Journal of Experimental Botany, Vol. 53, No. 376, pp. 1909-1918,
September 1, 2002
© 2002 Oxford University Press
Independent induction of two blue light-dependent monovalent anion transport systems in the plasma membrane of Monoraphidium braunii
Received 9 November 2001; Accepted 13 June 2002
Centro de Investigaciones Biológicas, CSIC, Velázquez 144, E-28006 Madrid, Spain
1 Present address and to whom correspondence should be sent: Centro de Ciencias Medioambientales, CSIC, Serrano 115bis, E-28006 Madrid, Spain. Fax: +34 564 86 79. E-mail: maquinones{at}ccma.csic.es
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Abstract |
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In the plasma membrane of the green alga Monoraphidium braunii there are at least two monovalent anion transport systems. One of them is specific for bicarbonate. This transport system is activated by blue light and its induction is triggered by a decrease in the external CO2 concentration. The second transport system is responsible for nitrate uptake at least. This transport system is also activated by blue light and its induction occurs when there is no ammonium in the external medium. Both transport systems are synthesized independently. Hence, when M. braunii cells grow with nitrate as the only nitrogen source under high CO2, they have a nitrate transport system but lack a bicarbonate transporter. Conversely, cells grown with ammonium under low CO2, have a bicarbonate transport system but lack a nitrate transporter. Both transport systems are induced in cells irradiated with white light in the absence of a carbon source, suggesting that there may be precursors in the plasma membrane that only need the synthesis and assembly of some component(s) to become fully active. The induction of nitrate and nitrite reductases, however, only takes place when a carbon source is supplied to the cells.
Key words: Key words: Alkalinization, bicarbonate, blue light, green alga, nitrate reductase, nitrite reductase, protein biosynthesis.
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Introduction |
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Photosynthetic organisms require large amounts of inorganic carbon (Ci) and nitrogen (N) sources for the synthesis of biomass. The most abundant source of Ci in the biosphere is the atmospheric CO2. However, since the equilibrium CO2
HCO3– has a pKa of 6.3, whenever the pH of the medium is above that value, bicarbonate is the most readily accessible Ci source for aquatic photoautotrophic organisms. Many green algae and cyanobacteria have developed mechanisms to concentrate Ci inside the cells and, more specifically, near the active site of Rubisco. These carbon concentrating mechanisms (CCM) apparently involve active CO2 and/or HCO3– transport systems and carbonic anhydrase (CA) isoforms (Badger and Price, 1992). Active transport systems for CO2 and/or HCO3–, whose activity is independent of CA, have been reported in the green microalgae Chlamydomonas reinhardtii (Sültemeyer et al., 1989; Palmqvist et al., 1994), Scenedesmus obliquus (Thielmann et al., 1990) and Monoraphidium braunii (Giráldez et al., 2000). Strong alkalinizations of the external medium can be measured when HCO3– is used as a Ci source by green algae (Findenegg, 1979; Giráldez et al., 1998) and aquatic plants (William et al., 1983). It is of particular interest that the HCO3–-dependent alkalinization observed in M. braunii is also blue light (BL)-dependent (Giráldez et al., 1998). When several species of green microalgae are exposed to low Ci conditions, the acclimation process involves protein biosynthesis. In some cases, the function of these proteins in the CCM remains unknown (Ramazanov et al., 1995; Satoh and Shiraiwa, 1996), but others have been identified as CA isoforms in C. reinhardtii (Coleman and Grossman, 1984; Fukuzawa et al., 1990) or a plasma membrane HCO3– transport system in M. braunii (Giráldez et al., 2000). Regarding the assimilation of inorganic N, the main reservoir of this element in the biosphere is the atmospheric N2. However, only certain cyanobacteria and bacteria, either free or in symbiosis with plants or fungi, can convert N2 into NH4+. As a consequence, the main sources of inorganic N available and ready for use by most plants and algae in land and aquatic