003Go). In this study, we investigated the nature of Ci sensing in the freshwater cyanobacterium Synechococcus PCC7942, focusing particularly on the possibility of a photorespiratory-linked sensory pathway or a sensory event based on direct perception of the size of the internal HCO3– pool. To this end, we have used a variety of inhibitors, physiological conditions, and CCM mutants, and monitored internal Ci-pool sizes and high-affinity Ci-transporter expression and activity. Our results indicate that sensing of Ci limitation is primarily related to monitoring of the size of the internal HCO3– pool, but that an oxygen-dependent pathway is necessary for full induction of a high-affinity CCM at low Ci.


   RESULTS
TOP
 ABSTRACT
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 LITERATURE CITED
 

Regulation of Ci-Transporter Genes in Mutants of Synechococcus PCC7942 with Altered Ci Pools

To assess whether Ci sensing in Synechococcus PCC7942 might involve monitoring of the size of the internal Ci pool, we investigated CCM regulation in two mutants predicted to maintain internal Ci pools that differ significantly from wild type across a range of exogenous Ci concentrations. The first mutant, a predicted underaccumulator of Ci, is a high CO2-requiring mutant unable to perform active CO2 uptake owing to deletion of the chpY and chpX genes ({Delta}chpX{Delta}chpY). These genes encode putative carbonic anhydrase-like moieties integral to the NDH-1 CO2-transport complexes (Ohkawa et al., 2000Goa, 2000Gob; Shibata et al., 2001Go; Maeda et al., 2002Go). A second high CO2-requiring mutant investigated, {Delta}ccmM, lacks carboxysomes due to deletion of the ccmM gene, which encodes a structural element of carboxysomes (G.D. Price, unpublished data). The physiology of other mutants lacking carboxysomes is well established (Price et al., 1993Go; Ludwig et al., 2000Go), and accordingly this mutant would be predicted to overaccumulate internal Ci, owing to a diminished capacity to utilize the existing Ci pool for CO2 fixation.

Previously we have shown that exponentially growing wild-type Synechococcus PCC7942 cells bubbled with 1% to 2% CO2 in air maintain a basal low-affinity CCM, exhibiting a photosynthetic affinity for Ci, as measured by K0.5(Ci), of around 200 µM (Woodger et al., 2003Go). Although the growth rates of wild type and {Delta}chpX{Delta}chpY were similar at 5% CO2 (doubling times of 7.5 and 8.5 h, respectively), it is clear that the {Delta}chpX{Delta}chpY mutant grown at 5% CO2 has a relatively low photosynthetic affinity for Ci (Fig. 1A). For the wild type, photosynthetic rates were maximal at 1 mM external Ci, while near wild-type rates of maximum photosynthesis were exhibited by the {Delta}chpX{Delta}chpY mutant between 10 and 100 mM external Ci. By contrast, when grown at 1% CO2, it was evident that the mutant had a high photosynthetic affinity for Ci, with a component of the biphasic response to Ci being saturable at less than 1 mM Ci (Fig. 1A). Consistent with these observations, the expression of transcripts encoding components of the high-affinity, low-flux HCO3– transporters, SbtA and BCT-1, was found to be strongly induced in a sustained manner in {Delta}chpX{Delta}chpY cells grown at 1% CO2, but not in the wild type or in {Delta}chpX{Delta}chpY cells grown at 5% CO2 (Fig. 1B). The clear implication is that at 5% CO2 passive entry of CO2 is sufficient to support near maximal rates of CO2 fixation without triggering the induction of HCO3– transporters. Significantly, the accumulation of a Ci pool was not detected in the {Delta}chpX{Delta}chpY mutant (Table I) at external Ci concentrations up to 1 mM (near the upper detection limit for this technique), whereas wild-type cells had a pool of 23.5 mM. When cells were transferred from 5% to 1% CO2 for analysis of mRNA induction (Fig. 1B), they were subjected to an initial external Ci concentration of 4 mM. By extrapolation, we assume that the Ci-pool size in {Delta}chpX{Delta}chpY cells was similarly very reduced compared with the wild type immediately after the switch to 1% CO2.



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  		Figure 1. Rates of O2 evolution (A) in wild-type (WT) and {Delta}chpX{Delta}chpY ({Delta}X{Delta}Y) cells bubbled continuously with 5% or 1% CO2 in air, and the relative abundance (B) of cmpA and sbtA transcript pools in the same genotypes after transfer from bubbling with 5% to 1% CO2 in air. Transcript changes were determined by real-time RT-PCR (n = 4), and symbols represent transcript expression relative to the wild-type 0-min amount (set at 100%) ± SE. Note the break in the y axis in B. Each experiment was independently replicated and representative data are shown.

