CO2-insensitive mutants of the green alga Chlorella ellipsoidea were previously shown to be unable to repress an inorganic carbon-concentrating mechanism (CCM) when grown under 5% CO2. When air-grown, wild-type (WT) cells were transferred to 5% CO2, an abrupt drop of Pmax to 43% the original level of air-grown cells was observed within the initial 12 h. Photosynthetic affinities of WT cells to dissolved inorganic carbon (DIC) were maintained at high levels for the initial 4 d of acclimation, and then decreased gradually to lower levels over the next 6 d. In contrast to WT cells, the CO2-insensitive mutant, ENU16, exhibited a constant Pmax at maximum levels and a low K1/2[DIC] throughout the acclimation period. The rapid Pmax drop within 12 h of acclimation in WT cells was significantly reduced by treatment with 0.5 mm of 6-ethoxybenzothiazole-2-sulphonamide (EZA), a specific membrane-permeable inhibitor of carbonic anhydrase (CA), suggesting the participation of internal CAs in the temporary drop in Pmax in WT cells. WT and ENU16 cells were grown in controlled equilibrium [CO2], and the photosynthetic rate of each acclimated cell type was measured under equilibrated growth [DIC] conditions. In WT cells acclimated to 0.14–0.4% [CO2], K1/2[DIC] values increased as [CO2] increased, and the photosynthetic rates at growth DIC conditions were shown to decrease to about 70% the Pmax level in this intermediate [CO2] range. Such decreases in the net photosynthetic rates were not observed in ENU16. These results suggest that algal primary production could be depressed significantly under moderately enriched CO2 conditions as a result of acquiring intermediate affinities for DIC because of their sensitive responses to changes in the ambient [CO2].
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It has been well established that a number of microscopic algae take up both CO2 and HCO3- from the surrounding medium to provide an ample flux of CO2 for photosynthesis even under extreme CO2 limitation (Badger, Kaplan & Berry 1980; Kaplan, Badger & Berry 1980; Miller, Espie & Canvin 1990). This process is termed as inorganic carbon-concentrating mechanism (CCM), which is thought to be composed of active uptakes of dissolved inorganic carbon (DIC) and flux controls of the intracellularly accumulated DIC (Badger et al. 1980; Espie & Colman 1986; Raven 1997; Mitra et al. 2004). In cyanobacteria, HCO3- transporters have been localized at the plasmalemma (Omata et al. 1999), whereas the putative HCO3- trapping systems by an efficient hydrolyzation of CO2 are localized at the thylakoid membrane (Shibata et al. 2001, 2002). In contrast, in eukaryotic algae, the molecular nature of DIC uptake has not been elucidated, and the location of the transporters is not known. The intracellularly accumulated DIC seems to be located in the chloroplast as HCO3-, which is dehydrated to CO2 only in close proximity to the carboxylase, and internal carbonic anhydrases (CAs) appear to participate in this process (Funke, Kovar & Weeks 1997; Raven 1997; Mitra et al. 2004). In the green alga, Chlamydomonas reinhardtii, a lumenal CA, Cah3, is thought to play a key role in providing CO2 to ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) (Funke et al. 1997; Raven 1997). It is also proposed in C. reinhardtii that a stromal CA, Cah6, functions as a leakage barrier of CO2 by rapidly hydrolyzing CO2 in the stroma to HCO3- using the alkaline environment of the stroma during active photosynthesis (Mitra et al. 2004).
Expression of most of the known CCM components in cyanobacteria and eukaryotic algae is regulated by the ambient CO2 concentration, and CCMs are usually repressed and derepressed, respectively, under enriched and depleted CO2 environments (Matsuda & Colman 1995b; Wang, Postier & Burnap 2004). It has been thought that the increase in [CO2] in the external medium would eliminate the requirement for a CCM by increasing the diffusive influx of CO2 as the substrate for photosynthesis. However, molecular details of the initial sensing process for [CO2] change have not been elucidated. There seems to be both activation and repression processes, respectively, under low and high CO2 conditions in algal CCM regulations. Numerous CO2-requiring mutants of the green alga C. reinhardtii have been isolated. It is known that a type of CO2-requiring mutant has an impaired region in a gene ccm1 (cia5), which encodes a putative zinc-finger protein, and exhibits a phenotype in which the expression of at least 47 low CO2-inducible proteins is lost even under air-levelCO2 (Miura et al. 2004). On the other hand, an active repression system of a CO2-regulated gene cah1 was also found in C. reinhardtii (Kucho, Ohyama & Fukuzawa 1999), and removal of the silencer region in the cah1 promoter resulted in the derepression of cah1 transcription under high CO2, suggesting that increments of CO2 itself could constitute a repressive signal to algal transcription systems.
