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BIOENERGETICS AND PHOTOSYNTHESIS

The Role of the C₄ Pathway in Carbon Accumulation and Fixation in a Marine Diatom¹

Department of Environmental Sciences, Rutgers University, New Brunswick, New Jersey 08901 (J.R.R.); and Department of Geosciences, Princeton University, Princeton, New Jersey 08544 (A.J.M., F.M.M.M.)

	ABSTRACT

TOP ABSTRACT RESULTS AND DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
The role of a C₄ pathway in photosynthetic carbon fixation by marine diatoms is presently debated. Previous labeling studies have shown the transfer of photosynthetically fixed carbon through a C₄ pathway and recent genomic data provide evidence for the existence of key enzymes involved in C₄ metabolism. Nonetheless, the importance of the C₄ pathway in photosynthesis has been questioned and this pathway is seen as redundant to the known CO₂ concentrating mechanism of diatoms. Here we show that the inhibition of phosphoenolpyruvate carboxylase (PEPCase) by 3,3-dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate resulted in a more than 90% decrease in whole cell photosynthesis in Thalassiosira weissflogii cells acclimated to low CO₂ (10 µM), but had little effect on photosynthesis in the C₃ marine Chlorophyte, Chlamydomonas sp. In 3,3-dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate-treated T. weissflogii cells, elevated CO₂ (150 µM) or low O₂ (80–180 µM) restored photosynthesis to the control rate linking PEPCase inhibition with CO₂ supply in this diatom. In C₄ organic carbon-inorganic carbon competition experiments, the ¹²C-labeled C₄ products of PEPCase, oxaloacetic acid and its reduced form malic acid suppressed the fixation of ¹⁴C-labeled inorganic carbon by 40% to 50%, but had no effect on O₂ evolution in photosynthesizing diatoms. Oxaloacetic acid-dependent O₂ evolution in T. weissflogii was twice as high in cells acclimated to 10 µM rather than 22 µM CO₂, indicating that the use of C₄ compounds for photosynthesis is regulated over the range of CO₂ concentrations observed in marine surface waters. Short-term ¹⁴C uptake (silicone oil centrifugation) and CO₂ release (membrane inlet mass spectrometry) experiments that employed a protein denaturing cell extraction solution containing the PEPCKase inhibitor mercaptopicolinic acid revealed that much of the carbon taken up by diatoms during photosynthesis is stored as organic carbon before being fixed in the Calvin cycle, as expected if the C₄ pathway functions as a CO₂ concentrating mechanism. Together these results demonstrate that the C₄ pathway is important in carbon accumulation and photosynthetic carbon fixation in diatoms at low (atmospheric) CO₂.

The question of the existence of enzymes necessary for a C₄ pathway in diatoms has now been largely resolved by the recently sequenced genome of Thalassiosira pseudonana (E.V. Armbrust, J.A. Berges, C. Bowler, B.R. Green, D. Martinez, N.H. Putnam, S. Zhou, A.E. Allen, K.E. Apt, M. Bechner, M. Brzezinski, B.K. Chaal, A. Chiovitti, A.K. Davis, M.S. Demarest, J.C. Detter, T. Glavina, D. Goodstein, M.Z. Hadi, U. Hellsten, M. Hildebrand, B.D. Jenkins, J. Jurka, V.V. Kapitonov, N. Kröger, W.W.Y. Lau, T.W. Lane, F.W. Larimer, J.C. Lippmeier, S. Lucas, M. Medina, A. Montsant, M. Obornik, M.S. Parker, B. Palenik, G.J. Pazour, P.M. Richardson, T.A. Rynearson, M.A. Saito, D.C. Schwartz, K. Thamatrakoln, K. Valentin, A. Vardi, F.P. Wilkerson, and D.S. Rokhsar, unpublished data). Genes coding for phosphoenolpyruvate carboxylase (PEPCase), phosphoenolpyruvate carboxykinase (PEPCKase), and pyruvate orthophosphate dikinase (PPDK, which catalyzes the synthesis of PEP in many C₄ plants) have been identified in the genome of this diatom. The intracellular localizations of all of these enzymes in diatoms, which are critical to a complete understanding of carbon metabolism in these organisms, are uncertain. The absence of upstream targeting sequences adjacent to the genes for PEPCase and PPDK in T. pseudonana is consistent with cytoplasmic localizations. The localization of PEPCase in the cytoplasm provides the necessary intracellular compartmentalization for simultaneous carbon fixation by PEPCase and Rubisco in a single cell (Magnin et al., 1997; Voznesenskaya et al., 2002). PEPCKase localization cannot be evaluated with certainty from the gene targeting sequence of T. pseudonana, but its activity was found to follow that of Rubisco in crude cell fractions of T. weissflogii (Reinfelder et al., 2000) and PEPCKase protein was immunolocalized within the chloroplast of the marine diatom Skeletonema costatum (Cabello-Pasini et al., 2001).