environments are NO3– and NH4+. The uptake of NO3– is a process highly regulated by light in green algae such as C. reinhardtii (Azuara and Aparicio, 1984), Chlorella fusca (Calero et al., 1980) and M. braunii (Aparicio and Quiñones, 1991). The photoregulation by low wavelength radiation sometimes involves the activation/inactivation of an NO3– transport system and, at other times, controls the induction and biosynthesis of this transport system. In M. braunii, blue light (BL) acts as a photoswitch on the activity of the NO3– transport system, so that the cells can take up this anion from the medium only when this radiation is present in the actinic light (Aparicio and Quiñones, 1991). The uptake of NO2– in M. braunii cells is also BL-dependent when they are in the absence of a Ci source, but when the cell suspensions are bubbled with air plus 2% CO2, the BL-dependence of NO2– transport is lost after 1 h and the process continues under red light (RL) only (Aparicio and Quiñones, 1991). On the other hand, the biosynthesis of the BL-dependent NO3– and NO2– uptake system(s) proceeds under RL in this green alga (Aparicio and Quiñones, 1991). Conversely, in C. reinhardtii the induction processes for two high affinity plasma membrane transport systems, system III (specific for NO2–) and system IV (bi-specific for NO3– and NO2–), require BL to take place (Quiñones et al., unpublished results). These C. reinhardtii transport systems, however, do not require continuous BL irradiation to remain active.
In M. braunii, NO3– and Cl– probably enter the cells through the same transport system, or at least their transport systems share some components, including the BL photoreceptor (Witt and Aparicio, 1995a). Both anions competitively inhibit each other’s uptake and their transport shows similar Ks values for both high and low affinity system(s), optimum pH under CO2-free conditions (Witt and Aparicio, 1995a), and almost identical action spectra (Witt and Aparicio, 1995b). Interestingly, this putative NO3––Cl– transporter also seems to be responsible for the uptakes of NO2–, Br– and I– (Aparicio et al., 1994). The uptake of NO3– in M. braunii produces a strong and continuous alkalinization of the external medium. This alkalinization is biphasic and consists of a first phase of fast alkalinization that lasts c. 3–5 min followed by a second phase in which the alkalinization is slower and lasts as long as BL is present (Aparicio et al., 1994). The first phase or phase of maximal alkalinization corresponds to the H+ consumption due to the fast initial accumulation of NO3– in the cells plus its reduction. The second phase, continuous alkalinization, corresponds to the NO3– taken up by the cells as the anions accumulated in the cytoplasm are reduced to NH4+ by NR and NiR. Therefore, the speed of alkalinization during the continuous phase depends on NR activity, since NiR activity is ordinarily much higher than that of NR on a cell basis (Aparicio et al., 1994). The uptake of Cl– always produces a transient alkalinization of the external medium, due to the fact that this anion is not metabolized, just like NO3– in the absence of NR (Aparicio et al., 1994). The uptake of HCO3– by M. braunii, on the other hand, produces a continuous, lineal alkalinization of the external medium and shows a slightly different action spectrum (Giráldez et al., 1998), suggesting that the entrance of this monovalent anion may be mediated by a different transport system and seems to be controlled by a different BL photoreceptor, and presumably is the limiting step in Ci assimilation.
In this paper, it is shown that there are at least two independent transport systems for monovalent anions in the plasma membrane of Monoraphidium braunii. One of them is specific for HCO3– and the other could be general for other inorganic monovalent anions, and the activities of both are BL-dependent. The induction of these two transport systems depends on the presence and concentration of different Ci or N sources in the external medium. The HCO3–-specific transport system is synthesized when the external concentration of CO2 is low, in the presence or absence of HCO3–. The NO3––Cl– transport system is induced when there is no NH4+ in the external medium, in the presence or absence of NO3–. None of the two induction processes requires BL.