In the green alga Chlorella ellipsoidea, CO2-insensitive mutants were previously isolated and were shown to exhibit a constitutive expression of the CCM which was fully derepressed under 5% CO2 (Matsuda & Colman 1996). Recent studies using the marine diatom Phaeodactylum tricornutum showed unequivocally that increases in ambient [CO2] constitute a repression signal, which is mediated by an increase in cAMP concentration, presumably in the cytosol, at the promoter region of the chloroplastic CA gene, ptca1 (Harada et al. 2006). The promoter region of the ptca1 possesses cAMP responsive elements, and the deletion of one of these elements from the ptca1 promoter resulted in derepression of this promoter under high CO2 conditions (Harada et al. 2006). These results suggest strongly that microalgae evolved repression systems for the CCM in response to increases in the ambient CO2 concentrations, and in some algae, the repression/derepression system seems to be a major mechanism in the control of CCM expression.
The physiological and evolutionary implications of acquiring repression machinery for the CCM, receiving high CO2 as a direct signal, are not clear, and the number of investigations on this aspect is extremely limited. There have been a few reports on the relationship between intracellular acidification under extreme high CO2 and intracellular CAs in the green algae, Chlorococcum littorale and Chlorella sp. UK001 (Satoh, Kurano & Miyachi 2001), and on phenotypic selection of C. reinhardtii under elevated [CO2] at about 2.5 times the present atmospheric level (Collins & Bell 2004). In the present study, acclimation processes of air-grown cells of the green alga C. ellipsoidea to elevated CO2 concentrations of up to 5% were investigated. Chlorella ellipsoidea, which lacks external CA activity (Rotatore & Colman 1991a), is known to possess active uptake systems for both CO2 and HCO3-, and is able to accumulate DIC intracellularly to about 80-fold the DIC concentration in the external medium (Rotatore & Colman 1991b; Matsuda & Colman 1995a). Photosynthetic parameters during acclimation processes were compared between wild-type (WT) cells and cells of a CO2-insensitive mutant.
MATERIALS AND METHODS
Algal culture and growth conditions
WT cells of C. ellipsoidea (UTEX 20) were obtained from University of Texas Culture Collection, and CO2-insensitive mutants were obtained by chemical mutagenesis of the WT C. ellipsoidea as described below. WT and mutant cells were grown axenically in Bold's basal medium as batch cultures with constant aeration by atmospheric air, with or without supplementary CO2 under constant illumination at 25 °C. For acclimation to 5% CO2, air-grown cells at mid-logarithmic phase were harvested and transferred to 5% (v/v) CO2 in air in the presence or absence of 0.5 mm 6-ethoxybenzothiazole-2-sulphonamide (EZA), a membrane-permeable inhibitor of CA, and allowed to acclimate for up to 10 d. The density of culture was maintained in the range 0.25–0.45 OD730 by daily dilutions. For acclimation to intermediate CO2 conditions below 0.6% CO2, 5% CO2-grown cells at mid-log phase were harvested and transferred to controlled CO2 conditions and allowed to acclimate for 6 h, which was previously shown to be the duration required for full expression of high-affinity photosynthesis in C. ellipsoidea (Matsuda & Colman 1995a). For controlled CO2 conditions, the pHs of the media were adjusted to 7.0 ± 0.1 by adding 0.5 M KH2PO4 or K2HPO4 at the initial stage of acclimation. The medium was aerated at each CO2-enriched air concentration using glass-fritted gas dispersion tubes at a rate of 0.002–0.003 m3 min−1 to establish equilibrium rapidly between gaseous and dissolved CO2 as described previously (Matsuda & Colman 1995b). Total concentrations of DIC in the medium were measured by gas chromatography (Shimadzu, Kyoto, Japan) as described by Birmingham & Colman (1979).
