Journal of Experimental Botany, Vol. 51, No. 345, pp. 807-815,
April 2000
© 2000 Oxford University Press
Limiting CO2 levels induce a blue light-dependent HCO3- uptake system in Monoraphidium braunii
Centro de Investigaciones Biológicas, CSIC, Velázquez 144, E-28006 Madrid, Spain
Received 16 July 1999; Accepted 30 November 1999
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Abstract |
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The in situ photoactivation of an
uptake system in the green alga Monoraphidium braunii requires the irradiation of the cell suspensions with short wavelength radiation (blue, UVA and/or UVC). Plasma membrane ATPase inhibitors block the uptake of this monovalent anion at pH 9. M. braunii cells grown in high CO2lack an uptake system in their plasma membrane, but those grown in low CO2can take up this anion at high rates. Cells grown in high CO2, transferred to CO2-limiting conditions in the light, start taking up in 30 min, although they take 90 min to reach maximum rates of transport. Therefore, this induction process seems to be triggered by low external CO2 concentration. In fact, increasing or decreasing the external concentration does not induce the uptake system and only a decrease in CO2 concentration in the medium triggers the induction process. The appearance of the transport activity is sensitive to cycloheximide, indicating that cytoplasmic protein biosynthesis is necessary for the induction of the uptake system. Photosynthetically active radiation, but not particularly blue light, is essential for induction of the uptake system to occur and the inhibition of photosynthesis by DCMU blocks it. From these results it can be inferred that when M. braunii cells detect a drop in CO2 concentration, they induce a blue light-dependent uptake system.
Key words:
Blue light, transport, CO2 concentration, Monoraphidium braunii, photosynthesis, protein biosynthesis.
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Introduction |
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It is well established that a number of cyanobacteria and microalgae, when grown photoautotrophically in CO2-limiting conditions, have the capacity to increase the CO2 concentration around the active site of Rubisco by inducing a carbon-concentrating mechanism (CCM). This mechanism that operates in the light comprises active transport systems for Ci species, mainly CO2 and/or
, and external and internal carbonic anhydrase (CA) enzymes (Aizawa and Miyachi, 1986
; Spalding, 1989; Badger and Price, 1992, 1994). In green algae, Ci transport systems have been found at the plasma membrane and the chloroplast envelope (Moroney et al., 1987; Goyal and Tolbert, 1989; Sultemeyer et al., 1989, 1991; Rotatore and Colman, 1991; Ramazanov and Cárdenas, 1992; Palmqvist et al., 1994; Amoroso et al., 1998). During acclimation to low Ci conditions a number of proteins are synthesized and, although the function of many of them in the CCM is not yet understood (Manuel and Moroney, 1988; Spalding and Jeffrey, 1989; Ramazanov et al., 1995a, b; Satoh and Shiraiwa, 1996), some of them have been identified as different CA isoenzymes (Coleman and Grossman, 1984; Fukuzawa et al., 1990; Eriksson et al., 1996). Another important question that remains to be clarified in the understanding of the CCM in algae is the identification of its regulatory environmental signal(s).
Blue light (BL) and UV radiation are important environmental signals which activate or stimulate many processes in algal and plant cells, and these organisms have developed specific photoreceptor systems in order to detect these radiations (Kaufman, 1993; Short and Briggs, 1994; Jenkins et al., 1995). In the green unicellular microalga Monoraphidium braunii, the uptake of the metabolically most significant anions, 
, 
, Cl-, and is under short wavelength radiation control (Aparicio et al., 1994; Witt and Aparicio, 1995; Quiñones et al., 1997; Giráldez et al., 1998). The uptake of could be measured either by monitoring the pH of the medium or by following the O2 evolution associated with the photosynthetic reduction of the CO2 derived from the taken up by the cells. The uptake of Ci associated with the increase in the external medium pH was earlier described for Scenedesmus obliquus (Findenegg, 1979) and for some aquatic plants (William, 1983). uptake in M. braunii was activated by irradiation of the cell suspensions with BL, UVA or UVC radiations (Giráldez et al., 1998). When the suspensions were irradiated only with strong red light (RL) in the presence of at pH 9, very small changes in the pH of the external medium or very small rates of O2 evolution were observed. The BL-stimulated O2 evolution rates were not affected by acetazolamide (AZA), a periplasmic CA inhibitor, suggesting that the -dependent O2 evolution was due to the photoactivation of a uptake system at the plasma membrane (Giráldez et al., 1998). The action spectrum for uptake, which showed three main bands in the blue, UVA and UVC regions, was similar to those reported for and Cl- in this alga, suggesting that the same photoreceptor triggered these three uptake processes. This photoreceptor might contain flavins and pterins, and may be located at the plasma membrane (Witt and Aparicio, 1995; Giráldez et al., 1998).