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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 NO3– or NH4+ as N source and MoO42– or WO42–, at pH 7.1, under a 14/10 h light/dark photoperiod, as described by Quiñones and Aparicio (1990). Cultures were bubbled with air (cells grown in low CO2) or air plus 5% CO2 (cells grown in high CO2) at 25 °C under a constant photon fluence density (PFD) of 150 µmol m–2 s–1 of white light (WL). Cells were collected 1 h after the beginning of the light period by centrifugation at 2000 g and washed three times with 30 mM 3-[cyclohexylamino]-2-hydroxy-1propanesulphonic acid (Capso) buffer, pH 9 (this pH was optimal for HCO3– uptake and almost optimal for the transport of NO3–). Then, the cells were resuspended in the same buffer at a concentration of 30 µg Chl ml–1, stirred with a magnetic bar and irradiated as indicated below, in a CO2-free atmosphere, at 25 °C, for 0–60 min before starting the O2 evolution measurements. Prior to the measurements with non-incubated cells, they were nevertheless bubbled with CO2-free air (synthetic air: 79% N2, 21%O2: Air Liquide, Spain) for a few minutes to remove the CO2 accumulated during their growth. For pH measurements, the cells were washed and resuspended in a 2 mM MgSO4 unbuffered solution, and incubated in the conditions described above. For nitrate reductase (NR) and nitrite reductase (NiR) measurements, the cells were resuspended in regular NO3–-containing growth medium or medium containing 20 µM WO42– instead of MoO42–, and sparged with air for 0–180 min. The suspensions used to determine in situ NR had a chlorophyll content of 60 µg Chl ml–1.
Chlorophyll measurements
Chlorophyll content in the cell suspensions was determined according to Marker (1972).
Photosynthetic O2 evolution measurements
O2 evolution was measured with a Clark-type electrode (Hansatech, England), using 3 ml cell suspensions placed in a temperature-controlled cuvette at 25 °C. Linear rates of O2 evolution were determined over periods of several minutes after the addition of KNO3 or NaHCO3.
pH measurements
pH measurements were carried out at 25 °C with a Titralab system from Radiometer (Denmark). The cell suspensions were kept in CO2-free air during the measurements. The initial pH of the suspensions was adjusted to 9 by adding diluted solutions of KOH or H2SO4. Changes in pH were measured after the addition of KNO3 or NaHCO3.
In situ NR activity
In situ NR activity was determined according to Corzo et al. (1991), by mixing 1 ml of cell suspension (60 µg Chl ml–1) with 5 ml of a solution containing 5% propanol-1, 100 mM KNO3, 50 mM K2HPO4 and 10 nM chloramphenicol, pH 7.5. This solution did not contain Na+. The mixture was slightly stirred in a water bath at 30 °C in darkness. After 10 min, a 1 ml aliquot was used to measure NO2– by diazotation (Snell and Snell, 1949).
NiR activity
This enzymatic activity was measured according to Losada and Paneque (1971) in crude extracts obtained as described by Balandín and Aparicio (1987) with few modifications: the cells were mixed with 0.3 mm diameter glass beads (Biospec Products, Bartlesville, USA) and vigorously shaken in a Mini-Beadbeater (also from Biospec Products) for seven periods of 50 s at 4 °C.
Irradiation conditions and radiation measurements
Cell suspensions were irradiated during the incubation periods with WL (300 µmol m–2 s–1) from a Zeiss Ikon slide projector (Braunscheweig, Germany) fitted with a 150 W tungsten–halogen lamp. For pH and O2 evolution measurements, cell suspensions were irradiated with either strong (200 µmol m–2 s–1) RL of 690 nm or the same strong RL plus weak (20 µmol m–2 s–1) BL of 450 nm. RL was obtained from a 500 W mercury–xenon arc lamp from Oriel (USA) and BL was obtained from a 150 W tungsten–halogen lamp in a housing with three glass-fibre light pipes. 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 (specified for measurements in the visible spectrum by the manufacturer) coupled to a LI-188B integrating quantum-radiometer-photometer from Li-Cor (USA).