Mutagenesis and isolation of CO2-insensitive mutants
WT cells of C. ellipsoidea were treated with a chemical mutagen N-nitroso-N-ethylurea (ENU) (Sigma, St. Louis, MO, USA) at a concentration of 0.5 mg cm−3 for 30 min, centrifuged at 5000 rpm for 2 min and resuspended in fresh Bold's medium to wash out the remaining ENU. Cells were plated on 9.0 cm Petri plates containing 1% agar–Bold's medium at a density of about 300 cells per plate, and colonies were allowed to form under continuous illumination at photon flux density of 100 µmol m−2 s−1 at room temperature. Approximately 6 × 103 cells were mutated, and each colony on the plates was then screened by the 14C-screening method as described by Matsuda & Colman (1996). As a result, 13 CO2-insensitive mutants, which are unable to repress high-affinity photosynthesis under enriched CO2 condition (Matsuda & Colman 1996), were isolated. One CO2-insentive mutant, designated ENU16, was used for further experiments.
Determination of photosynthetic parameters
Cells acclimated to 5% CO2 were washed with water and CO2-free 50 mm Na+–K+–phosphate buffer pH 7.8, and resuspended in the same buffer at a chlorophyll (Chl) concentration of 40 µg cm−3. The rate of photosynthetic O2 evolution of the cell suspension was measured in a Clark-type oxygen electrode (Hansatech Instruments, Norfolk, UK) at various initial NaHCO3 concentrations under saturating light intensity (1600 µmol m−2 s−1). High-CO2 cells acclimated to intermediate equilibrium [CO2] conditions were harvested after 6 h acclimation, washed with water and then with CO2-free 50 mm Na+–K+–phosphate buffer, pH 7.0. Cells were resuspended in the same buffer at a Chl concentration of 40 µg cm−3, and the photosynthetic rate was measured as described earlier with added 10 µg cm−3 bovine CA under illumination at a photon flux density of 1600 µmol m−2 s−1 at 25 °C. DIC concentrations in culture media, which were measured every hour during acclimation, were found to be stable in a range of calculated [DIC] with an SD less than 5%. The mean values of measured [DIC] in the growth media were employed as the substrate DIC concentrations for measurements of the net rates of photosynthesis under growth conditions, and NaHCO3 was added to the oxygen electrode assay to the final concentrations equal to the measured [DIC] in growth conditions in the presence of bovine CA to maintain equilibrium at pH 7.0. After measuring the net rates of photosynthesis at the growth condition, NaHCO3 was further added to a final concentration of 5 mm, and the Pmax value of each acclimated cell sample was determined. The ratios of the net rate of photosynthesis to the Pmax rates were calculated for cells from each growth condition. The compensation CO2 concentrations were determined gas chromatographically as described byBirmingham & Colman (1979). The intracellular accumulation of DIC was determined using the silicone oil centrifugation technique as described by Matsuda & Colman (1995a).
Measurement of CA activity
Internal CA activities were measured using a modification of the technique of Wilbur & Anderson (1948). An aliquot of cell extract (usually 100 mm3) was added to 1.4 cm3 of ice-cold 2 mm veronal buffer (pH 8.3) in a water-jacketed chamber maintained at 4 °C. The pH of the veronal buffer was constantly monitored with a pH electrode, and the reaction was started by the addition of ice-cold CO2-saturated water (0.5 cm3). The time taken for the pH drop from pH 8.3 to 8.0 was measured, and units of activity were calculated as Wilbur–Anderson unit using the formula (Tc/T) − 1, where Tc and T are the time taken in the absence or presence of sample, respectively. Activity was standardized to the total amount of Chl in the assay.
