Induction of the carbon concentrating mechanism (CCM) has been investigated during the acclimation of 5% CO2-grown Chlamydomonas reinhardtii 2137 mt + cells to well-defined dissolved inorganic carbon (Ci) limited conditions. The CCM components investigated were active HCO3− transport, active CO2 transport and extracellular carbonic anhydrase (CAext) activity. The CAext activity increased 10-fold within 6 h of acclimation to 0·035% CO2 and there was a further slight increase over the next 18 h. The CAext activity also increased substantially after an 8 h lag period during acclimation to air in darkness. Active CO2 and HCO3− uptake by C. reinhardtii cells were induced within 2 h of acclimation to air, but active CO2 transport was induced prior to active HCO3− transport. Similar results were obtained during acclimation to air in darkness. The critical Ci concentrations effecting the induction of active Ci transport and CAext activity were determined by allowing cells to acclimate to various inflow CO2 concentrations in the range 0·035–0·84% at constant pH. The total Ci concentration eliciting the induction and repression of active Ci transport was higher during acclimation at pH 7·5 than at pH 5·5, but the external CO2 concentration was the same at both pHs of acclimation. The concentration of external CO2 required for the full induction and repression of Ci transport and CAext activity were 10 and 100 μM, respectively. The induction of CAext and active Ci transport are not correlated temporally, but are regulated by the same critical CO2 concentration in the medium.
If you can't find a tool you're looking for, please click the link at the top of the page to "Go to old article view". Alternatively, view our Knowledge Base articles for additional help. Your feedback is important to us, so please let us know if you have comments or ideas for improvement.
Many micro-algal species respond to limitations in extracellular dissolved CO2 by the induction of a carbon concentrating mechanism (CCM). The function of the micro-algal CCM is to elevate the CO2 concentration in the vicinity of the main carboxylating enzyme, Rubisco, which is located in the algal cell chloroplast ( Moroney & Somanchi 1999). Rubisco isolated from various micro-algal species has a low affinity (high Km) for CO2 in comparison with the whole cell affinity (K1/2) for CO2 ( Badger et al. 1998 ). CCM derepression in high CO2-grown Chlorella spp. is in response to low external dissolved CO2 concentration ( Matsuda & Colman 1995b; Colman, Bozzo & Matsuda 1998). It has been proposed that the CCM is induced when the CO2 concentration in the medium is reduced to a critical level. Matsuda, Bozzo & Colman (1998) have suggested the possibility of a CO2 sensor at the green algal cell surface which, under high CO2 growth conditions would cause repression of the CCM, whereas under CO2 depletion the sensing mechanism would initiate a signalling cascade culminating in de novo protein synthesis, and the derepression of the CCM.
The CCM of green algae such as Chlorella ellipsoidea and Chlorella kessleri consists of a high-affinity inorganic carbon (Ci) uptake system ( Matsuda & Colman 1995a; Matsuda et al. 1998 ; Matsuda, Williams & Colman 1999), which gives rise to a low CO2 compensation concentration, an accumulation of intracellular Ci, and reduced photorespiration. Chlamydomonas reinhardtii is a biflagellate unicellular green alga with similar high-affinity Ci uptake system. Palmqvist, Sjöberg & Samuelsson (1988) report an increased affinity for Ci and CO2 by C. reinhardtii during acclimation to low Ci conditions. This increased affinity in C. reinhardtii cells is due to active transport of CO2 and HCO3− ( Sültemeyer et al. 1989 ). During acclimation to low CO2, the affinities for Ci transport by protoplasts and isolated chloroplasts increase significantly in C. reinhardtii cells ( Amoroso et al. 1998 ) indicating that active CO2 and active HCO3− transport occur at both the plasmalemma and chloroplast envelope. In another green algal species, C. ellipsoidea, active Ci transport is localized at the plasma membrane, but not at the chloroplast envelope ( Rotatore & Colman 1991). Active CO2 and HCO3− transport are induced in C. ellipsoidea ( Matsuda & Colman 1995a) and C. kessleri ( Matsuda et al. 1998 ) during acclimation to low CO2.