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Induction of a HCO3–-specific transport system
M. braunii cells, grown in a medium containing NO3– as the only N source, synthesized the NO3– transport system and were able to take up this anion upon irradiation with BL. On the other hand, cells grown in high CO2 lacked the HCO3–-specific transport system. When M. braunii cells, grown in both NO3– as the only N source and high CO2, were collected and resuspended in an unbuffered solution with NO3– as the only monovalent anion, a strong alkalinization of the external medium could be observed immediately after the irradiation of the cell suspension with a weak BL added to a strong background of RL (Fig. 1A). This result implied that the cells synthesized the NO3– transport system during their previous growth. However, when the cells were resuspended with HCO3– as the only monovalent anion, no BL-dependent alkalinization could be observed (Fig. 1A), indicating that they lacked the HCO3– transport system. Similar results were obtained when the NO3–- or HCO3–-dependent O2 evolutions were measured (Fig. 1C), showing that only the NO3– transport system was present in the cells. All responses observed were BL- and monovalent anion-dependent, since the controls in the absence of monovalent anions showed no responses to BL (Fig. 1A, C).
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Cells grown with NO3– and high CO2, that is lacking the HCO3– transport system, were incubated for 1 h in the absence of a N source, under WL irradiation and in a CO2-free atmosphere. After the incubation period, the NO3– transport system remained functional and the cells showed BL- and NO3–-dependent alkalinization of the external medium (Fig. 1B) and O2 evolution (Fig. 1D), as before. On the other hand, during the 1 h incubation in the absence of CO2, the cells synthesized the HCO3– transport system and, therefore, acquired the ability to take up this anion from the medium. Thus, they showed both BL- and HCO3–-dependent alkalinization of the external medium (Fig. 1B) and O2 evolution (Fig. 1D), when HCO3– was the only monovalent anion present in the cell suspensions. Incubation for periods shorter than 1 h resulted in the absence of HCO3– transport activity (data not shown), supporting that there was de novo synthesis of the transport system and not activation of a pre-existing transporter.
Induction of a NO3– transport system
M. braunii cells, grown in a medium with low CO2 synthesized the HCO3–-specific transport system and were able to take up this anion upon irradiation with BL (Fig. 2). On the other hand, cells grown with NH4+ as the only N source lacked the NO3– transport system. When M. braunii cells, grown in both NH4+ as the only N source and low CO2, were collected and resuspended in an unbuffered solution with HCO3– as the only monovalent anion, a strong alkalinization of the external medium could be observed upon irradiation of the cell suspension with weak BL over the strong RL background (Fig. 2A). This indicated that the cells synthesized the HCO3– transport system during their growth with low CO2. However, when the cells were resuspended with NO3– as the only monovalent anion, no BL-dependent alkalinization could be observed (Fig. 2A), suggesting that they lacked the NO3– transport system. When the HCO3–- or NO3–-dependent O2 evolutions were measured, the results were the same (Fig. 2C), that is, the cells only had the HCO3– transport system. As mentioned above, the cells in the absence of monovalent anions showed no responses to BL (Fig. 2A, C).
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Cells pretreated with NH4+ and low CO2, that is with a HCO3– transport system but lacking a NO3– transport system, were incubated for 1 h in 2 mM MgSO4 or Capso buffer in the absence of a N source, under WL irradiation and in a CO2-free atmosphere. After the incubation, these cells could take up and assimilate HCO3– as before, showing BL-dependent alkalinization of the external medium (Fig. 2B) and O2 evolution (Fig. 2D) in the presence of this anion. On the other hand, during the incubation in the absence of NH4+, the cells were able to synthesize the NO3– transport system, thus acquiring the capacity to take NO3– from the medium. When these cells were resuspended in an unbuffered solution with NO3– as the only monovalent anion and a small PFD of BL was added to the strong RL background, a transient alkalinization of the external medium could be observed (Fig. 2B). This alkalinization increased when the NO3– concentration was raised in the medium (Fig. 2B, inset), resembled that described for M. braunii cells that had a NO3– transport system but lacked NR (Witt and Aparicio, 1995b) and was due to the cytoplasmic accumulation of NO3– until the influx and efflux of this anion were equal, leaving the pH stationary (Aparicio et al., 1994). The lack of NR in the cells is the reason why there was no continuous alkalinization (Fig. 2B) or O2 evolution when they were irradiated with BL in the presence of NO3– (Fig. 2D).