Stimulation of photosynthesis by BL has been observed in several brown algae, but it seems to occur through different BL activating mechanisms (Schmid and Dring, 1996). In Ectocarpus siliculosus, BL ctivates a mechanism for Ci acquisition, producing a stimulation of photosynthesis which occurs in the absence of extracellular or intracellular CA activity (Schmid, 1998). The author suggests that BL activates the release of CO2 from an intermediate metabolite. However, in Laminaria saccharina, the presence of AZA prevented the BL-dependent stimulation of photosynthesis indicating that the BL activating mechanism relies on the activity of an extracellular CA (Schmid et al., 1996). It has been suggested that pterins may be involved in this BL response of L. saccharina (Maier and Schmid, 1997).
The results presented in this paper indicate that in M. braunii, the operation of the BL-dependent uptake system requires the activity of a plasma membrane ATPase. M. braunii cells grown in high CO2 were unable to take up from the external medium while those grown in low CO2 did so at high rates. When cells grown in high CO2 were exposed to Ci-limiting conditions in the light, an uptake system was induced in these cells. BL was not essential for this induction process. A decrease in the CO2 concentration of the external medium seems to be the environmental signal that promotes the induction of the uptake system, which only operates under BL irradiation.
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Materials and methods |
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Algal growth and general experimental conditions
Monoraphidium braunii (Legnerová, 202–7d) cells from the Universität Göttingen (Germany) were grown in a mineral medium with
as N source, at pH 7.1, under a 14/10 h light/dark photoperiod. Cultures were bubbled with air (cells grown in low CO2) or air plus 5% CO2 (cells grown in high CO2) at 28 °C under a constant PFD of 120 µmol m-2 s-1 of white light (WL). Cells were collected during the light period by centrifugation at 2000xg and washed three times with either CO2-free 25 mM HEPES, pH 7, or CO2-free 25 mM 3-[cyclohexylamino]-2-hydroxy-1 propanesulphonic acid (Capso), pH 9, buffers. Then, the cells were resuspended in the corresponding buffer, stirred with a magnetic bar and irradiated as indicated below, in an argon atmosphere for 60–120 min before starting the O2 evolution measurements. For pH measurements, the cells were washed, resuspended in a 2 mM MgSO4 unbuffered solution and incubated in the conditions described above.
Photosynthetic O2 evolution measurements
O2 evolution was measured with a Clark-type electrode (Hansatech, England), using 3 ml cell suspensions (40 µg Chl ml-1) placed in a temperature-controlled cuvette at 25 °C. Linear rates of O2 evolution were measured over periods of several minutes after the addition of 100 µM NaHCO3.
pH measurements
pH measurements were carried out at 25 °C with a Titralab system from Radiometer (Denmark). The cell suspensions (40 µg Chl ml-1) were kept in CO2-free air during the measurements. The initial pH of the suspensions was adjusted by adding diluted solutions of NaOH or H2SO4. Changes in pH were measured after the addition of 100 µM NaHCO3.