Chlamydomonas reinhardtii has an external carbonic anhydrase which is largely repressed by growth on high CO2. During the acclimation of C. reinhardtii cells to low external CO2 concentrations, there is a marked increase in activity of various carbonic anhydrases (CA), but particularly in the periplasmic or external carbonic anhydrase (CAext) ( Aizawa & Miyachi 1986; Coleman 1991; Badger & Price 1994). It is thought that the function of CAext is to maintain the equilibrium between HCO3− and CO2 at the cell surface. The importance of the periplasmic CA in the Chlamydomonas CCM has been debated in the past: Moroney, Husic & Tolbert (1985) demonstrated that CAext is essential for the utilization of Ci at low external CO2 concentrations and alkaline pH, whereas studies using CA inhibitors and cell wall-less mutants have demonstrated that CAext is not required for active Ci uptake under these conditions ( Williams & Turpin 1987). Furthermore, Van & Spalding (1999) have generated a null mutant (Cah1) of C. reinhardtii which has no Cah1 gene expression, which encodes for CAext during growth in air, but maintains similar photosynthetic characteristics when compared to wild-type cells. These results suggest that CAext is not an essential component in the CCM.
Expression of CAext depends on the pH of the medium during growth and acclimation. In the green alga, Chlorella ellipsoidea C-27, Shiraiwa, Yokoyama & Satoh (1991) demonstrated that external CA activity was highest in cells acclimating to low CO2 in the pH range of 7·0–8·0, and lower in cells acclimating at pH 5·5. Gehl, Colman & Sposato (1990) demonstrated that CAext activity in Chlorella saccharophila is suppressed by growth at acid pH. These results suggest that CAext activity is induced under conditions where the ratio of dissolved HCO3− concentration to dissolved CO2 concentration is high. Similarly, Williams & Colman (1996) found that CAext activity increased with a decrease in CO2 availability. However, the critical inorganic carbon concentration effecting the induction of CAext has not been determined in C. reinhardtii or any other micro-alga.
In this study we examined the correlation between the induction of active Ci transport and increase in CAext activity during the acclimation of C. reinhardtii cells to low external CO2 concentrations. The critical Ci species and concentration effecting the induction of these CCM components were also determined.
MATERIALS AND METHODS
An axenic culture of wild-type Chlamydomonas reinhardtii Dangeard (2137 mt +) cells was obtained from the Chlamydomonas Genetics Center, Duke University. Cells were grown axenically in batch culture under a constant light fluence (100 μmol m−2 s−1) as described previously ( Gehl et al. 1990 ), in Sager–Granick medium (SGM; Sager & Granick 1953) without acetate or citrate. The medium was modified by replacing 0·038 m M ammonium nitrate with 0·042 m M ammonium chloride. Cultures grown under high-CO2 conditions were aerated with 5% (v/v) CO2 in air at a rate of 3·6 L min−1.
The time course of induction of extracellular CAext activity was determined during the acclimation of high CO2-grown cells to air. High CO2-grown cells were harvested at mid-logarithmic growth phase (A730 0·4) by centrifugation at 5000 g for 3 min at room temperature, resuspended in SGM at pH 6·6, and allowed to acclimate to air level CO2. During the acclimation process, samples of cell suspension were taken periodically for the CAext activity measurement; the cells were harvested, washed in 20 m M Na+-barbital buffer (pH 8·3), resuspended in 1·5 mL of the same buffer and placed in a water-jacketed chamber (2·0–4·0 °C) containing a pH electrode. The CA activity of the intact cells was determined by a potentiometric assay ( Williams & Colman 1996) in which 0·5 mL of ice-cold CO2-saturated water was injected into the sample and the time taken for the pH to drop from 8·3 to 8·0 was recorded. Units of activity were calculated using the formula (Tc/Ts) – 1, where Ts and Tc are the times recorded in the presence and absence, respectively, of the cell sample. The chlorophyll concentration of the cell suspension was between 20 and 50 μg Chl mL−1, and was determined as described previously ( Williams & Colman 1993).