Conditions required for the induction of NR and NiR
NR, the enzyme that reduces NO3– to NO2–, was not induced in substantial amounts when M. braunii cells grown with NH4+ as the only N source were incubated in a MgSO4 solution (Table 1). However, when the cells were resuspended in growth medium with NO3– as the only N source during the incubation period, they were able to synthesize NR in an air atmosphere and under WL irradiation (Table 1). After a 2–3 h incubation, the cells suspended in MgSO4 showed a small NR activity, but those suspended in growth medium reached maximum NR activities (Table 1). NiR, the enzyme that reduces NO2– to NH4+, was also induced when the cells were suspended in growth medium but not in a MgSO4 solution (Table 1).
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The incubation of NH4+ and low CO2-grown M. braunii cells for 1–2 h in growth medium led to the biosynthesis of the NO3– transport system and NR. Consequently, when they were resuspended in a MgSO4 solution with NO3– as the only monovalent anion and irradiated with weak BL over a strong RL background, they caused strong and continuous alkalinizations of the external medium (Fig. 3). The BL- and NO3–-dependent alkalinization was always slightly faster in cells incubated for 2 h than in cells incubated for 1 h (Fig. 3).
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One of the components of the growth medium whose presence is essential for the biosynthesis of a fully active NR is Mo. NR has a Mo cofactor that is necessary for the catalytic activity of the enzyme. Some experiments were done in which Mo was substituted for W in the growth medium where the NH4+ and low CO2-grown cells were incubated. When M. braunii cells were incubated with W-containing medium they could synthesize the NO3– transport system and, therefore, show a transient alkalinization of the external medium upon irradiation with BL in the presence of NO3– (Table 2). There was no continuous alkalinization under BL irradiation, indicating that the cells lacked an active NR that would allow them to reduce NO3– to NO2–. The alkalinization was continuous only when the cells were incubated with Mo-containing medium (Table 2). In these cells, the maximal rate of alkalinization was equal to the sum of the alkalinization that depended on NO3– transport (seen in W-incubated cells), plus the alkalinization that depended on the activity of NR (continuous alkalinization in Mo-incubated cells).
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Simultaneous restoration of the NO3– and Cl– uptake capacities in NH4+-grown cells
M. braunii cells grown with NH4+ as the only N source lack both the NO3– transport system and NR. When these cells were resuspended in a MgSO4 solution with NO3– or Cl– as the only monovalent anions and irradiated with weak BL over a background of strong RL, no alkalinizations of the external medium could be observed (Fig. 4A). However, the incubation of these cells in MgSO4 for 3 h led to the biosynthesis of the NO3– transport system and, therefore, there was a transient alkalinization of the external medium when the cells were illuminated with BL in the presence of NO3– (Fig. 4B). Correspondingly, when the cells were resuspended with Cl– as the only monovalent anion, they also showed a transient alkalinization of the external medium upon irradiation with BL (Fig. 4B). Finally, the incubation of the cells in growth medium for 3 h led to the biosynthesis of both the NO3– transport system and NR. As a result, the irradiation of the cell suspension with BL in the presence of NO3– as the only monovalent anion produced a strong and continuous alkalinization of the external medium, while in the presence of Cl– the alkalinization remained transient (Fig. 4C).
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Further evidence for the simultaneous recovery of the capacity of M. braunii cells to transport NO3– and Cl– is provided by the results shown in Table 2. When NH4+-grown cells were transferred to a NO3–-containing medium, always in the absence of Mo, they synthesized a non-functional NR and, therefore, showed transient alkalinizations of the external medium in the presence of NO3– and Cl–, consistent with the biosynthesis of the transport system responsible for the uptake of both anions. When the cells were transferred to regular growth medium, containing NO3– as the only N source and Mo, they recovered the capacity to take up NO3– and Cl– (Table 2), as well as NR.
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Anion transport through the plasma membrane of green microalgae is a process of great importance for their survival and proliferation. Most of the main inorganic nutrients that these organisms take up from the aquatic medium, including Ci and N sources, are anions. Therefore, the regulation of anion transport through their membranes is a key step in their metabolism. Microalgae have a negative membrane potential and, consequently, they have developed active transport mechanisms that allow them to take anions up from the medium.