 

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  		Table I. Internal Ci-pool size in Synechococcus PCC7942 wild-type, {Delta}ccmM, and {Delta}chpX{Delta}chpY cells grown with bubbling at 5% CO2 in air

Values are averages of at least three separate measurements (±SE) at 1.0 mM or 0.1 mM external Ci and 150 µM O2. n.d., Not detectable.

 
We subjected wild type and {Delta}ccmM to a rapid transition between 5% CO2 and air and measured the resulting changes in internal Ci-pool size and the abundance of transcripts that encode components of high-affinity Ci-transport complexes, namely, cmpA, sbtA, and chpY (Fig. 2). The size of the internal Ci pool was determined for high-Ci cells of both genotypes at two concentrations of Ci: 0.1 mM, the initial concentration of external Ci when cells are first transferred to air (McGinn et al., 2003Go), and 1 mM Ci, close to the upper detection limit for this type of MIMS assays and saturating for the active uptake of Ci in high-Ci wild-type cells (Table I; Fig. 1A). At 0.1 mM external Ci, the {Delta}ccmM mutant had more than a 2-fold larger internal Ci pool compared with wild type (29 mM versus 13 mM). The same relationships held at 1 mM external Ci where the {Delta}ccmM mutant had a pool size of 68 mM while wild type accumulated a pool of 29 mM. In the {Delta}ccmM mutant, the air induction of cmpA and sbtA transcript pools over 4 h was substantially suppressed compared with wild type, while chpY induction was also suppressed at the later time points but to a lesser degree (Fig. 2). The kinetics of Ci-transporter gene induction in air-induced wild-type cells (initial peak followed by intermediate steady-state levels) differ from those observed previously in very severely Ci-limited cells (transcripts are maintained at elevated levels; Woodger et al., 2003Go).



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  		Figure 2. Relative abundance of cmpA (A), sbtA (B), and chpY (C) transcript pools in wild-type (WT) and {Delta}ccmM cells transferred from aeration with 5% CO2 in air to air bubbling for 4 h. Transcript changes were determined by real-time RT-PCR (n = 4), and symbols represent transcript expression relative to the wild-type 0-min amount (set at 100%) ± SE. Note the break in the y axis in A. The experiment was independently replicated and representative data are shown.

 

Oxygen Dependence of CCM Induction

Previously we found that the treatment of Synechococcus PCC7942 cells with GLY, an inhibitor of RuBP regeneration, suppressed low-Ci induction of Ci-transporter gene expression (Woodger et al., 2003Go). This result is consistent with a signal-response pathway that is activated by the depletion of the internal Ci pool (since the Ci pool would not be depleted in this instance owing to low rates of CO2 fixation). However, it is also consistent with the idea that an active photorespiratory pathway is necessary for sensing of Ci limitation, as RuBP regeneration is also necessary for the oxygenase reaction of Rubisco. Since the ratio of CO2:O2 is the primary determinant of photorespiratory activity within a wild-type cyanobacterial cell, we investigated the oxygen dependence of CCM induction in wild-type cells subject to Ci limitation. Cells were switched from aeration with 2% CO2 in air to sparging with a gas mix containing 0.015% (v/v) CO2 and oxygen concentrations ranging between 2% (v/v) and 40% (v/v) for 30 min. The induction of transcript pools encoding high-affinity Ci-transporter components—cmpA, sbtA, and chpY—was determined and found to be strongly oxygen dependent (Fig. 3). Induction of these transcripts at 2% O2 was only 20% to 30% of the maximum increase, which occurred between 15% and 40% O2.



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  		Figure 3. Relative abundance of cmpA, sbtA, and chpY transcript pools in wild-type cells transferred for 30 min from aeration with 2% CO2 in air to 0.015% ppm CO2 containing various O2 concentrations. Transcript changes were determined by real-time RT-PCR (n = 4), and symbols represent the extent of induction after the shift to low Ci as a percentage of the maximum response (set at 100%) ± SE. The maximum response (100%) equates to an approximately 800-fold, 250-fold, or 12-fold change for cmpA, sbtA, and chpY, respectively. The experiment was independently replicated and representative data are shown.