The time course of induction of active Ci transport during the acclimation of high CO2-grown cells to air was assessed as described previously ( Matsuda & Colman 1995a). The Ci concentration in the medium was monitored periodically. During the acclimation process, 25–30 mL of cell culture suspension was harvested periodically, as described above. The O2 evolution rates were measured with a Clarke-type O2 electrode apparatus (Hansatech Instruments Ltd, Norfolk, UK). The capacity of the cells to take up HCO3− actively was assessed by comparing the O2 evolution rate at 100 μMCi, pH 8·0 and 25 °C, with the uncatalyzed rate of CO2 formation from the available HCO3− in the medium, calculated according to the method of Miller & Colman (1980). The presence of a catalyst would invalidate this technique of active HCO3− transport determination therefore since we find that high CO2-grown C. reinhardtii cells always contain trace activity of CAext, this determination was done in the presence of 5 μM acetazdamide (AZA), a CA inhibitor.
Stimulation of the O2 evolution rate at 100 μMCi upon the addition of bovine CA (10 μg mL−1) was used as a measure of active CO2 uptake. In the presence of added CA there will be no limitation in the supply of substrate to the CO2 transporter and photosynthetic O2 evolution will therefore be supported by active CO2 uptake, HCO3− uptake and diffusive CO2 uptake. However, the bulk of Ci uptake will be mediated by the CO2 transporter since micro-algae have a higher affinity for CO2 than HCO3− ( Matsuda et al. 1999 ).
The critical Ci conditions corresponding to the induction of active Ci transport and CAext activity were determined by the procedure of Matsuda & Colman (1995b). High CO2-grown cells were harvested at mid-log phase, and resuspended in SGM (buffered with phosphate at pH 5·5 and 7·5). Cell suspensions were axenically transferred to 0·5 L cylindrical culture vessels equipped with a sampling port plugged with a rubber serum stopper, and aerated with defined inflow CO2 concentrations in the range of 0·035–0·84%. The dissolved CO2 concentration in the medium was maintained constant by adjusting the pH to ± 0·1 units, by injections of 2·0 M HCl or 2·0 M NaOH and by controlling the inflow CO2 concentration. The inflow CO2 concentration and the Ci concentration of the medium were measured by gas chromatography ( Birmingham & Colman 1979). Equilibrium conditions between HCO3− and CO2 in the culture medium were verified by comparing the calculated concentrations of Ci at the pH of the acclimating medium and inflow CO2 concentration ( Buch 1960; Stumm & Morgan 1981) with the measured concentration of Ci in the medium. Acclimating cells were harvested after 2 h of acclimation at the defined CO2 concentration. At this point, rates of photosynthetic oxygen evolution were measured at 100 μMCi, pH 8·0 and 25 °C with AZA, followed by measurements with excess bovine CA. Acclimating cells were also harvested after 6 h at the defined CO2 concentration, and CAext activity was measured.
Changes in extracellular carbonic anhydrase activity during acclimation to low CO2
Chlamydomonas reinhardtii cells grown under high CO2 conditions were acclimated to ambient CO2 conditions. CAext activity was measured periodically and was found to increase markedly within the first 5 h of acclimation to 0·035% CO2. Within 6 h, there was a 10-fold increase in CAext activity, when compared with the basal level of activity measured in high CO2-grown cells ( Fig. 1). There was a slight subsequent increase in activity between 6 h and 24 h of acclimation. Changes in CAext activity were also measured with cells acclimating to air in darkness ( Fig. 1). Within 8 h of acclimation in darkness, there was a three-fold increase in comparison with high CO2-grown cells, which represented a slight lag in the induction of CAext activity, as compared to that in light. After 10 h of acclimation under low CO2 and darkness, CAext activity was approximately two-fold greater than cells acclimated for 8 h. There was no significant increase in CAext activity in the dark between 10 h and 24 h of acclimation.
In order to assay for the induction of active HCO3− transport in C. reinhardtii cells during acclimation to low CO2, it was necessary to block CAext activity. At alkaline pH conditions, the presence of CAext activity maintains the CO2-HCO3− equilibrium, and therefore comparison of the measure of photosynthetic O2 evolution rates with the calculated uncatalyzed rate of CO2 formation is not a valid assessment of active HCO3− uptake. AZA (5 μM) was found to completely inhibit CAext activity in air-grown cells harvested at mid-log phase.