Irradiation conditions and radiation measurements
Cell suspensions were irradiated during the incubation period with WL (600 µmol m-2 s-1) from a slide projector (Zeiss Ikon, Braunscheweig, Germany) fitted with a 150 W tungsten-halogen lamp. In some of the experiments, cell suspensions were irradiated during the incubation period on one side with 300 µmol m-2 s-1 of 690 nm RL from a 200 W lamp from Oriel (USA) and on the other side with 45 µmol m-2 s-1 of 450 nm BL from a slide projector. For pH and O2 evolution measurements, cell suspensions were irradiated with either strong (150 µmol m-2 s-1) RL of 690 nm or this strong RL plus weak (18 µmol m-2 s-1) BL of 450 nm. RL was obtained from a 150 W tungsten-halogen lamp in a housing with three glass-fibre light pipes and BL was obtained from a 500 W mercury-xenon arc lamp from Oriel (USA). Interference filters of 10 nm bandwidth from Oriel and Schott (Germany) were used to obtain monochromatic radiations (450 and 690 nm). The PFD of the different radiations was measured with a LI-190SB quantum sensor coupled to a LI-188B integrating quantum-radiometer-photometer from Li-Cor (USA).
Inhibitors
Diethylstilbestrol (DES), cycloheximide (CHI), chloramphenicol (CAP), and DCMU were purchased from SIGMA. DES, CAP and DCMU were dissolved in ethanol; the final concentration of ethanol in the treated cell suspensions was never higher than 1%. Control experiments with only 1% ethanol gave the same results as those performed in its absence. Vanadium oxide, V2O5, was purchased from Merck and was used as a source of vanadate (Gallagher and Leonard, 1982; Thielmann et al., 1990).
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Results |
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Inhibition of the
-dependent photosynthetic O2 evolution by vanadate and DESIn the green alga M. braunii, the
-dependent BL-stimulated photosynthetic O2 evolution at pH 9 does not depend on the activity of a periplasmic CA (Giráldez et al., 1998). Therefore, it is very likely that the is transported as such in an energy-dependent process. In order to demonstrate this, the effect of two ATPase inhibitors, vanadate and DES, on the -dependent photosynthetic O2 evolution at pH 9 was tested using M. braunii cells grown in low CO2 (Fig. 1). Vanadate has a strong affinity for the active site, preventing the formation of the covalent phosphoenzyme intermediate of ATPase (Cantley et al., 1978). DES is also an effective inhibitor of ATPases (Balke and Hodges, 1979; Gallagher and Leonard, 1982). As the concentration of these inhibitors increased in the external medium of the cell suspensions kept at pH 9 in the presence of , both of them inhibited the O2 evolution rates sustained under RL or under RL plus BL. As shown in Fig. 1, the BL-stimulated O2 evolution rates decreased to a level similar to or slightly lower than the values observed under RL irradiation in the absence of inhibitors. On the other hand, there also was inhibition of the RL-stimulated O2 evolution rates. However, at high concentrations of inhibitors (>200 µM vanadate or >50 µM DES), the differences between O2 evolution rates obtained in RL or RL plus BL were not significant (see overlapping error bars), suggesting that both vanadate and DES probably inhibited the BL-dependent component completely. Since at pH 9 there is almost no CO2 available in the medium, these results mainly indicate that the BL-dependent plasma membrane transport of requires an active ATPase. The fact that the RL-dependent O2 evolution rates were also slightly inhibited by vanadate and DES suggests that some Ci was also actively taken up by the cells under these irradiation conditions.