In this study, we examine the importance of the C₄ pathway in diatom photosynthesis by measuring the effects of PEPCase inhibition and C₄ organic carbon-C_i competition on whole cell O₂ evolution and inorganic carbon fixation in T. weissflogii. We also examine the form of carbon concentrated during short-term ¹⁴C uptake (silicone oil centrifugation) and CO₂ release (membrane inlet mass spectrometry) experiments to evaluate the role of the C₄ pathway in the diatom CCM. To provide a benchmark to differentiate C₄ and C₃ pathways, we conduct parallel experiments with the marine Chlorophyte Chlamydomonas sp.

	RESULTS AND DISCUSSION

TOP ABSTRACT RESULTS AND DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

The Importance of C₄ Carbon Fixation in Diatom Photosynthesis

If the C₄ pathway is quantitatively important to photosynthesis in diatoms, then the inhibition of PEPCase should have a major effect on photosynthetic O₂ evolution. To test the role of PEPCase in diatom photosynthesis, we used the PEPCase-specific inhibitor 3,3-dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate (DCDP), an analog of PEP that inhibits PEPCase from a range of C₄ and C₃ plants, but does not inhibit enzymes that catalyze other reactions in which PEP is a substrate (Jenkins et al., 1987). The addition of DCDP to suspensions of T. weissflogii cells grown in air-equilibrated medium caused a more than 90% decrease in photosynthetic O₂ evolution compared with uninhibited cells (Fig. 1). Thus, in diatoms acclimated to 10 µM CO₂, a major fraction of net carbon fixation depends on the synthesis of C₄ organic carbon by PEPCase. In Chlamydomonas sp. grown under identical conditions as T. weissflogii, DCDP had a small effect on whole cell photosynthetic O₂ evolution (Fig. 1). Chlamydomonas sp. are C₃ photoautotrophs in which PEPCase serves an anaplerotic role replacing TCA cycle carbohydrates used in amino acid synthesis (Huppe and Turpin, 1994; Rivoal et al., 1998). The minimal effect in this organism provides confidence that the major depression of photosynthesis by DCDP observed in the diatom was not due to the inhibition of anaplerotic PEPCase activity. If the inhibition of PEPCase in T. weissflogii impairs the ability of the diatom cells to concentrate CO₂, but not carbon fixation, then the addition of a high concentration of CO₂ to the external medium should restore photosynthesis in DCDP-inhibited diatoms to the level of uninhibited cells. Indeed the addition of 150 µM CO₂ to DCDP-inhibited T. weissflogii cells restored photosynthesis to the level of the control (Fig. 1), demonstrating that DCDP did not interfere with the Calvin cycle and that the shutdown of photosynthesis resulting from PEPCase inhibition in T. weissflogii is linked to the supply of inorganic carbon to the cell. Thus DCDP-inhibited cells behave as C₃ autotrophs lacking a mechanism to actively concentrate inorganic carbon for photosynthesis. Such cells should be particularly susceptible to the negative effects of O₂ on photosynthesis as a consequence of photorespiration and show increased photosynthesis rates when the concentration of O₂ is lowered. Decreasing the O₂ concentration in the medium did in fact result in an increase in photosynthetic O₂ production of DCDP-inhibited diatom cells to the levels of the uninhibited control (Fig. 1). The complete recovery of photosynthesis in response to lowering the O₂ concentration in these experiments is likely due to the high carboxylation to oxygenation ratio of diatom Rubisco compared with that from other eukaryotic microalgae (Badger et al., 1998). This effect may have been pronounced due to the somewhat elevated CO₂ concentrations (15–30 µM) at the experimental pH.

View larger version (18K): [in this window] [in a new window]   Figure 1. Effects of PEPCase inhibition on net photosynthetic O2 evolution in (A) the marine diatom T. weissflogii and (B) the marine chlorophyte Chlamydomonas sp. Graphs depict whole cell O2 evolution in control incubations (thick lines; 15–30 µM CO2 and 300–400 µM O2) or in the presence (thin lines) of the PEPCase inhibitor DCDP (750 µM). For T. weissflogii, photosynthesis rates of PEPCase-inhibited cells with 150 µM CO2 plus 300 to 400 µM O2 (+DCDP high CO2) and 80 to 180 µM O2 plus 15 to 30 µM CO2 (+DCDP low O2) are also shown.

View larger version (16K): [in this window] [in a new window]   Figure 2. Oxaloacetic acid-dependent O2 evolution (thick line) and dark O2 consumption (thin line) in T. weissflogii cells at the Ci compensation point given 1 mM OAA at 120 s.