 
The time-dependent course of Ci-transporter gene induction at high and low oxygen was also determined. Wild-type cells were switched from aeration with 2% CO2 in air to sparging with a gas mix containing 0.015% CO2 and either 2% or 21% oxygen for 3 h. The induction of cmpA and sbtA transcript pools was suppressed in a sustained fashion at low oxygen compared with high oxygen (Fig. 4). This was also the case for the chpY transcript pool up until 120 min after the transition to low Ci. Between 120 and 180 min after the switchover, chpY transcript pools in the low and high oxygen treated cells began to converge. To assess the physiological significance of this result, high-Ci cells were subjected to the same high and low oxygen treatment (above) over 24 h. As shown in Figure 5, by 24 h after the switch to low Ci, the low and high oxygen treated cells exhibited a K0.5(Ci) of 44 µM and 10 µM (medium- and high-affinity states, respectively). These values were relatively unchanged from the values of 62 µM and 13 µM observed at 3.5 h.



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  		Figure 4. Relative abundance of cmpA (A), sbtA (B), and chpY (C) transcript pools in wild-type cells transferred from aeration with 2% CO2 in air to 0.015% ppm CO2 containing 2% or 21% O2 over 180 min. Transcript changes were determined by real-time RT-PCR (n = 4), and symbols represent the extent of induction after the shift to low Ci at each time point as a percentage of the maximum response (set at 100%) ± SE. The maximum response (100%) equates to approximately a 1,300-fold, 350-fold, or 21-fold change for cmpA, sbtA, and chpY, respectively. The experiment was independently replicated and representative data are shown.

 


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  		Figure 5. Changes in photosynthetic affinity for Ci, as K0.5(Ci), in wild-type cells grown with 2% CO2 in air and switched to aeration with 0.015% CO2 containing either 2% or 21% O2 for 1.5, 3.0, 4.5, and 24 h. Symbols represent the average K0.5(Ci) ± SE after the shift to low Ci at each time point (n ≥ 3). Note the break in the x axis.

 

Effect of Oxygen Concentration on Ci Accumulation

The influence of oxygen concentration on the internal Ci-pool size of high-Ci Synechococcus PCC7942 cells during the transition to Ci limitation was evaluated. Internal Ci-pool sizes were assayed in wild-type cells grown at 5% CO2 at an external Ci concentration of 0.1 mM and an external O2 concentration of either 25 or 150 µM (Table II). These conditions approximate the initial conditions in culture during the experiments where high-Ci cells were first transferred to low Ci at either 2% or 15% O2 (Fig. 3). As previously observed in air-grown Synechococcus UTEX 625 cells (Li and Canvin, 1997aGo), no statistically significant difference in pool size was observed at high or low O2 with pool sizes measured at 13 or 13.3 mM, respectively. Although full induction of a high-affinity CCM appears to be oxygen dependent (Figs. 3 and 4), this result indicates Ci-pool size is not itself strongly oxygen dependent.


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  		Table II. Internal Ci-pool size in Synechococcus PCC7942 wild-type cells grown with bubbling at 5% CO2 in air

Values are averages of at least three separate measurements (±SE).

 

Regulation of Ci-Transporter Genes in Synechococcus PCC7942 Cells Treated with Inhibitors of Ci Accumulation and Photorespiratory Activity

We tested whether potentially photorespiratory activity or, alternatively, internal Ci-pool size is more significant in determining the degree of CCM induction under Ci limitation. Synechococcus PCC7942 cells were transferred from high CO2 to air sparging for 30 min in the presence of the CO2-uptake inhibitor EZ, which prevents strong accumulation of an internal Ci pool (Price and Badger, 1989aGo), or the Calvin cycle inhibitor GLY, which would inhibit photorespiration (Sicher, 1984Go). Both inhibitors were also used in combination. The EZ treatment was applied additionally to {Delta}ccmM cells, which overaccumulate internal Ci (Table I). In agreement with previous results (Woodger et al., 2003Go), GLY treatment strongly suppressed induction of high-affinity Ci-transporter gene expression in wild-type cells (Fig. 6, A and B). By contrast, EZ treatment enhanced this induction. The net effect of a combined treatment was the restoration of Ci-transporter gene induction to wild-type (or higher) amounts. In these experiments, the relative induction of expression of Ci-transporter genes reflected more closely decreases in the size of internal Ci pool rather than increases in photorespiratory activity. Although the relative photorespiratory activity of the {Delta}ccmM mutant is assumed to be low (Schwarz et al., 1995Go), EZ treatment also restored the induction of high-affinity Ci-transporter transcripts in this background, presumably by preventing accumulation of the ordinarily high internal Ci pool within this mutant. This result is consistent with a mode of Ci sensing dependent on depletion of the internal Ci pool.



