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BIOENERGETICS AND PHOTOSYNTHESIS

Evidence for K⁺-Dependent HCO₃^– Utilization in the Marine Diatom Phaeodactylum tricornutum¹

Department of Biology and Hubei Key Laboratory of Bioanalytical Technique, Hubei Normal University, Huangshi 435002, Hubei, China

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Photosynthetic utilization of inorganic carbon in the marine diatom Phaeodactylum tricornutum was investigated by the pH drift experiment, measurement of K_1/2 values of dissolved inorganic carbon (DIC) with pH change, and comparison of the rate of photosynthesis with the rate of the theoretical CO₂ formation from uncatalyzed HCO₃^– conversion in the medium. The higher pH compensation point (10.3) and insensitivity of the photosynthetic rate to acetazolamide indicate that the alga has good capacity for direct HCO₃^– utilization. The photosynthetic rate reached 150 times the theoretical CO₂ supply rate at 100 µmol L^–1 DIC (pH 9.0) in the presence of 10 mmol L^–1 K⁺ and 46 times that in the absence of K⁺, indicating that for pH 9.4-grown P. tricornutum, HCO₃^– in the medium is taken up through K⁺-dependent and -independent HCO₃^– transporters. The K_1/2 (CO₂) values at pH 8.2 were about 4 times higher than those at pH 9.0, whereas the K_1/2 (HCO₃^–) values at pH 8.2 were slightly lower than those at pH 9.0 whether without or with K⁺, providing further evidence for the presence of the two HCO₃^– transport patterns in this alga. Photosynthetic rate and affinity for HCO₃^– in the presence of K⁺, respectively, were about 2- and 7-fold higher than those in the absence of K⁺, indicating that K⁺-dependent HCO₃^– transport is a predominant pattern of HCO₃^– cellular uptake in low DIC concentration. However, as P. tricornutum was cultured at pH 7.2 or 8.0, photosynthetic affinities to HCO₃^– were not affected by K⁺, implying that K⁺-dependent HCO₃^– transport is induced when P. tricornutum is cultured at high alkaline pH.

The characterization of the DIC species utilized from the medium by various phytoplankton has been extensively studied. Most organisms examined to date can use CO₂ or HCO₃^– or both, but species-specific preferences are observed (Kaplan et al., 1991; Espie and Kandasamy, 1992; Rotatore et al., 1995; Raven, 1997; Matsuda et al., 2001; Chen and Gao, 2004).

Transfer of HCO₃^– into the cell can be directly or indirectly performed in many phytoplankton. Low-CO₂-induced carbonic anhydrase (CA) in periplasmic space is widely present in many freshwater and marine microorganisms (Badger and Price, 1994; Elzenega et al., 2000; Hobson et al., 2001; Chen and Gao, 2003). Indirect HCO₃^– utilization can be performed by the periplasmic CA-catalyzed conversion of HCO₃^– to CO₂. Two direct HCO₃^– uptake systems are proposed in cyanobacteria (Kaplan and Reinhold, 1999; Giordano et al., 2005): an ATP-binding cassette-type transporter with a high affinity for HCO₃^– (Bonfil et al., 1998; Ohkawa et al., 1998; Omata et al., 1999) and a Na⁺-dependent transporter (Miller et al., 1984; Espie and Kandasamy, 1992, 1994; Gao and Zou, 2001; Qiu and Liu, 2004). Many eukaryotic photosynthetic organisms, both freshwater and marine, are capable of direct utilization of HCO₃^– from the medium (Rotatore et al., 1995; Amoroso et al., 1998; Matsuda et al., 2001; Colman et al., 2002). An anion-exchange HCO₃^– transporter and a H⁺-driven HCO₃^– transporter were suggested in marine macroalga and seagrasses (Larsson and Axelsson, 1999; Beer et al., 2002; Colman et al., 2002).