Photosynthetic parameters in cells of WT and ENU16 grown on air and 5% CO2
As described in detail previously (Matsuda & Colman 1995a), 5% CO2-grown WT cells showed significantly depressed photosynthetic affinities (K1/2 values of about 1000 µm DIC), and the affinity was not increased significantly by the addition of 10 µg cm−3 bovine CA to the external medium (Fig. 1a) (WT). In contrast, air-grown cells of the WT showed a much lower K1/2 [DIC] (275 µm), and this value decreased to about 75 µm by the addition of 10 µg cm−3 bovine CA (Fig. 1a) (WT), whereas Pmax was maintained unchanged, indicating that the CCM was repressed in 5% CO2 but induced in air, and the operation of the CCM at low external inorganic concentrations was limited by the rate at which CO2 was spontaneously supplied from HCO3- in the bulk medium. In sharp contrast to WT cells, in the CO2-insensitive mutant ENU16, high-affinity photosynthesis was not repressed but operated at maximum rate even when grown in 5% CO2 (Fig. 1b) (ENU16), and K1/2 [DIC] values were about 250 and 75 µm, respectively, without and with added 10 µg cm−3 bovine CA in the assay system, irrespective of growth CO2 conditions (Fig. 1b) (ENU16).
Acid-labile carbon accumulated in 5% CO2-grown WT cells at initial DIC of 100 µm was 18 nmol mg−1 Chl, which was increased to 250 nmol mg−1 Chl by acclimation of WT cells to air, whereas those in ENU16 were 135 and 180 nmol mg−1 Chl, respectively, in 5% CO2- and air-acclimated cells. These indicate that the CCM is expressed constitutively in ENU16 in a wide range of growth CO2 concentrations from air level to 5%. Pmax values were fairly stable in both cells at about 105 µmol O2 mg−1 Chl h−1 irrespective of growth CO2 concentrations (Fig. 1).
Changes in photosynthetic parameters during acclimation from air to 5% CO2
Air-grown cells of WT and ENU16 were transferred to 5% CO2, and changes in Pmax and K1/2 [DIC] values were monitored over 10 d acclimation to 5% CO2. Cell growth was arrested for the initial 2–4 d and then started again over the next 6–8 d. Cell density was maintained between 0.25 and 0.45 OD730 by daily dilution. Interestingly, an abrupt drop of Pmax in air-grown cells to a rate about 41% of that of the initial was observed in WT cells within 1 d of acclimation to 5% CO2 (Fig. 2a) (WT), and Pmax values gradually recovered to the original maximum level over the next 4 d of acclimation (Fig. 2a) (WT). K1/2 [DIC] values slowly increased in the initial 4 d of acclimation, which was followed by a rapid increase of K1/2 [DIC] during 4–10 d of acclimation to 5% CO2 (Fig. 2a) (WT). In contrast to the WT cells, no significant decrease in Pmax was observed in ENU16, and photosynthetic affinities were stable at maximum levels throughout the acclimation to 5% CO2 (Fig. 2b) (ENU16).
Changes in internal CA activities during acclimation from air to 5% CO2
Intact cells of WT and ENU16 showed little measurable CA activity using the method employed in this study, i.e. there was no external CA. CA activity in whole cell lysate of air-grown WT cells was about 125 WAU mg−1 Chl, whereas that in air-grown ENU16 was about 85 WAU mg−1 Chl (Fig. 3). During acclimation to 5% CO2 over 10 d, CA activities in WT–cell lysates decreased gradually and reached 70 and 35 WAU mg−1 Chl after 4 and 10 d of acclimation, respectively (Fig. 3a) (WT), whereas those in ENU16–cell lysates were stable at around 50 WAU at the late stage of acclimation although they underwent a significant drop within 12 h of acclimation (Fig. 3b) (ENU16).
Changes in Pmax during acclimation from air to 5% CO2 in the presence of EZA
An abrupt Pmax drop to about 53% the original levels of Pmax was observed initially (within 6 h acclimation) when both WT and ENU16 cells were acclimated from air to 5% CO2 in the presence of 0.5 mm EZA (Fig. 4). The acclimation profiles of WT and ENU16 cells under EZA treatment were strikingly similar, and the reduced Pmax at the initial acclimation stage quickly returned to the original levels within 24 h of acclimation, which was a much faster recovery than that of WT cells without EZA treatment (Fig. 4).