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  		Figure 6. Relative abundance of cmpA and sbtA (A) and chpY (B) transcript pools in wild-type (WT) and {Delta}ccmM cells transferred from aeration with 5% CO2 in air to air bubbling for 30 min in the presence of 300 µM EZ or 10 mM GLY, or with both inhibitors combined. Transcript changes were determined by real-time RT-PCR (n = 4), and bars represent the extent of induction after the shift to low Ci at each time point as a percentage of the wild type + EZ response (set at 100%) ± SE. The maximum response (100%) equates to approximately a 1,000-fold, 600-fold, or 14-fold change for cmpA, sbtA, and chpY, respectively. The experiment was independently replicated and representative data are shown.

 

Oxygen Dependence of Ci-Transporter Gene Induction in the {Delta}chpX{Delta}chpY Mutant

To examine more closely whether a lowered Ci pool is necessary, as well as sufficient, for induction of the expression of high-affinity Ci-transporter genes, gene expression was monitored in {Delta}chpX{Delta}chpY cells transferred from high CO2 to aeration with 0.01% CO2 containing 2% or 21% oxygen (Fig. 7). Like the wild type, full induction of Ci-transporter genes was strongly oxygen dependent, despite the significantly reduced internal Ci pool that exists under these conditions in the mutant (Table I). A strong depletion of the Ci pool therefore does not appear sufficient for full Ci-transporter gene induction; oxygen is required for the maximal response. This result also suggests that functional NDH-1 complexes (which encompass the ChpX or ChpY subunits) are not directly involved in sensing of Ci limitation through some oxygen-dependent process, such as electron transport to a terminal oxidase.



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  		Figure 7. Relative abundance of cmpA and sbtA transcript pools in wild-type (WT) and {Delta}chpX{Delta}chpY cells transferred from aeration with 5% CO2 in air to bubbling with 0.1% CO2 containing either 1% or 21% O2 for 30 min. Transcript abundance was determined by real-time RT-PCR (n = 4). Bars represent the extent of induction after the shift to low Ci as a percentage of the maximum response (set at 100%) ± SE. The maximum response (100%) equates to approximately a 350-fold or 100-fold change for cmpA and sbtA, respectively. The experiment was independently replicated and representative data are shown.

 

Effect of SHAM on Ci-Transporter Gene Induction in Synechococcus PCC7942 Cells

Although its precise function is unknown, O2 photoreduction occurs at significant rates in cyanobacteria (Badger et al., 2000Go). These rates are dependent on the availability of both oxygen and light, and also on Ci accumulation (Miller et al., 1988Go, 1991Go; Badger and Schreiber, 1993Go; Li and Canvin, 1997Goa, 1997Gob). Given its dependence on Ci accumulation, a process such as O2 photoreduction might potentially be linked to the signaling of Ci limitation. Salicylhydroxamic acid (SHAM), used extensively as an inhibitor of plant mitochondrial alternative oxidase (Schonbaum et al., 1971Go), has been noted to inhibit O2 photoreduction in cyanobacterial cells, including Oscillatoria chalbea and Synechococcus sp. RF-1 (Bader and Schmid, 1989Go; Weng and Shieh, 2004Go). However, the effect of SHAM on Synechococcus PCC7942 is, to our knowledge, untested.

The effect of SHAM on dark-equilibrated cells transferred into the light was tested (Fig. 8A). Increasing concentrations of SHAM between 1 and 5 mM progressively inhibited maximum rates of net O2 evolution, gross O2 uptake (an indirect measure of O2 photoreduction), and CO2 uptake by up to 60% compared to the untreated control amounts. This maximal inhibition was not recoverable by addition of high concentrations of total Ci, up to 100 mM (data not shown). SHAM treatment of high-Ci cells transferred to air enhanced the induction of high-affinity Ci-transporter gene expression at air levels of CO2. This effect was maximal at 2 mM SHAM, with the air induction of cmpA, sbtA, and chpY expression accentuated by 6- to 10-fold relative to the untreated control. To better define the effect of SHAM on Synechococcus PCC7942 cells, the size of the internal Ci pool was measured in high CO2-grown cells treated with 2 mM SHAM (Table III). This concentration was chosen since it was found to have a maximal effect on Ci-transporter gene induction without overt inhibition of photosynthesis (Fig. 8B). At 1 mM Ci, with or without GLY, SHAM treatment reduced the internal Ci-pool size by approximately 60%. Subsequent measurements were conducted in the presence of 10 mM GLY, which increases the resolution of the assay at low Ci, and a similar effect was observed at 0.2 mM external Ci. Given the joint inhibition by SHAM of both Ci accumulation and O2 photoreduction (Fig. 8A; Table III), it is difficult to ascribe the enhancement of CCM transcript induction at low Ci (Fig. 8B) to just one of these processes. Nonetheless, the result is broadly consistent with our prevailing hypothesis—that depletion of the Ci-pool activates the signal-response pathways leading to up-regulation of CCM activity.