The marine diatom, Phaeodactylum tricornutum, has been extensively investigated with respect to inorganic carbon utilization mechanisms (Rotatore et al., 1995; Nimer et al., 1997; Burkhardt et al., 2001; Matsuda et al., 2001, 2002). All data tested indicate that the marine alga has the capacity to take up HCO₃^– from the medium, although the quantitative analyses of HCO₃^– utilization are different in different experiments (because of the use of different methods). However, up to now there has been little information on the mechanism of active HCO₃^– uptake in P. tricornutum. In this study, we reexamined the inorganic carbon utilization of P. tricornutum, and gave evidence that K⁺ takes part in HCO₃^– uptake in the marine alga.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

pH Compensation Point

The pH values of seawater in a closed system containing cells of P. tricornutum increased with increasing incubation time (Fig. 1 ). There had been little difference in the pH drift of seawater in the presence and in the absence of 100 µmol L^–1 acetazolamide (AZ); the same maximal values of 10.3 were obtained with and without AZ (Fig. 1).

View larger version (7K): [in this window] [in a new window]   Figure 1. Time courses of pH drift of seawater by cells of P. tricornutum in a closed system. Black circle (), –AZ; black square (), +AZ. Values are means ± SE (n = 3).

To determine whether algae use HCO₃^– or CO₂, a useful approach is to compare the rate of photosynthesis with the rate of spontaneous formation of CO₂ from HCO₃^– in the medium (Raven et al., 1995; Matsuda et al., 2001, 2002). If photosynthetic rate can exceed spontaneous CO₂ formation, then that indicates that cells can use HCO₃^–.

When photosynthetic oxygen evolution was measured at pH 9.0 and 100 µmol L^–1 DIC for pH 9.4-grown cells (Table I ), the rate decreased from 48.9 ± 6.8 to 15.9 ± 2.0 µmol L^–1 min^–1 in the absence of K⁺, which decreased by 67%, indicating that a K⁺-dependent DIC transport system exists in the alga and plays an important role in its photosynthetic activity. The observed rates of O₂ evolution were significantly higher than the spontaneous CO₂ formation rate at 100 µmol L^–1 DIC (pH 9.0; Table I). In the presence of K⁺, the photosynthetic O₂ evolution rate was 150-fold the spontaneous CO₂ formation rate, whereas that in the absence of K⁺ was 46-fold the CO₂ formation rate (Table I). There was little difference in photosynthetic O₂ evolution in the presence and in the absence of AZ (Table I). The experiments of the extracellular CA inhibitor AZ showed that extracellular CA had little contribution to photosynthetic inorganic carbon utilization in P. tricornutum. These results indicated that K⁺-dependent and K⁺-independent direct HCO₃^– uptake both exist in pH 9.4-grown P. tricornutum cells.

View larger version (11K): [in this window] [in a new window]   Figure 2. Photosynthetic O2 evolution as a function of DIC for P. tricornutum cells grown at pH 9.4 and 2.0 mmol L–1 total DIC. Photosynthetic rates were measured at pH 8.2 (A) or 9.0 (B) without () or with () 10 mmol L–1 KCl. Values are means ± SE (n = 3).

Photosynthetic rate for pH 9.4-grown P. tricornutum increased with increasing KCl concentrations when measured at pH 9.0 and 100 µmol L^–1 DIC (Fig. 3 ). K_1/2 values of KCl (the KCl concentration required to give half-maximal photosynthetic rate) was 0.32 mmol L^–1, which was determined by fitting rates of photosynthetic oxygen evolution at various KCl concentrations to the Michaelis-Menten formula. Photosynthetic rate was saturated at 4 mmol L^–1 KCl and did not exhibit any decrease in the highest level (10 mmol L^–1 KCl). In natural seawater, potassium, a stable element, not a trophic element, is about 10 mmol L^–1. The result presented here suggested that K⁺ concentration in natural seawater is enough for P. tricornutum to keep up K⁺-dependent HCO₃^– use.

View larger version (8K): [in this window] [in a new window]   Figure 3. Photosynthetic O2 evolution as a function of KCl concentrations for P. tricornutum cells grown at pH 9.4 and 2.0 mmol L–1 total DIC. Photosynthetic rates were measured at pH 9.0 and 100 µmol L–1 DIC. Values are means ± SE (n = 3).