The monovalent anions, HCO3– and NO3–, are the main Ci and N sources available for green microalgae in natural aquatic environments. Through evolution, these organisms have developed strategies to obtain the necessary nutrients from the medium. Depending on the sources of Ci and N that are most abundant in the medium at any given time, they synthesize the corresponding proteins needed for the uptake (transport systems) and assimilation (enzymes) of those nutrients. For example, green algae grown with NH4+ as the only N source lack NR and NiR, the enzymes that reduce NO3– to NO2– and NO2– to NH4+ respectively (Hipkin and Syrett, 1977; Guerrero et al., 1981; Díez and López-Ruiz, 1989). Furthermore, NH4+-grown green algae lack a NO3– transport system (Florencio and Vega, 1983; Witt and Aparicio, 1995b). On the other hand, green algae grown under high CO2 lack a HCO3– transport system. Their transfer to low CO2 conditions is a signal that leads to the induction of proteins that play a role in the CCM, such as the HCO3– transport system (Palmqvist et al., 1994; Thielmann et al., 1990; Giráldez et al., 2000).
The different conditions in which the NO3– or the HCO3– transport systems are induced in M. braunii showed that there are at least two BL-dependent monovalent anion transport systems in the plasma membrane of this green microalga. When the cells were grown with NO3– as the only N source and air plus 5% CO2, they had the NO3– transport system but lacked the HCO3– transporter (Fig. 1A, C). The incubation under WL in a CO2-free atmosphere led to the induction of the HCO3– transport system, but did not affect the NO3– transporter induced before (Fig. 1B, D). This confirmed that a decrease in the external CO2 concentration is the environmental signal that causes the induction of the BL-dependent HCO3– transport system, independently of changes in HCO3– concentration or in pH (Giráldez et al., 2000).
On the other hand, when the cells were grown with NH4+ as the only N source and air (only 0.03% CO2), they had the HCO3– transport system but lacked the NO3– transporter (Fig. 2A, C). The incubation of these cells in the absence of a N source under WL in a CO2-free atmosphere resulted in the induction of the NO3– transport system, but did not affect the HCO3– transporter (Fig. 2B). This result indicated that the main requirement for the induction of the NO3– transport system in the plasma membrane of M. braunii was the absence of NH4+ in the culture medium. The presence of NO3– in the medium was not necessary for the induction to take place. Furthermore, the absence of a N source greatly increased the capacity of the cells to take up NO3– immediately after the incubation period, in a cycloheximide-sensitive process (Ullrich et al., 1981), suggesting that under those conditions a larger amount of transporter protein was synthesized. These experiments also showed that the absence of a Ci source during the incubation period did not hinder the biosynthesis of the NO3– transport system in M. braunii, although it remains to be established whether the presence of a Ci source would accelerate this process in this alga.