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  		Figure 8. Effect of various concentrations of SHAM on maximum rates of gross O2 uptake, CO2 uptake, and maximum O2 evolution in wild-type cells grown continuously with 2% CO2 (A), or on the relative induction of cmpA, sbtA, and chpY transcripts in wild-type cells transferred from aeration with 2% CO2 in air to air bubbling for 30 min (B). Transcript abundance was determined by real-time RT-PCR (n = 4), and symbols represent the extent of induction or repression after the shift to low Ci as a percentage of the 0 mM SHAM value (set at 100%) ± SE. The 0 mM values (i.e. 100%) equate to average rates of gross O2 uptake, CO2 uptake, and maximum O2 evolution of 270, 190, 480 µmol mg–1 h–1, respectively (n ≥ 3), and approximately 55-fold, 65-fold, or 7-fold increases in cmpA, sbtA, and chpY transcript pools, respectively.

 

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  		Table III. Effect of SHAM on internal Ci-pool size in Synechococcus PCC7942 wild-type cells grown by aeration with 2% CO2 in air

Values are averages of at least three separate measurements (±SD) at ≥150 µM O2 at the specified external concentrations of Ci, GLY, and SHAM. n.d., Not detectable.

 

   DISCUSSION
TOP
 ABSTRACT
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 LITERATURE CITED
 
In this study, we have investigated the transition between basal, low-affinity CCMs and inducible, high-affinity states in the freshwater cyanobacterium Synechococcus PCC7942. We were particularly interested in the question of whether sensing of Ci limitation, and the up-regulation of high-affinity Ci-transporter gene expression and activity, might involve monitoring of fluctuations in the size of the internal Ci pool or, alternatively, the activity of the photorespiratory pathway. Both of these sensory mechanisms seem feasible in principle—in high-Ci cells swapped to Ci limitation, it would be predicted that a transient drop in the size of the internal Ci pool would occur, as well as a transient increase in photorespiratory activity. These modes of Ci sensing are congruent with previous results (Woodger et al., 2003Go).

When high CO2-requiring mutants with altered capacities to accumulate Ci were transferred to moderately limiting concentrations of Ci, initial internal Ci-pool sizes were proportional to the subsequent induction of high-affinity Ci-transporter genes (Figs. 1 and 2; Table I). When {Delta}chpX{Delta}chpY cells were switched from growth at 5% to 1% CO2 in air, high-affinity Ci-transporter gene expression was enhanced some two orders of magnitude compared to wild type (Fig. 1B). Accumulation of an internal Ci pool in the {Delta}chpX{Delta}chpY mutant grown at 5% CO2 was not detectable with up to 1 mM external Ci (wild-type pools were 23.5 mM). By contrast, at 0.1 mM external Ci, equivalent to the "Ci shock" received by cells during the early phase of a high Ci to air transition, wild-type cells grown at 5% CO2 had a Ci pool of 13 mM while {Delta}ccmM cells had an elevated pool size of 29 mM (Table I). Air induction of two high-affinity Ci-transporter genes, sbtA and cmpA, was substantially suppressed in the {Delta}ccmM mutant (Fig. 2).

In favor of a photorespiratory-based mechanism for Ci sensing, Ci-limited wild-type cells exhibited a sustained requirement for oxygen (approaching ambient levels) to fully induce a high-affinity CCM (Figs. 3–5GoGo). This requirement was apparent for the maximal induction of both high-affinity Ci-transporter genes and, at the physiological level, for the maximal induction in cellular photosynthetic affinity for Ci (i.e. to approach a K0.5(Ci) of 10–15 µM). These results are broadly consistent with the idea that photorespiratory activity might normally signify Ci status within a cell since such a pathway would be sensitive to the prevailing CO2:O2 ratio.