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

There are contradictory reports on the effects of extracellular CA on photosynthetic inorganic carbon utilization in P. tricornutum. Nimer et al. (1997) reported that a pH drift was blocked by dextran-bound sulfonamide (200 µmol L^–1), an inhibitor of extracellular CA, in a closed system containing carbon-limited (pH 8.7, 1.0 mmol L^–1 DIC) cells of P. tricornutum (strain CCAP 1052/6). On the other hand, Burkhardt et al. (2001) demonstrated that inhibition of extracellular CA activity by dextran-bound sulfonamide (100 µmol L^–1) had no effect on photosynthetic rate in P. tricornutum (strain CCAP 1052/1A) grown at 36 µL L^–1 CO₂ (pH 9.1, 2.0 mmol L^–1 DIC), and addition of CA had no effect on the photosynthetic rate. Matsuda et al. (2001) reported that photosynthetic rate and affinity to DIC was not affected with added CA in P. tricornutum (UTEX 640). It suggested that differences between reports on extracellular CA in P. tricornutum were ascribed not only to methodological differences, but also to strain differences.

In cyanobacteria, studies have presented evidence for the presence of at least two HCO₃^– transport systems in the plasmalemma (Giordano et al., 2005), including a low-CO₂-induced ATP-binding cassette-type HCO₃^– transporter, named BCT1, with a high affinity for HCO₃^– and a Na⁺-dependent SbtA-type HCO₃^– transporter with a lower affinity for HCO₃^– than BCT1 (Omata et al., 1999; Shibata et al., 2002). Some suggestions were proposed that the Na⁺-dependent HCO₃^– transporter may be present in the marine -cyanobacteria (Badger et al., 2002; Shibata et al., 2002). P. tricornutum can take up both CO₂ and HCO₃^– as substrates for photosynthesis, and CO₂ to HCO₃^– uptake ratios decreased with decreasing CO₂ concentration in medium (Burkhardt et al., 2001; Matsuda et al., 2001). These findings suggested that HCO₃^– would be a predominant species when P. tricornutum cells acclimated to high alkaline pH. Rees (1984) has reported that photosynthetic O₂ evolution in P. tricornutum is dependent on the presence of Na⁺ and suggested Na⁺ increased inorganic carbon uptake by facilitating the use of HCO₃^–. At present, we gave evidence for the presence of K⁺-dependent HCO₃^– in this alga.

With comparing the O₂ evolution rate with the spontaneous CO₂ formation rate, our data indicated that photosynthetic rates at low DIC concentration were much higher than the CO₂ supply in the medium. The photosynthetic rate reaches 150 times the theoretical CO₂ supply rate at 100 µmol L^–1 DIC (pH 9.0) in the presence of K⁺ and 46 times in the absence of K⁺, indicating that HCO₃^– in the medium is taken up by this alga through K⁺-dependent and K⁺-independent transporters. These conclusions are supported by the results from Figure 2 and Table II. The K_1/2 values of CO₂ at pH 8.2 are about 4 times higher than the values at pH 9.0, whereas the K_1/2 values of HCO₃^– at pH 8.2 are slightly lower than those at pH 9.0 whether in the presence or in the absence of K⁺, providing further evidence for the presence of the two HCO₃^– transport patterns in this alga.

Meanwhile, photosynthetic rates in the absence of K⁺ account for 32.5% of those in the presence of K⁺, indicating that the K⁺-dependent HCO₃^– transporter is a predominant part of HCO₃^– uptake in low DIC levels by the alga. On the other hand, the K_1/2 value for HCO₃^– in the absence of K⁺ is more than 7-fold higher than that in the presence of K⁺, indicating that the K⁺-dependent HCO₃^– transporter has a significantly higher affinity to HCO₃^– than the K⁺-independent HCO₃^– transporter, and implying that K⁺-dependent HCO₃^– transport is a more effective and predominant pattern of HCO₃^– cellular uptake in low DIC concentrations.

It should be noted that the presence of K⁺-dependent and K⁺-independent HCO₃^– transporters is when P. tricornutum cells were grown at high alkaline pH (pH 9.4). As P. tricornutum cells were cultured at pH 7.2 or 8.0 and 2.0 mmol L^–1 DIC, our results showed that photosynthetic affinities to DIC are not affected by K⁺, suggesting that pH 7.2- or 8.0-grown P. tricornutum cells had poor capacity for K⁺-dependent DIC use and implying that K⁺-dependent HCO₃^– use was induced when P. tricornutum was cultured at high alkaline pH.