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Effect of the external pH on the uptake
The external pH determines the ratio CO2/ (pKa 6.3) in the medium and, therefore, variations in pH could lead M. braunii cells to develop different strategies to satisfy their carbon requirements. To study this problem, M. braunii cells grown in low CO2 were incubated in the absence of a carbon source under WL for 120 min at pH 7 or pH 9. Aliquots of these cell suspensions were subsequently exposed to either pH 7 or pH 9 and irradiated with RL or RL plus BL to measure the -dependent O2 evolution rates. Even though only was supplied as Ci, at pH 7 the CO2/ equilibrium generates enough CO2 to support photosynthesis, whereas CO2 concentrations at pH 9 are too low to do so. As shown in Table 1, M. braunii cells incubated at pH 7 had a slightly lower ability to take up and reduce than cells incubated at pH 9. In both, pH 7- and pH 9-incubated cells, the RL-dependent O2 evolution rates were higher when the assays were conducted at pH 7 than at pH 9. However, when assayed at pH 7 none of them showed significant increases in O2 evolution in response to the additional irradiation with BL. On the other hand, at pH 9 both pH 7- and pH 9-incubated cells increased their O2 evolution rates by 100% and 200%, respectively, when additionally irradiated with BL. The vanadate and DES sensitivities seemed quite different depending on the history of the cells. In cells incubated at pH 9, the ATPase inhibitors affected both the RL- and BL-dependent O2 evolution rates at pH 9 but not at pH 7. In cells incubated at pH 7, these compounds only inhibited BL-stimulated O2 evolution rates at pH 9. However, at pH 7, conditions when very high rates of O2 evolution under RL and small BL stimulation of these rates were common, the inhibitors had almost no effect.
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Induction of the uptake system in M braunii cells
The effect of low and high CO2 on the induction of an uptake system was studied. For this, M. braunii cells grown in low and high CO2, at pH 7, were collected and resuspended in a CO2-free buffer at pH 9. The two cell suspensions were incubated in the absence of a carbon source (argon atmosphere) for several hours under WL. At different times, aliquots were taken out and assayed for uptake activity, measured indirectly as O2 evolution, under RL or RL plus BL.
Aliquots of cells grown in low CO2 collected at the beginning of the incubation time showed, under RL and in the presence of , fair rates of O2 evolution and those rates were substantially stimulated by BL (Fig. 2A). So, cells grown in low CO2 were already capable of taking up . However, during the incubation in WL and in the absence of a carbon source, their ability to take up and reduce increased with time, this increment being shown only under bichromatic irradiation. It took 45 min of incubation to reach a maximum rate of O2 evolution. On the other hand, cells grown in high CO2 collected at the beginning of the incubation period did not evolve detectable O2 either under RL or RL plus BL when resuspended at pH 9 in the presence of (Fig. 2B). As incubation under WL irradiation proceeded, these cells began to take up and reduce (Fig. 2B). After 30 min under bichromatic irradiation and 60 min under RL, -dependent O2 evolution was detected. The maximum O2 evolution rates were achieved after 90 min of incubation. At this time, O2 evolution rates under RL plus BL were more than five times higher than those observed under RL only.
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These results suggested that cells grown in high CO2 needed to activate or synthesize the
uptake system. To check whether protein synthesis was involved, the effect of CHI or CAP on the -dependent O2 evolution at pH 9 was tested. The results of Fig. 3 clearly show that CHI completely suppressed this induction, while CAP only slightly affected the process (see values in the absence of inhibitors in Fig. 2B
). Hence, protein synthesis in the cytosol is necessary for the induction of the uptake system during the incubation of M. braunii cells under Ci-limiting conditions.
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Light quality and the induction of the uptake system
Figure 4 shows that both incubation with RL or RL plus BL induced the uptake system in cells previously grown in high CO2. transport activity was assayed under RL or RL plus BL. The maximum rates were reached after 60 min of incubation. However, in cells incubated at both light regimes, the final O2 evolution rates obtained under bichromatic irradiation were lower than those measured in cells incubated with WL (Fig. 2B). On the other hand, the cell suspensions irradiated with RL plus BL during the incubation period showed significantly higher rates of -dependent O2 evolution, when assayed >only with RL, than those irradiated exclusively with RL during the incubation period.
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Photosynthetic activity requirement for the induction of the uptake system
It has been reported previously that the uptake of can also be monitored by the pH increase in the external medium of unbuffered M. braunii cell suspensions in a CO2-free atmosphere (Giráldez et al., 1998). The effects of darkness and DCMU, an inhibitor of photosynthetic electron transport, on the induction of the uptake system were tested in order to study the energy requirements for this process. As expected, when cells grown in high CO2 at pH 7 were transferred to an unbuffered solution at pH 9, the external pH remained unchanged when they were irradiated either with RL or RL plus BL in the presence of (Table 2). However, after incubation in an unbuffered solution, at pH 9, irradiated with WL in a CO2-free atmosphere for 120 min, a significant -dependent pH increase could be observed under RL and, to a much higher extent, under RL plus BL (Table 2). The induction of the uptake system occurred during the incubation with WL in Ci-limiting conditions, which was in full agreement with the results described above (Fig. 2). When these cell suspensions were incubated in darkness or irradiated in the presence of DCMU, no change in the external pH was detected when the cell suspensions were irradiated in the presence of (Table 2). Thus, energy from dark respiration did not seem to be enough to sustain the induction of this uptake system.