Previously we have observed that GLY represses the induction of high-affinity Ci-transporter genes in Synechococcus PCC7942 at low Ci (Woodger et al., 2003Go). This may occur by one of two routes: (1) by repression of photorespiratory activity (due to reduced Rubisco oxygenase activity in the absence of RuBP regeneration) or (2) inhibition of CO2 fixation, leading to maintenance of a high internal Ci pool. Conversely, we have also found that EZ, a carbonic anhydrase inhibitor that inhibits CO2 uptake in cyanobacteria (Price and Badger, 1989aGo), has the opposite effect to GLY and enhances the induction of high-affinity Ci-transporter genes at low Ci (Woodger et al., 2003Go). Accordingly, in this study, to simultaneously decrease photorespiratory activity (by blocking RuBP regeneration) and reduce the size of the Ci pool (by blocking CO2 uptake), EZ and GLY were applied jointly to wild-type Synechococcus PCC7942. The net effect was restoration of the induction of high-affinity Ci-transporter gene expression to control (or higher) levels at low Ci. This result indicates that induction of a high-affinity CCM in Synechococcus PCC7942 correlates more closely with depletion of the Ci pool than it does with increases in photorespiratory activity. In support of this idea, EZ treatment to deplete the large Ci pool in the {Delta}ccmM mutant (Table I) restored the low-Ci induction of high-affinity Ci-transporter genes to near wild-type amounts (Fig. 6).

There are further reasons to doubt that increased photorespiratory activity triggers CCM induction in a Ci-limited Synechococcus PCC7942 cell. Although Synechococcus PCC7942 contains a nearly complete set of higher plant-like photorespiratory enzymes (Codd and Stewart, 1973Go; Renstrom et al., 1989Go; Helman et al., 2003Go), the existence of a fully active photorespiratory pathway is controversial (Han and Eley, 1973Go; Renstrom and Bergman, 1989Go). Indeed, in Synechocystis PCC6803 the Gly decarboxylase complex is dispensable for growth at limiting Ci (Hagemann et al., 2005Go). Without knowledge of the specific fluctuations in pool sizes of photorespiratory metabolites under Ci limitation or the precise effect of GLY on these pools, we cannot completely exclude a role for photorespiratory activity in Ci sensing, but our results to date fail to lend support to this idea.

Our data are instead consistent with a primary Ci-sensing mechanism in Synechococcus PCC7942 that involves perception of a transient decline in the size of the internal Ci pool. Nonetheless, while a reduction in the Ci pool is necessary for stimulating the signaling pathways that lead to induction of a high-affinity CCM, it is not a sufficient condition since there is a secondary requirement for oxygen to fully activate these same pathways (Figs. 3–5GoGo and 7). Such a requirement can be understood in physiological terms since, at low O2, a less potent CCM would be required to deliver the same rate of CO2 fixation owing to reduced competition for Rubisco active sites. However, at a mechanistic level, it is not obvious how the proposed sensing of a decline in the internal Ci pool might also be integrated with some oxygen-dependent cellular process (i.e. photorespiration, terminal oxidase activity, O2 photoreduction, or respiration). Based on emerging evidence that SHAM inhibits O2 photoreduction in cyanobacteria (Bader and Schmid, 1989Go; Weng and Shieh, 2004Go), we applied this inhibitor to wild-type cells, but nonspecific effects on Ci accumulation complicated the interpretation of the resulting increase in Ci-transporter gene induction (Fig. 8, A and B; Table III). However, A-type flavoprotein mutants deficient in O2 photoreduction are viable at low Ci (Helman et al., 2003Go), suggesting O2 photoreduction is not crucial to Ci sensing.

In conclusion, future work to dissect Ci sensing and regulation of the CCM in unicellular cyanobacteria may usefully be focused on the potential role of a signaling pathway that is triggered primarily by a decline in the size of the internal Ci pool. Such a model might encompass the activity of a HCO3–-binding sensory protein, such as the HCO3–-activated soluble adenylate cyclases that are involved in cAMP-mediated signal transduction in mammalian systems (Zippin et al., 2001Go) and that are represented also in cyanobacteria (Chen et al., 2000Go; Cann, 2004Go). Alternatively, transcription factors such as CmpR/NdhR/CcmR that are involved in the control of the expression of Ci-transporter genes may themselves be regulated by binding of HCO3–. Certainly, small metabolites and anions, including Calvin cycle intermediates, have been implicated in the regulation of other LysR transcription factors (Dubbs et al., 2004Go). Our work suggests a model for Ci sensing must encompass a secondary input from some oxygen-dependent process that controls the magnitude of the response. The nature of that potential process is unclear, although our work has thus far failed to implicate O2 photoreduction or photorespiration in Ci sensing. The enhanced transcription of an alternative sigma factor in Rhodobacter sphaeroides in response to singlet oxygen is an intriguing development (Anthony et al., 2005Go) since Wang et al. (2004)Go have identified a number of sigma factors from Synechocystis PCC6803 that exhibit Ci-responsive expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
LITERATURE CITED
 