The incubation of NH4+-grown M. braunii cells in a MgSO4 solution in the absence of N and Ci sources allowed the cells to synthesize the NO3– transport system (Fig. 2B), but not NR or NiR (Table 1). This was the reason why cells irradiated with a weak BL in addition to a strong RL background in the presence of NO3– did not evolve O2 (Fig. 2D). It is well known that green algae grown with NH4+ as the only N source lack NR activity (Guerrero et al., 1981). However, Hipkin and Syrett (1977) showed that some NH4+-grown green algae had distinct levels of NR mRNA, suggesting that NH4+ represses the biosynthesis of NR at a post-transcriptional level. In M. braunii, the changes in NR activity induced by the presence of NH4+ concur with changes in NR protein (Díez and López-Ruiz, 1989). The biosynthesis and degradation of NiR are closely related to those of NR in most plants and algae (Vennesland and Guerrero, 1979). According to the results shown in Table 1, this seems to be the case for the regulation of the biosynthesis of NR and NiR in M. braunii. The induction processes of both enzymes required the incubation of the cells in a complete growth medium for at least 1 h, although maximum levels of enzymatic activities were not reached until 2–3 h into the incubation periods (Table 1). At those times, the slope of the NO3–-dependent continuous alkalinization of the medium reached its maximum value (Fig. 3). NR, the enzyme that reduces NO3– to NO3–, has prosthetic groups that contain Fe, such as a cytochrome, and Mo, such as a molybdo-pterin (Johnson et al., 1980; (De la Rosa et al., 1981). Correspondingly, NiR, the enzyme that reduces NO2– to NH4+, has Fe-containing prosthetic groups, such as a sirohaem and an iron–sulphur centre (Murphy et al., 1974; Aparicio et al., 1975). Therefore, the availability of Fe and Mo (only for NR) in the external medium is essential for the cells to proceed with the synthesis and assembly of the fundamental components that form the enzymatic complexes of NR and NiR. The results demonstrate that, when NH4+-grown M. braunii cells were incubated in an NO3–-containing Mo-free growth medium, where this micronutrient had been substituted by W, they were unable to synthesize an active NR and, therefore, although they could take up NO3– from the medium (producing a transient alkalinization), they could not reduce this anion (Table 2). Likewise, the absence of Fe from the medium would have an equally negative effect on the biosynthesis of both NR and NiR.
The presence of a plasma membrane-bound NR (PM-NR) in Chlorella sorokiniana was the starting point for an hypothesis that closely related NO3– uptake and reduction by suggesting that PM-NR was involved in NO3– uptake in green algae (Tischner et al., 1989). Furthermore, the use of anti-NR IgG fragments led to the inhibition of both NR and NO3– transport. Shortly after, however, Corzo et al. (1991) suggested that NO3– uptake and reduction were independent processes in M. braunii. The results of this work further support this conclusion by showing NO3– uptake in cells that lacked the ability to reduce this anion.
As mentioned above, the incubations that led to the induction of NR and NiR were carried out in cell suspensions bubbled with air (0.03% CO2) and not in a CO2-free atmosphere. In the absence of a Ci source, M. braunii cells synthesized neither NR nor NiR (data not shown), probably due to the lack of the carbon skeletons required for the biosynthesis of amino acids. On the other hand, NR and NiR were induced when the cells were incubated in growth media with or without NO3– (for comparison see Table 1 and Fig. 4). In view of these results, it may be concluded that the induction of NR and NiR in M. braunii require the absence of NH4+ in the external medium (but not the presence of NO3– or NO3–) and the presence of a Ci source, even if it is at a low concentration. It is important to bear in mind that the induction of the NO3– and HCO3– transport systems did occur when the cells were incubated for 1 h in the absence of Ci sources (Figs 1, 2), since those results may indicate that very little protein biosynthesis is necessary for the cells to achieve the necessary monovalent anion transport systems. One possible explanation for this could be that there are precursors of both transport systems in the plasma membrane of M. braunii that only need the synthesis and/or assembly of some (small) component(s) to become fully active upon the removal of NH4+ or a decrease in CO2 concentration.
It has been proposed that NO3– and Cl– are taken up from the medium through transport systems that share at least some components in M. braunii (Witt and Aparicio, 1995a). This conclusion was based mainly on the following facts: NO3– and Cl– competitively inhibit each other’s uptake, they have matching pH optima and response time to BL, and both show almost identical action spectra (Witt and Aparicio, 1995a, b). It has been shown here that NH4+-grown cells lack the NO3– transport system and when such a system is induced in a medium lacking a N source, the cells recover the ability to take up NO3– and Cl– from the medium at the same time (Fig. 4) (cf. Aparicio et al., 1994; Witt and Aparicio, 1995b). Similar results were obtained when the cells were grown and incubated in a medium where Mo had been replaced by W (Table 2). This evidence further supports the conclusion that the NO3– and Cl– transport systems of M. braunii share some, if not many, components.