Net rate of photosynthesis under culturing conditions in cells acclimated to various equilibrium [CO2]
In this experiment, both WT and ENU16 cells were initially grown in 5% CO2 and transferred to controlled [CO2], in which gaseous CO2 was equilibrated with dissolved CO2 in the bulk medium. The equilibrium was confirmed by comparing the measured [DIC] in the bulk medium to the calculated [DIC] during acclimation (data not shown). The photosynthetic rates of each cell type were measured by adding NaHCO3 at the [DIC] measured in the bulk medium in the presence of bovine CA. Pmax values of acclimated cells of WT and ENU16 were maintained between 100 and 110 µmol O2 mg−1 Chl h−1. In WT cells acclimated to 0.14–0.4% [CO2], K1/2[DIC] values remained stable at intermediate levels ranging from 50 to 200 µm, which are correlated with the [CO2] increase in the bulk medium (Fig. 5), and the photosynthetic rates at culturing DIC conditions were shown to be reduced to about 70% of the Pmax level over this intermediate [CO2] range (Fig. 5). In contrast, in ENU16 cells, photosynthetic affinity was maintained at a maximum at all growth CO2 concentrations (Fig. 5). The net photosynthetic rates under culturing conditions in ENU16 remained stable at Pmax levels when grown in 0.14–0.4% [CO2] (Fig. 5). In both strains, net photosynthetic rates in cells grown in pCO2 below 0.15% decreased as [CO2] in the bulk medium decreased (Fig. 5), which is simply caused by DIC limitation in the bulk medium. Net photosynthesis of both strains reached to a Pmax rate of about 105 µmol O2 mg−1 Chl h−1 at around 0.15% CO2. The doubling rate of cells grown in 0.25% [CO2] was found to be about 1.3 ± 0.04 (n = 2) times doubling per day in WT cells, whereas that of ENU16 was 1.55 ± 0.02 (n = 2) times doubling per day.
The CO2-insensitive phenotype of C. ellipsoidea has previously been characterized as the constitutive operation of the CCM over a growth CO2 range from air level to 5% (Matsuda & Colman 1996), which confers a high-affinity photosynthesis on cells even when grown on high CO2 (Fig. 1). In ENU16, all the photosynthetic parameters, except K1/2[DIC] values in 5% CO2-grown cells, were similar to those in WT cells, indicating that the photosynthetic apparatus was intact but that mutagenesis had caused lesions in the mechanisms controlling the expression of the CCM.
An abrupt drop in the Pmax value at the initial stage of acclimation of air-grown cells to 5% CO2 occurred specifically in WT cells (Fig. 2). At this stage of acclimation, both WT and ENU16 cells exhibited maximum photosynthetic affinity for DIC (Fig. 2), implying that this Pmax drop is not related directly to the operation of high-affinity photosynthesis. A similar Pmax drop caused by a high CO2 shock was previously reported in a hyper-CO2-tolerant strain of the green alga C. littorale (Satoh et al. 2001). Suppression of photosynthesis by high CO2 shock in C. littorale lasted for 1–4 d after transferring cells from air to more than 20% CO2, and cell growth was arrested in this period (Satoh et al. 2001). Because these physiological effects of high CO2 shock were effectively mitigated by the addition of membrane-permeable inhibitor of CA, EZA, the extreme CO2 syndrome in C. littorale, was ascribed to intracellular acidosis (a pH drop from 7.0 to 6.4) because of internal CA activity in air-grown cells (Satoh et al. 2001). Because C. littorale is a hyper-CO2-tolerant strain, there is a huge difference in threshold toxic concentrations of CO2 between the two algal species, C. littorale and C. ellipsoidea, i.e. C. ellipsoidea cannot grow in CO2 above 15% (data not shown). It is therefore probable that a disruption of homeostasis similar to that in C. littorale was induced by 5% CO2 in the less CO2-tolerant species, C. ellipsoidea. From the fact that no suppression of photosynthesis occurred in ENU16 (Fig. 2) (ENU16), it is also noteworthy that a CO2-sensing system could potentially be a negative factor in the response of cells to high-CO2 shock in addition to its key role in CCM regulation.