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Induction of the uptake system in M. braunii cells grown in high CO2 by Ci-limiting conditions
M. braunii cells grown in high CO2 at pH 7 were unable to take up from the external medium at pH 9 but, after incubation at this high external pH in the absence of a carbon source, they were able to incorporate this anion. According to this, Ci-limiting conditions may be the signal promoting the induction of the uptake system. In order to investigate if a drop in the external CO2 concentration could induce the uptake system, M. braunii cell suspensions were transferred from high to low CO2 conditions. To do that, cells grown in high CO2 were either transferred to low CO2 in media buffered at pH 7 and 9, or incubated in high CO2 at both pHs. After a 120 min incubation, their ability to take up and reduce was measured to see if the signal inducing the uptake system was an increase in pH (as suggested by the results described in Table 1) or a decrease in CO2 concentration.
Table 3 shows that only cultures transferred from high to low CO2 conditions were able to take up and reduce , independently of the pH. Furthermore, the -dependent O2 evolution rates measured at pH 9 under RL plus BL were only slightly higher in the cells incubated at pH 9 than in the ones incubated at pH 7.
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Discussion |
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M. braunii cells grown in Ci-limiting conditions, develop an uptake system to take up
from the external medium, which is under BL control. M. braunii cells grown in low CO2 had extremely low levels of periplasmic CA activity and the BL-induced -dependent O2 evolution rates were not affected by AZA (Giráldez et al., 1998). Therefore, M. braunii had to transport from the external medium in an active process. This uptake system depended on the activity of a plasma membrane ATPase, since vanadate and DES completely suppressed the BL-stimulated -dependent O2 evolution (Fig. 1; Table 1). However, the RL-dependent O2 evolution rates were also affected by these ATPase inhibitors suggesting that some Ci was also actively taken up under RL, probably as a consequence of the pH decrease caused by the plasma membrane ATPase in the periplasmic space. This pH decrease would lead to the production of some CO2, particularly if some CA activity remained associated to the plasma membrane, and this CO2 could be taken up by the cells. In this sense, it may be considered that some CO2 can be actively transported under RL. Nevertheless, in the absence of monovalent anions in the outer medium, no changes in pH were observed upon irradiation of unbuffered M. braunii cell suspensions (Giráldez et al., 1998).
The requirement of plasma membrane ATPase activity for the uptake of both and CO2 has been previously suggested for some green algae (Thielmann et al., 1990; Rotatore and Colman, 1991; Rotatore et al., 1992; Karlsson et al., 1994). Hence, M. braunii cells seem to have a transporter for and its operation depends on the activity of a plasma membrane ATPase. ATPase-dependent transport mechanisms have also been observed in other species of green algae grown in low CO2 (Palmqvist et al., 1988; Thielmann et al., 1990; Karlsson et al., 1994) and aquatic plants (William, 1983). In Dunaliella tertiolecta, a low CO2-inducible 100 kDa protein was found to immunoreact with a polyclonal antibody raised against the plasma membrane ATPase of Zea mays (Ramazanov et al., 1995b). The authors suggested that this protein could be responsible for the vanadate inhibition of the photosynthetic transport observed in this green alga.