Cyanobacterial Strains and Culture Conditions

Cells of the cyanobacterium Synechococcus PCC7942 ({Delta}ccmM mutant and wild-type strains) were cultured in modified BG-11 medium (Price and Badger, 1989bGo) containing 20 mM HEPES-KOH, pH 8.0, at 28°C with a light intensity of approximately 85 µmol photons m–2 s–1. For the {Delta}chpX(cupB){Delta}chpY(cupA) mutant, kanamycin and chloramphenicol were included in the media at final concentrations of 12 µg mL–1 and 5 µg mL–1, respectively. Aeration to all cultures was delivered through a pipette with a 1-mm annulus at a flow rate of approximately 0.15 L min–1. To rapidly induce a medium-high-affinity CCM in wild type, exponentially growing cells (OD730 of 0.3–0.4) that had been bubbled with an air-CO2 mixture containing 2.0% to 5.0% CO2 were harvested by centrifugation at 4,800g for 6 min and resuspended in an equivalent volume of modified BG-11 medium containing a final concentration of 0.1 mM Ci, or, in experiments were oxygen concentration was varied, in medium that had been preequilibrated with the intended new gas mixture for 5 to 10 min. Cell cultures were returned to the original conditions (unless otherwise specified) but aerated instead with air or specific CO2/O2/N2 mixes delivered through mass flow controllers (MKS Instruments). To rapidly induce a medium-affinity CCM in the {Delta}chpX{Delta}chpY mutant, exponentially growing cells (OD730 of 0.3–0.4) that had been bubbled with an air-CO2 mixture containing 5.0% CO2 were harvested as above and resuspended in medium containing 4 mM Ci, then bubbled with 1% CO2 in air under culture conditions otherwise as described above. For SHAM treatments in culture or in the mass spectrometer, cells were always preincubated in the dark with the inhibitor for at least 5 min prior to being returned to the light.

The {Delta}chpX{Delta}chpY deletion mutant was constructed sequentially. First, a pUC18-based construct consisting of the 0.99-kb and 1.00-kb regions immediately upstream and downstream of chpX was assembled. The primers for amplifying the upstream sequence were forward 5'-AGAATTCTCCCACTCCACTAGTCGTTTC and reverse 5'-AGGATCCTCGCGGTTCAGGGGTGAAC, introducing EcoRV and BamHI sites at the 5' and 3' ends, respectively. The primers for amplifying the downstream flanking sequence were forward 5'-TGGATCCACGCCCTTTGCTAACGCTC and reverse 5'-TAAAAGCTTGCTGCCAAACAGGCCACC, introducing BamHI and HindIII sites at the 5' and 3' ends, respectively, for assembly inside pUC18. A chloramphenicol resistance marker gene (Dzelzkalns et al., 1984Go) was cloned into the resulting BamHI site in the same transcriptional orientation as the chpX flanking sequences. The cartridge was used to transform wild-type Synechococcus PCC7942 cells as described by Price and Badger (1989b)Go, and segregation was confirmed by PCR analysis. The primers for amplifying the chpY upstream sequence were forward 5'-CTCCCTCGGAATTCATTTCAGCC and reverse 5'-TAGGATCCGTGGAAGACGCCAGAGTC, introducing EcoRI and BamHI sites at the 5' and 3' ends, respectively. The primers for amplifying the downstream flanking sequence were forward 5'-ATGGATCCAAAGCCAAGACTACCGCTAG and reverse 5'-AAATAAGCTTCGCCAACGCAAGACAACAAAG, introducing BamHI and HindIII sites at the 5' and 3' ends, respectively, for assembly inside puC18. A kanamycin resistance marker (Klughammer et al., 1999Go) was cloned into the resulting BamHI site in the same transcriptional orientation as the chpY flanking sequences. The cartridge was used to transform the {Delta}chpX mutant (as above), and segregation was confirmed by PCR analysis.

To construct the {Delta}ccmM mutant, the ccmM gene was removed from the ccmKLMNO operon while preserving the polycistronic message for ccmKLNO. In brief, a pUC18-based construct consisting of the 0.66-kb region immediately upstream of ccmM and a 0.48-kb region immediately downstream of ccmM flanking a kanamycin resistance/SacB cartridge from pRL250 (Cai and Wolk, 1990Go) was used to transform cells of Synechococcus PCC7942. Primers used for the upstream fragment were forward 5'-GGAAGTTCAAGCTTCTGTCTCTG and reverse 5'-TTGGGCTCGGATCCCTCTAACCTCC. Primers used for the downstream fragment were forward 5'-TCGCCCTTGGATCCATGGATCTACCG and reverse 5'-CTGCTGTGGAATTCTTAGCGATCG. Following segregation, the Kan-SacB cartridge was removed by the SacB exchange method (Cai and Wolk, 1990Go). The markerless ccmM deletion was named {Delta}ccmM and verified as a high CO2-requiring mutant that could be complemented by expression of ccmM from a shuttle vector (G.D. Price, unpublished data).