Induction of active Ci transport during acclimation to air
Photosynthetic O2 evolution rates were measured periodically during the acclimation of high CO2-grown C. reinhardtii cells to ambient CO2. The measurement of O2 evolution at 100 μMCi, pH 8·0 and 25 °C in the presence of 5 μM AZA was compared with the maximum rate at which CO2 is formed by the spontaneous dehydration of HCO3−, which, under these conditions, is 7·3 nmol O2 mL−1 min−1. In high CO2-grown cells, O2 evolution rates measured in the presence of AZA are significantly lower than the spontaneous dehydration rate, which suggests that there is no active HCO3− uptake ( Fig. 2). Within 2 h of acclimation to air, there was an increase in O2 evolution in the presence of AZA, which was 15-fold greater than the spontaneous dehydration rate ( Fig. 2). This indicates that active HCO3− uptake is induced within 2 h of acclimation to low CO2.
In 30-min acclimated cells the addition of bovine CA resulted in stimulation of O2 evolution, which was approximately three-fold greater than the O2 evolution rate in the presence of AZA. This indicates the induction of active CO2 uptake. Active CO2 uptake appeared to be fully induced within 2 h of acclimation to low CO2 ( Fig. 2). O2 evolution rates measured with bovine CA were not inhibited in the presence of AZA (data not shown). At 2 h there was a five-fold increase in the O2 evolution rate in comparison to that of high CO2-grown cells.
The time course of CCM induction was also assessed during the acclimation of C. reinhardtii cells to 0·035% CO2 in darkness. O2 evolution measured in the presence of AZA was significantly greater than the spontaneous dehydration rate within 2 h of acclimation to air ( Fig. 3), indicating that active HCO3− uptake is induced in darkness. The addition of bovine CA resulted in a stimulation of O2 evolution: measured rates were approximately two-fold greater than the rate of O2 evolution supported by active HCO3− uptake within 2 h of acclimation to low CO2 ( Fig. 3). This indicates that active CO2 uptake is also induced during acclimation in darkness.
Determination of the critical Ci concentration eliciting the CCM response
Chlamydomonas reinhardtii cells were acclimated to various external CO2 concentrations in the range of 0·035–0·84% for 6 h. After 2 h of acclimation, a small volume of the acclimating cell culture was harvested in order to measure photosynthetic O2 evolution. For each external CO2 concentration at which C. reinhardtii cells were acclimated, O2 evolution rates were measured in the presence of AZA, followed by measurements in the presence of bovine CA. With an increase in the external CO2 concentration during acclimation, there was a concomitant decrease in photosynthetic O2 evolution ( Figs 4 & 5). In cells acclimating at pH 5·5, HCO3− transport was fully induced at approximately 11 μMCi, whereas at pH 7·5 HCO3− transport was fully induced at approximately 160 μMCi ( Figs 4 & 5). Regardless of the pH of the culture medium in which the cells were acclimated, HCO3− transport was fully induced at approximately 10 μM dissolved CO2 external. The maximum rates of photosynthetic O2 evolution, with AZA and with CA, in cells acclimated to CO2-free air were not significantly different from those acclimated in media containing 10 μM CO2 (data not shown). HCO3− transport was fully repressed during acclimation at pH 7·5 and 5·5 at approximately 1600 and 100 μMCi, respectively. The concentration of CO2 at these total Ci concentrations is the same, HCO3− uptake was fully repressed at 98 μM dissolved CO2.
The same phenomenon was apparent with O2 evolution measurements in the presence of bovine CA. Active CO2 transport was fully induced in cells at approximately 13 and 192 μMCi during acclimation at pH 5·5 and 7·5, respectively ( Figs 4 & 5). At both pHs, active CO2 transport was fully induced at 12 μM dissolved CO2 in the external medium. Transport of this inorganic carbon species was fully repressed at when cells were acclimated at 100 μM dissolved CO2, at both pHs.
In order to compare CA activity under different concentrations of CO2, cells were allowed to acclimate for 6 h, which has been found to give maximum CA activity in time course experiments. CAext activity was measured in C. reinhardtii cells acclimated to various external CO2 concentrations at pH 5·5 or 7·5. CAext activity increased concomitantly with a decrease in the external CO2 concentration during the 6 h acclimation period ( Fig. 6). The highest level of CAext activity was approximately 68 W-A units mg Chl−1. Regardless of the pH at which the cells were acclimated, CAext was fully induced during acclimation at 10 μM dissolved CO2. Basal CAext activity (< 10 W-A units mg Chl−1) was apparent after acclimation to dissolved external CO2 concentrations above 100 μM CO2 ( Fig. 6).