According to the availability of Ci, M. braunii cells developed different strategies to satisfy their carbon requirements. With high CO2 dissolved in the medium, the cells lacked an uptake system. However, with low or no CO2 supply, the cells developed an uptake system, particularly at high pH. In fact, at pH 7, their ability to take up was lower than at pH 9 (Table 1). Therefore, M. braunii cells seemed to be able to develop a more efficient uptake system at high external pH. Some components of the CCM in green algae have been reported to be induced by changes in external pH. When Scenedesmus cells grown in high CO2 were transferred to air, they developed a system for utilizing CO2 in media with pH 5 to 8 and another system for utilizing in media with pH 7 to 11 (Thielmann et al., 1990). In Chlorella regularis, the induction of CA was suppressed when cells grown in high CO2 were transferred to air under acidic pH conditions (Shiraiwa et al., 1991). In addition, one polypeptide was found to be induced in Chlorella regularis in low CO2 conditions when exposed to pH 5.5, but this polypeptide was not induced at pH 8 (Satoh and Shiraiwa, 1996). In fact, these authors suggested that under acidic conditions this alga induced a mechanism, not involving CA, to transport and incorporate Ci, in which this polypeptide could be involved.
M. braunii cells grown in low CO2 at pH 7, were able to transport some when collected and resuspended at pH 9 and the rates of O2 evolution measured under RL were stimulated by BL (Fig. 2A). This fact suggested that cells grown in low CO2 were equipped with an uptake system although not enough to reach maximum rates of uptake. On the contrary, when M. braunii cells grown in high CO2 at pH 7, were collected and resuspended at pH 9, they were completely unable to take up from the external medium. But after incubation in the absence of a carbon source at pH 9 under WL irradiation, M. braunii cells were able to take up and reduce this anion particularly when assayed under bichromatic irradiation (Fig. 2B). The presence of CHI during the incubation completely inhibited the induction of the uptake system (Fig. 3), suggesting that this system or some essential component of it or a factor involved in the regulation of its activity, was synthesized during this period in Ci-limiting conditions. The fact that CHI inhibited the induction of the uptake system, but CAP did not (Fig. 3), indicates that the necessary protein(s) was(were) encoded by a nuclear gene(s).
The uptake system of M. braunii was activated by BL to a high level and maybe by RL to a lower level. This could be because the pigment that is responsible for the BL effect also has some absorption in RL, or because of cross-talk between the RL and BL signal transduction pathways. It also could be due to a photosynthetic component, which would be further activated and/or synthesized under BL. In microalgae there are many enzymes related to photosynthesis which have been reported to be under BL control (Ruyters, 1984). In addition, it has been observed that BL was also required for the induction of a periplasmic CA activity in Chlamydomonas reinhardtii (Dionisio et al., 1989).
Table 3 shows that M. braunii cells are able to sense changes in the external CO2 concentration. When the cells were grown in high CO2, they were unable to take up and remained so when incubated in high CO2 at either pH 7 or 9. It should be pointed out that at pH 9, there was a substantial accumulation of in the medium. Hence itself did not induce its own uptake system. However, when the cells were transferred to low CO2 for 120 min, they developed an uptake system for this anion both at pH 7 and 9. The fact that in low CO2 conditions at pH 7 the cells developed a uptake system, apparently suggested that a decrease in Ci concentration in the external medium was the signal that triggered the induction process. However, even in low CO2 there was a substantial accumulation of at pH 9, since the suspensions were constantly bubbled with air. Thus, low external CO2 concentrations promote the induction of the uptake system, even in the presence of significant concentrations of Ci (mostly ) in the medium. At high concentration of CO2 in the medium, this carbon species appears to be always preferred as carbon source by the cells, particularly considering that the transport of has a net energy cost.
According to the results described in Table 3, in low CO2 conditions, the external pH did not significantly affect the ability of the cells to transport under bichromatic irradiation. This apparent contradiction with the results of Table 1 may be due to differences in the experimental conditions. In the experiment summarized in Table 3, the cells were transferred to a mineral medium, instead of a buffer, and those conditions were probably more physiological for the cells and in no way comparable. In addition, M. braunii cells were transferred from high (air plus 5% CO2) to low (0.03% CO2) conditions while in the experiments summarized in Table 1, cells grown in low CO2 were incubated in an argon atmosphere, where theoretically there was no CO2.