The induction processes of the NO3– and HCO3– transport systems require photosynthetically active radiation, but not particularly BL, in the green microalga M. braunii. The biosynthesis of the NO3– transport system in cells grown with NH4+ took place when they were incubated in the absence of a N source under strong RL irradiation (Witt, 1995). Likewise, the biosynthesis of the HCO3– transport system in cells grown with high CO2 took place when they were incubated in low CO2 or a CO2-free atmosphere under strong RL (Giráldez et al., 2000). However, it has been hypothesized that the addition of a weak BL to the strong RL background used during the incubation has a stimulating effect on the biosynthesis of the transport systems. Therefore, the incubation of the cells under WL would have an analogous enhancing effect. The activation of both transport systems, on the other hand, required the blue component of the spectrum in the actinic light (Aparicio et al., 1994; Giráldez et al., 2000). Furthermore, the NO3– and HCO3– transport systems remained active only as long as the cells were irradiated with BL, so that this radiation acted as a photoswitch for NO3– and HCO3– uptake in M. braunii (Aparicio and Quiñones, 1991). The biosynthesis of NR in M. braunii can also proceed in the absence of BL, when NH4+-grown cells are incubated in the absence of a N source under strong RL. However, the NR accumulated is inactive and requires the irradiation of the cells with a low PFD of BL for its activation (Quiñones and Aparicio, 1990). Again, it has been proposed that the incubation of NH4+-grown cells with RL plus BL or with WL not only produces an active enzyme, but a larger amount of protein is synthesized. Conversely, the induction of NiR requires a BL signal to take place in this green microalga, since incubation of NH4+-grown M. braunii cells in the absence of an N source, or even in the presence of NO3– or NO2–, under a strong RL does not result in the biosynthesis of this enzyme (Quiñones and Aparicio, 1990).
The set of data shown here, together with those published before (Aparicio and Quiñones, 1991; Aparicio et al., 1994; Witt and Aparicio, 1995a; Giráldez et al., 1998), reflect a good correlation between environmental factors such as pH, light, Ci, and N availability, and the strategy adopted by sun-adapted microalgae to avoid photodamage. Under non-saturating light conditions, most water bodies containing phytoplankton show high dissolved Ci concentrations due to compensation between global respiration and low photosynthetic activity in those aquatic systems. Under these conditions, the values of pH are close to the pKa of the CO2HCO3– equilibrium, the former being the main Ci source for phytoplankton. NO3– transport into microalgal cells is slow, due to energy shortage, and limited to that necessary to assimilate N for biosynthetic purposes. NO2– assimilation, like that of CO2, is independent of BL under these conditions (Aparicio and Quiñones, 1991).
At high light intensities, algal metabolism (anion uptake) causes the pH of their immediate surrounding to increase (up to 10 or more), leading to the conversion of most of the CO2 around the cells to HCO3–. A BL-dependent HCO3– transport system is then readily synthesized (Giráldez et al., 2000). This transporter is of utmost importance at high pH values, like the optimum pH for the BL-dependent NO3– transport system. Algae use the HCO3– transporter to capture any available Ci and concentrate it close to the active site of Rubisco, while NO3– is taken up and reduced much in excess and released to the external medium mainly as NH4+ (Azuara and Aparicio, 1984). In fact, NR and NiR are present in green algae at levels in considerable excess of the rates of nitrate assimilation (Guerrero et al., 1981). This apparently futile electron flow through the NO3– assimilation pathway allows the photosynthetic apparatus to proceed without collapse during high irradiance intervals (Azuara and Aparicio, 1984). Sun-adapted algal genera like Chlamydomonas or Monoraphidium have developed this mechanism, that once synthesized is governed and co-ordinated only by BL, pH and Ci, to act at high pH as a photosynthetic electron bypass that protects the algae against destructive processes associated with high irradiation regimes, while Ci and N assimilation can proceed. The co-ordination of the two independent transport systems described in this paper, that use BL as a photoswitch, is important to ensure a coupling between C/N assimilation and the protection of the photosynthetic apparatus.
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Acknowledgements |
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The authors wish to thank Lucas Oya for his excellent technical assistance. This work was supported by DGICYT grants PB 96-0554-CO1 and PB 96-0807.
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