The CCM in C. reinhardtii is induced during acclimation to a continuous exposure to a low concentration of CO2. The development of the micro-algal CCM includes the induction of high-affinity Ci transport activity, and in some species, the induction of various CAs. It is apparent in this C. reinhardtii strain that the induction of active Ci transport ( Fig. 2) occurs well in advance of the full induction of CAext activity ( Fig. 1) during acclimation to ambient CO2 conditions. At the time that active Ci transport is fully induced (2 h), CAext activity is only 50% fully induced. Induction of these two CCM components do not seem to be correlated. This suggests that CAext activity has an accessory, rather than a central, role in the CCM of C. reinhardtii, and it is likely that the function of the CAext is to maintain a supply of CO2 to high-affinity CO2 transporters at the cell surface. This is consistent with the report of Van & Spalding (1999) that a C. reinhardtii mutant which does not express periplasmic CA has the ability to acclimate to low CO2 in the same manner as wild-type cells. The induction of both components, CAext and CO2 transport, seems to be in response to the same critical CO2 concentration in the external bulk medium ( Figs 4, 5 & 6).
The time course of induction of active Ci transport seems to vary among green algal species and between strains of the same species. The induction of high-affinity HCO3− and CO2 transport in C. reinhardtii was shown to occur within 2 h of acclimation to low CO2 ( Fig. 2). In contrast, Sültemeyer, Fock & Canvin (1991) report that the induction of high-affinity photosynthesis in C. reinhardtii (strain 11/32b) occurs after 4 h acclimation to low CO2. These time courses of induction were more rapid than those in C. kessleri ( Matsuda et al. 1998 ) and C. ellipsoidea ( Matsuda & Colman 1995a), which occurred within 5·5 h under the same conditions. A low level of active CO2 transport exists in high CO2-grown C. reinhardtii cells ( Fig. 2, Sültemeyer et al. 1989 ) and a three-fold increase is induced prior to the induction of active HCO3− transport in C. reinhardtii. The temporal separation in the induction of the two uptake systems is also a characteristic of Chlorella cells acclimating to low CO2 ( Matsuda et al. 1998 ).
In C. reinhardtii, active CO2 and active HCO3− uptake are repressed at approximately 100 μM dissolved CO2 ( Figs 4 & 5), although active CO2 transport is not totally repressed, even when cells are grown at high CO2 conditions ( Figs, 2, 3, 4 & 5), and while HCO3− transport cannot be detected there may be a low-affinity HCO3− transport system present in these cells. HCO3− transport can be detected at approximately 60 μM ( Figs 4 & 5). In C. ellipsoidea, the CO2 concentration at which HCO3− transport starts to be induced is approximately 60 μM ( Matsuda & Colman 1995b), whereas in C. kessleri this occurs at a dissolved CO2 concentration of approximately 70 μM. This suggests that bicarbonate transport is induced in response to a lower dissolved CO2 concentration in C. reinhardtii than in Chlorella spp. Active CO2 uptake is 50% fully induced in Chlorella spp. at dissolved CO2 concentrations that are greater than those required to initiate the induction of HCO3− transport during acclimation ( Matsuda & Colman 1995b; Colman et al. 1998 ). High-affinity active CO2 transport in green algae is very effective in depleting the external medium of CO2 ( Sültemeyer et al. 1991 ), and in the acclimation of C. reinhardtii cells to low CO2, this might explain the lag in the induction of active HCO3− uptake which is induced at a lower CO2 concentrations than CO2 transport. In Chlorella spp., the critical Ci species effecting the induction of HCO3− and CO2 transport is the dissolved CO2 in the external medium ( Matsuda & Colman 1995b).