Comparing the K_1/2 (HCO₃^–) values measured without K in pH 9.4-acclimated cells with that measured in pH 7.2- and 8.0-acclimated cells is more representative of K⁺-independent HCO₃^– transport. It appears that the K_1/2 (HCO₃^–) values measured in pH 7.2- and 8.0-acclimated cells were lower than those measured without K in pH 9.4-acclimated cells, suggesting that K⁺-independent HCO₃^– transport was suppressed when K⁺-dependent HCO₃^– transport was induced at high alkaline pH.

It is difficult to find available data that can be used to assess the spontaneous CO₂ formation rate in seawater at the exact conditions you want. Matsuda et al. (2001) has reported that the spontaneous CO₂ formation rate calculated by Equation 1 (see equations in "Materials and Methods") at 100 µmol L^–1 HCO₃^– and pH 8.2 is 0.6 µmol L^–1 min^–1 at 25°C and 31.65 salinity (S), where the constants used are the published value of k₁ (Johnson, 1982), 1.17 x 10^–4 s^–1 , 3.98 x 10^–4, and calculated k₃ of 3.31 s^–1, K₁ of 1.22 x 10^–6, K₂ of 9.0 x 10^–10. From Equations 4, 8, and 9, it can be calculated that the constants are k₁ of 1.37 x 10^–4 s^–1, k_d of 2.08 x 10⁴ at 25°C, and 31.65 S, and then the recalculated rate of CO₂ formation is 1.4 µmol L^–1 min^–1 at 100 µmol L^–1 HCO₃^– and pH 8.2, which is about 1-fold the CO₂ formation rate calculated by Matsuda et al. (2001). Comparing rates of the calculated CO₂ formation at 100 µmol L^–1 HCO₃^– and pH 8.2 or pH 9.0 (see "Materials and Methods") based upon Equations 4, 8, and 9 with those described by Matsuda et al. (2001), it appeared that the calculated CO₂ formation rates from Equation 8 are higher than those obtained by Matsuda et al. (2001). The difference will affect the ratios of photosynthetic rates to the estimated CO₂ formation rate, but will not affect the conclusion that the rates of photosynthesis are by far higher than the rate of spontaneous CO₂ supply in the medium of 100 µmol L^–1 HCO₃^– (pH 9.0).

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Materials and Growth Conditions

Phaeodactylum tricornutum (strain 2038) from the Institute of Oceanography, Chinese Academy of Sciences, Qingdao City, China, was cultured in filtered seawater enriched with f/2 medium under continuous illumination of 180 µmol m^–2 s^–1 at 20°C, with 2.0 mmol L^–1 DIC (pH 7.2, 8.0, and 9.4). The DIC concentrations of the medium were controlled by adding NaHCO₃ solution into CO₂-free seawater. Changes in the DIC levels in the medium were kept within 10% by renewing the medium every 24 h. The CO₂-free seawater was adjusted according to Gao et al. (1993). The cells for experiments were collected during the midexponential growth phase.

pH Drift Experiment

For the closed-system pH drift experiment, the cells (about 1.0 ± 10⁷ cells/mL) were washed and resuspended in a closed bottle (80 mL) in unbuffered fresh medium of filtered natural seawater (2.0 mmol L^–1 DIC). The pH drift was monitored at 20°C and 180 µmol m^–2 s^–1. AZ (Sigma-Aldrich) was used at a final concentration of 100 µmol L^–1. The pH compensation points were determined as the pH value when it no longer increased.

Measurements of Photosynthetic Parameters

Photosynthetic oxygen evolution was measured with a Clark-type oxygen electrode (Chlorolab 2; Hansatech Instruments) at 20°C and 400 µmol m^–2 s^–1. Cells were harvested, washed, and resuspended in CO₂-free, K⁺-free artificial seawater (0.4 mol L^–1 NaCl, 20 mmol L^–1 MgSO₄, 20 mmol L^–1 MgCl₂·6H₂O, 10 mmol L^–1 CaCl₂, 2 mmol L^–1 Na₂SiO₃·9H₂O, 4 mmol L^–1 H₃BO₃) buffered with 25 mmol L^–1 Tris-HCl at pH 8.2 or 9.0. Following the addition of NaHCO₃, the rates of oxygen evolution were measured at defined DIC concentrations with the addition of 10 mmol L^–1 KCl or NaCl. For the measurement of photosynthetic rates at 100 µmol L^–1 DIC and pH 9.0 to compare with the maximal rate of spontaneous CO₂ formation in artificial seawater, cells were collected at a chlorophyll a concentration of 10 µg/mL. AZ was used at a final concentration of 100 µmol L^–1. For the measurement of photosynthetic rates as a function of DIC concentrations or KCl concentrations at pH 8.2 or 9.0, cells were harvested at 2 µg chlorophyll a/mL.