The mentioned considerations prompted us to assess internal CA activity during the acclimation processes. Chlorella ellipsoidea does not possess measurable external CA activity (Rotatore & Colman 1991a) and therefore CA activities detected in cell lysates represent internal CA activity. As compared to the expected down-regulation of internal CAs in WT cells, that of ENU16 cells underwent an initial steep-drop but was maintained at higher, relatively stable, levels than those in WT cells in the late stages of acclimation (Fig. 3). Because the type and localization of internal CAs in C. ellipsoidea have not been established, the reason for this difference is unclear. However, it is most likely that some of internal CA activities are controlled by factors mutated in ENU16 cells, and this lesion seems to confer on ENU16 cells a significant tolerance to the shock with the treatment by 5% CO2. The temporary decrease in Pmax in WT cells in the initial stages of acclimation to 5% CO2 was significantly mitigated by EZA, i.e. a relatively small drop in Pmax was recovered in a day, resembling the response in ENU16 cells, whereas in WT cells in the absence of EZA, a strong inhibition of photosynthesis lasted for at least 2 d (Fig. 4). It is probable that the CO2-insensitive mutation in ENU16, not only causes derepression of high-affinity photosynthesis, but also blocks the CO2-responsive induction of some subtypes of internal CAs, which in turn, would protect cells from intracellular acidification caused by internal CA under high CO2. Although such a CA protein is yet to be specified biochemically, protein fingerprint analysis done with in vitro translation products of mRNAs extracted from WT and ENU16 cells showed clearly that one major soluble protein at molecular mass of 41 kDa and isoelectric point (pI) of 5.0 was induced in air only in WT cells, but no induction was observed in ENU16 cells (data not shown), indicating that both repressible and inducible responses are impaired in the CO2-insensitive mutant, ENU16.
Acclimation of cells to intermediate CO2 between 0.14 and 0.4% resulted in WT cells acquiring an intermediate affinity for DIC (Fig. 5). Interestingly, at this range of culturing cells at equilibrium DIC concentrations, net photosynthetic rates in WT cells decreased significantly (Fig. 5). In contrast to the direct limitation of external [DIC] below 0.14% CO2, this drop of net photosynthesis at intermediate CO2 range would occur as a result of acclimated cells not being saturated by [DIC] in the culture media because of their decreased photosynthetic affinity for DIC. This result indicates that the CO2 response system does not necessarily lead to the acquisition of a maximum net rate of photosynthesis, but sometimes reduces the rate significantly under moderately elevated CO2. The doubling rate of ENU16 was about 1.2 times faster than that in WT cells under [CO2] between 0.24 and 0.3% presumably because of the difference in net photosynthetic rates between WT and ENU16 cells in these CO2 range. The phenotype of CO2-insensitive mutants is thus likely to be superior to the WT both in tolerating high-CO2 shock and maintaining growth rate under moderately enriched CO2. The physiological significance of having the CO2 response system is thus so far unclear.
The results of the present study indicate that the response of WT cells to [CO2] does not confer any survival value under conditions of CO2 shock or those of intermediate [CO2] between air level and 5%, because the CO2-insensitive mutant, ENU 16, maintained a stable photosynthetic rate under changing growth conditions of [CO2] and grew at a doubling rate 1.2 times that of WT cells. However, the cells in this study were cultured under optimal conditions of nutrient supply, and this does not reflect the conditions of the natural habitat of the alga in which the supply of required inorganic nutrients, other than DIC, may be limited. The cost of synthesizing and maintaining active DIC transport systems may therefore be a disadvantage to cells growing under nutrient-limited conditions. The CO2-sensing response of WT cells, which allows some repression of active DIC uptake, may therefore allow the cells to acclimate to conditions of high [CO2] and low availability of other nutrients. It is also possible that high CO2 condition could readily occur because of a bacterial respiration in natural soil environment, and the CO2 sensing system in WT cells of C. ellipsoidea might reveal a significant survival value under CO2 higher than 5%.
This work was supported in part by a grant from the Natural Sciences and Engineering Research Council of Canada to B.C., and in part by a grant from Invitation for Research Institute of Innovative Technology for the Earth Research Proposal to Y.M.