Real-Time Quantitative RT-PCR Assays

All Synechococcus PCC7942 sequences were obtained from the draft genome sequence located at the U.S. Department of Energy Joint Genome Institute (http://genome.jgi-psf.org/finished_microbes/synel/synel.home.html) or from GenBank. First-strand cDNA synthesis from normalized total RNA, isolated according to McGinn et al. (2003)Go, was conducted as described by Woodger et al. (2003)Go. Real-time reverse transcription (RT)-PCR assays using primer pairs specific to cmpA, sbtA, and chpY and incorporating SYBR Green I to monitor product formation were performed as described by Woodger et al. (2003)Go, except a Rotorgene 3000 Thermocycler (Corbett Research) was used and fold changes in transcript abundance relative to a basal condition were calculated after the method of Liu and Saint (2002)Go. Amplification efficiencies for each primer pair were determined as the average across any run. All reactions were carried out in quadruplicate, and the error was propagated using standard methods.


Mass Spectrometric Measurements

Cells were prepared and analyzed in the mass spectrometer as described previously (Badger et al., 1994Go; Sültemeyer et al., 1995Go). Assays were performed in 4-mL volumes in a thermostatted (30°C) mass spectrometer cuvette allowing membrane inlet analysis of 16O2 (mass 32), 18O2 (mass 36), and CO2 (mass 44). For measurements of the photosynthetic affinity for Ci, cells were assayed at a chlorophyll density of 2 µg mL–1 in BG-11 media buffered with 50 mM BisTris-propane-HCl, pH 7.9, where NaNO3 had been replaced with 20 mM NaCl. A light intensity of 700 µmol photons m–2 s–1 was used. The maximum rate of net O2 evolution (Vmax) was measured in the presence of at least 1 mM NaHCO3 (up to 150 mM Ci for the {Delta}chpX{Delta}chpY mutant), and the photosynthetic affinity for Ci was determined as K0.5(Ci), that is, the Ci concentration required to reach half the maximum rate of net O2 evolution. Measurements at low levels of Ci were initiated at around 25 µM O2 and allowed to progressively increase throughout the Ci range.

Measurements of internal Ci-pool size were conducted using a modified version of the method of Sültemeyer et al. (1995). Assays were conducted at a chlorophyll density of 2 µg mL–1 and at a light intensity of 100 µmol photons m–2 s–1 except in experiments utilizing SHAM, where chlorophyll density and light intensity were increased to 20 µg mL–1 and 700 µmol photons m–2 s–1, respectively. To further increase sensitivity in assays with SHAM at low Ci, a dark incubation for 7 min in the presence of 10 mM GLY was included prior to illuminating the cells (meaning that Ci accumulated from the medium of illuminated cells was maximally accumulated in the internal Ci pool rather than utilized for CO2 fixation). In all assays the rate of change in CO2 was monitored and the Ci-pool size was estimated by integrating the area under the rate versus time curve generated after cells were subjected to a dark-light or light-dark transition.

For 18O2 uptake assays, cells at a chlorophyll density of 4 µg mL–1 were preincubated in the light (700 µmol photons m–2 s–1) for 10 min. The assay medium was flushed with nitrogen, and a bubble of 18O2 was introduced to the cuvette and removed when total oxygen reached 150 µM. Ci from 0.1 or 1 M stocks was added to the desired concentration. Cells were illuminated to commence the assay. Calculations of rates of 18O2 uptake were as described previously (Franklin and Badger, 2001Go). Measurements of the Ci concentration in cell-free culture medium were performed according to McGinn et al. (2003)Go.


   ACKNOWLEDGMENTS
 
Ms. L. Tucker and Ms. B. Dixon provided excellent technical assistance. Thanks to Dr. B. Long for comments on the manuscript.

Received July 28, 2005; returned for revision September 25, 2005; accepted September 26, 2005.


   FOOTNOTES
 
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: G. Dean Price (dean.price{at}anu.edu.au).

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.069146.

* Corresponding author; e-mail dean.price{at}anu.edu.au; fax 61–2–61255075.


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