The photosynthetic K_1/2 values of DIC (the DIC concentration required to give the half-maximal photosynthetic rate) were determined by fitting rates of photosynthetic oxygen evolution at various DIC concentrations to the Michaelis-Menten formula. The K_1/2 values of HCO₃^– and CO₂ were estimated from K_1/2 (DIC). The HCO₃^– and CO₂ concentrations at defined DIC levels were calculated according to Chen (1999).

Chlorophyll a concentration was determined by the spectrophotometric method described by Jeffrey and Humphrey (1975).

Calculation of the Maximal Rate of Spontaneous CO₂ Formation in Artificial Seawater

The rate of spontaneous formation of CO₂ from HCO₃^– was determined by the following formula according to Miller and Colman (1980) and Matsuda et al. (2001):

(1)

where K₁ and K₂, respectively, are the first and second dissociation constants of CA and were derived by the following equations described by Goyet and Poisson (1989):

(2)

(3)

where T and S are the absolute temperature and salinity, respectively. The salinity of the artificial seawater used in the study was calculated to be 31.81%. K₁ and K₂ were calculated to be 1.18 x 10^–6 and 9.05 x 10^–10 at 20°C and 31.81% of salinity (31.81 S), respectively, where k₁ is the rate constant of the following reaction:

The rate constant of k₁ was derived by the following equation described by Johnson (1982):

(4)

The value for k₁ was calculated to be 0.74 x 10^–4 s^–1 at 20°C and 31.81 S, where is the true dissociation constant of carbonic acid and k₃ is the rate constant of the following reaction:

There have been few reported values of k₃ and in seawater at the exact condition you want (e.g. at 20°C and 31.81 S). According to Matsuda et al. (2001), when the value at 25°C ranges from 1.58 x 10^–4 – 3.98 x 10^–4 (Stumm and Morgan, 1995), the k₃ value at 25°C and 31.65 S was estimated to be 1.32 to 3.31 s^–1, and then a ratio value of k₃ to at 25°C and 31.65 S was approximately 0.83 x 10⁴. When using the k₃ to value of 0.83 x 10⁴ as the approximate value at 20°C and 31.81 S, the other constants described above the CO₂ formation rate at 20°C and 31.81 S were calculated to be 0.26 µmol L^–1 min^–1 at 100 µmol L^–1 DIC (pH 9.0).

According to Johnson (1982), the rate law for the reaction of CO₂ with water is:

(5)

Equation 5 can be simplified following the procedure of Lucas (1975), by making the assumption that photosynthesizing cells scavenge all CO₂ molecules as rapidly as they are formed by the dehydration of HCO₃^–. Namely, the rate of concern is the gross dehydration of HCO₃^– to CO₂ and so the back reaction was ignored; therefore,

(6)

The equilibration between DIC is almost instantaneous in seawater. The HCO₃^– concentration during HCO₃^– dehydration is, thus,

(7)

The rate of spontaneous formation of CO₂ from HCO₃^– was, therefore, determined as:

(8)

The k_d value can be derived by the following equation (Johnson, 1982).

(9)

The k_d value was calculated to be 2.6 x 10⁴ at 20°C and 31.81 S and then the CO₂ formation rate was 0.31 µmol L^–1 min^–1 at 100 µmol L^–1 DIC (pH 9.0), which is slightly higher than the rate calculated by using the k₃ to value of 0.83 x 10⁴. In this study, the rate of 0.31 µmol L^–1 min^–1 was used.

Received February 23, 2006; returned for revision April 1, 2006; accepted April 2, 2006.

	FOOTNOTES

 
¹ This work was supported by the Young Foundation of the Hubei Education Office.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Xiongwen Chen (xiongwenchen{at}eyou.com).

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.106.079616.

^* Corresponding author; e-mail xiongwenchen{at}eyou.com; fax 86–0714–6515772.

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