The mechanisms by which algal cells detect changes in external CO2 concentration, responsible for initiating the induction or repression of the adaptation processes, are not known. It has been suggested that the concentration of various metabolites of the photosynthetic carbon assimilation pathway may play a role in the induction of the CCM in microalgae. There are some data to support this hypothesis. In Chlamydomonas reinhardtii wild-type cells, the CA activity increased in the light due to photosynthetic activity, since in mutants defective in photosynthesis there was no stimulation by light of this enzymatic activity (Spalding and Ogren, 1982). In addition, it has been reported that the presence of DCMU inhibited the induction of the CA activity by low CO2 in the light (Ramazanov and Semenenko, 1988; Dionisio-Sese et al., 1990) and that no induction took place in darkness (Spencer et al., 1983). However, it has been demonstrated that the changes in the periplasmic CA transcript abundance that took place by lowering the CO2 concentration occurred both in the presence and absence of light (Bailly and Coleman, 1988), suggesting that the induction of the periplasmic CA in C. reinhardtii is not a strictly light-requiring mechanism. On the other hand, photosynthesis is not essential for the induction of CA in Chlorella vulgaris cells (Shiraiwa and Miyachi, 1983). The induction of the uptake system in M. braunii only took place in low CO2 conditions and in irradiated cell suspensions. There seems to be a light requirement for this process to occur since both darkness and the presence of DCMU in the light are able to block it completely (Table 2), suggesting that photosynthetic activity is required for the induction of the uptake system in this green alga. Nevertheless, DCMU and darkness could simply cause an increase in Ci concentration by producing the accumulation of Ci when DCMU inhibits photosynthesis or when the cells rely on dark respiration for their metabolic energy supply. This is supported by the fact that there is induction of the uptake system under a CO2-free atmosphere, where photosynthesis should be almost completely suppressed. However, there is always some photosynthetic activity in the absence of external Ci, due to CO2 generated by respiration, and this low CO2 concentration could be the signal promoting the induction of the uptake system.
An alternative model is that the transport systems themselves could also act as direct sensors of Ci concentration (Coleman, 1991) but, at the moment, there are no data to support this hypothesis. In fact, induction of CA synthesis in Chlorella took place not only by lowering the CO2 concentration, but also by increasing the O2/CO2 ratio in cells grown in high CO2 (Ramazanov and Semenenko, 1984, 1986). Furthermore, it has been demonstrated that the induction of three low CO2-inducible polypeptides in C. reinhardtii cells was also determined by the O2/CO2 ratio in the growth medium (Villarejo et al., 1996). It has also been suggested (Marcus et al., 1983) that phosphoglycolate was the messenger involved in the induction of the CCM in Anabaena variabilis, while other authors proposed that the photorespiratory N cycle or some aspect of N metabolism may be related to the regulation of CA induction in Chlorella regularis (Umino and Shiraiwa, 1991).
In conclusion, data from this paper show that M. braunii cells are able to sense a decrease in external CO2 concentration and, in response, they induce a BL-dependent uptake system. BL is not required for its induction, but it is essential for its activation once induced. Further studies are required to clarify how M. braunii cells sense changes in external CO2 and the factors involved in the induction of this uptake system. One of the aims of future studies is to identify the protein involved in the transport of across the plasma membrane of M. braunii.
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Acknowledgments |
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We would like to thank Dr IM Møller for critical reading of the manuscript and Lucas Oya for excellent technical assistance. This work was supported by DGICYT grants PB 96–0554-CO1 and PB 96–0554-0807.
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Notes |
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1 To whom correspondence should be addressed. Fax: +34 91 564 86 79. E-mail:maquinones{at}cib.csic.es
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Abbreviations |
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AZA, acetazolamide; BL, blue light; CA, carbonic anhydrse; CAP, chloramphenicol; CCM, carbon concentrating mechanism; CHI, cycloheximide, Chl, chlorophyll; Ci, inorganic carbon; DES, diethylstilbestrol; PFD, photon fluence density; RL, red light; WL, white light..
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