The induction of high-affinity Ci transport activity has been shown to require de novo protein synthesis in Chlorella spp. ( Matsuda & Colman 1995a; Colman et al. 1998 ). A variety of polypeptides are synthesized in C. reinhardtii in response to low CO2 acclimation. One of the polypeptides synthesized during the low CO2 response is a 37 kDa polypeptide, which is a CAext ( Coleman et al. 1984 ). Two genes encoding for CAext in C. reinhardtii have been isolated ( Fujiwara et al. 1990 ). The genes, CAH1 and CAH2, are differentially regulated by the external CO2 concentration in the bulk medium: CAH1 expression increases during acclimation to low CO2, whereas CAH2 expression increases under high CO2 growth. The CAH1 gene consists of a silencer and an enhancer region upstream of a minimal promoter ( Kucho, Ohyama & Fukuzawa 1999). Under low CO2 conditions in the presence of light, the enhancer element activates the CAH1 promoter, whereas under high CO2-conditions the silencer region represses transcription.
In the present study, the change in CAext activity during low CO2 acclimation was investigated. CAext is a low CO2-responsive CA, which is most likely encoded by CAH1. CAext activity reached its maximum after 6 h acclimation to ambient CO2 ( Fig. 1). The time course of induction was used to determine the time needed for acclimation to various external CO2 concentrations at pH 5·5 and 7·5. Under equilibrium conditions, at pH 5·5, approximately 95% of the total Ci exists in the form of CO2. At pH 7·5, approximately 6·6% of the total Ci exists in the form of CO2. In previous studies, the principal question about the induction of CAext was whether there was a response to total Ci, HCO3− concentration or CO2 concentration in the bulk medium. Regardless of the pH of the medium during acclimation, CAext activity in C. reinhardtii starts to increase when the dissolved CO2 concentration in the external medium is lower than 100 μM ( Fig. 6). Maximum CAext activity in C. reinhardtii was observed during acclimation to 10 μM dissolved CO2. These results suggest that the increase in CAext activity is initiated at the dissolved CO2 concentration triggering the induction of active CO2 uptake.
Previous studies on the induction of CAext have maintained that the process requires light ( Spalding & Ogren 1982; Umino, Satoh & Shiraiwa 1991). For example, Dionisio-Sese, Fukuzawa & Miyachi (1990) reported the requirement for light in the induction of CAext and found specifically that blue light at 460 nm was most effective ( Dionisio, Tsuzuki & Miyachi 1989). However, our results indicate that CAext activity increases after a significant lag period during acclimation to air in darkness ( Fig. 1). This has not been observed before, possibly because, in previous studies, CAext expression in the dark was only monitored in cells acclimated for 4–6 h ( Dionisio-Sese et al. 1990 ). Our findings are in agreement with the results of Rawat & Moroney (1995) who found that high CO2-grown C. reinhardtii produced CAH1 transcript when allowed to acclimate to air in the dark.
Light is not an absolute requirement for the induction of the CCM in green algae. Active Ci transport is induced in Chlorella spp. during acclimation to low CO2 in darkness ( Matsuda & Colman 1995b; Matsuda et al. 1998 ). It is also apparent that active Ci uptake in C. reinhardtii is induced during acclimation to air in darkness ( Fig. 3). It has been proposed that the build-up of photorespiratory products in the green algal cell served as the trigger for induction of the CCM ( Marcus, Harel & Kaplan 1983), but this is not consistent with the induction of active Ci uptake in the dark. This conclusion is supported by studies with photorespiratory mutants of this species: phosphoglycolate phosphatase (pgp-1) mutants of C. reinhardtii characteristically accumulate phosphoglycolate regardless of Ci conditions, and it has been suggested that the intracellular accumulation of phosphoglycolate triggers CCM induction ( Suzuki, Marek & Spalding 1990). However, Suzuki, Mamedov & Ikawa (1999) reported an increased CCM efficiency in double suppressor mutants of pgp-1 that maintain the defect in phosphoglycolate phosphatase activity but have a greater affinity for Ci than the original mutant, which suggests that photorespiratory product build-up is not a trigger for CCM induction in C. reinhardtii.
Matsuda et al. (1998) proposed that the trigger for induction of the CCM in Chlorella is a critical dissolved CO2 concentration in the bulk medium, which serves as a substrate for a CO2-sensing moiety on the cell surface. The induction of the CCM and CAext in C. reinhardtii cells is also triggered by a critical CO2 concentration in the external bulk medium.
This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada to B.C.