View larger version (19K): [in this window] [in a new window]   Figure 3. Effects of C4 organic acids on carbon fixation (shaded bars) and O2 evolution (open bars) in the marine diatom T. weissflogii. Measurements were made in the presence or absence of 2 mM malic acid or OAA. Error bars represent propagated 14C counting errors for carbon fixation rates and SEs for O2 evolution rates.

Unequivocal evidence that marine diatoms possess a CCM has been obtained by a number of researchers (Rotatore et al., 1995; Burkhardt et al., 2001; Tortell and Morel, 2002), but the biochemical mechanism of the diatom CCM is unknown. The production and decarboxylation of C₄ organic carbon to supply Rubisco with CO₂ represents a carbon storage and delivery mechanism that may function as a CCM to support diatom photosynthesis in low CO₂ surface waters. Thus, the CCM and the C₄ pathway in diatoms may be one and the same. This is supported indirectly by the observation that C₄ carbon fixation (PEPCase activity) in T. weissflogii increases over the range of CO₂ concentrations (10 µM and lower; Reinfelder et al., 2000) where CCM activity increases in marine diatoms (Rotatore et al., 1995; Burkhardt et al., 2001; Tortell and Morel, 2002).

If the C₄ pathway serves as the CCM in diatoms, transported carbon should be stored as an organic (C₄) rather than inorganic (presumably ) compound before fixation in the Calvin cycle in these organisms. In this case, the inorganic carbon that has been measured as intracellular in previous studies of the CCM in diatoms should have resulted from rapid decarboxylation of a C₄ compound. We thus attempted to prevent such decarboxylation in the course of the same types of experiments—using the silicon oil centrifugation or the membrane inlet mass spectrometry (MIMS) methods—that have been used to demonstrate the existence of a CCM in diatoms.

In the silicone oil centrifugation method, inorganic carbon is measured in cells that have been spiked with inorganic ¹⁴C and then collected within seconds by centrifugation into a lower layer of an organic extraction and inorganic carbon trapping solution containing methanol and NaOH. In an attempt to inactivate all intracellular enzymes after centrifugation, we tried various extraction-trapping solutions containing cell membrane and protein denaturants (TriReagent, SDS) at low osmotic strength. In experiments run with SDS in the trapping solution, the amount of intracellular ¹⁴C measured as inorganic carbon (acid volatile) in T. weissflogii during short-term uptake was lower than that obtained with the normal trapping solution of methanol and NaOH (Fig. 4). The lowest amount of short-term carbon accumulation measured as inorganic carbon was observed with a low osmotic strength trapping solution containing 1% SDS and 20 mM mercaptopicolinic acid (MPA). MPA is an inhibitor of PEPCKase that catalyzes the decarboxylation of OAA in some C₄ plants (Rathnam and Edwards, 1977) and is hypothesized to catalyze C₄ decarboxylation in the chloroplast of the marine diatom T. weissflogii (Reinfelder et al., 2000). With the SDS-MPA metabolic inactivation solution, approximately 70% of the ¹⁴C taken up by T. weissflogii in 10 s was present as acid-stable organic carbon compared to only 24% with the standard methanol-NaOH trapping solution (Fig. 4). Since total ¹⁴C activities—organic plus inorganic—were the same (average residual within 6% of the mean) in all treatments with T. weissflogii, this result indicates that, in experiments with the standard trapping solution, a substantial portion of newly formed organic carbon was rapidly converted to inorganic carbon and that the trapping solution routinely used in the silicone oil centrifugation method does not immediately stop all metabolic activity after cells are removed from ¹⁴C-C_i. The most potent trapping solution for T. weissflogii (SDS-MPA) had no effect on the amount of intracellular ¹⁴C measured as inorganic carbon during short-term ¹⁴C exposures in Chlamydomonas sp. (Fig. 4). Thus a large fraction of the carbon taken up in 10 s is stored as organic carbon prior to fixation in the Calvin cycle in the diatom T. weissflogii, but not in the C₃ microalga Chamydomonas sp.

View larger version (58K): [in this window] [in a new window]   Figure 4. The fraction of short-term carbon accumulation measured as inorganic carbon (acid volatile 14C activity) by the silicon oil centrifugation technique in the marine diatom T. weissflogii and the marine chlorophyte Chlamydomonas sp. Treatments correspond to various extraction-trapping solutions. Numbers above bars are total (organic plus inorganic) 14C activities (kBq) collected after the cells were transferred to the trapping solutions by centrifugation. Error bars are SDs of means (n = 3). Note that in such experiments the entrainment of medium with the cells during centrifugation results in a background inorganic carbon in the trapping solution of up to 15% (dashed line) of the total (Tortell et al., 2000).

View larger version (17K): [in this window] [in a new window]   Figure 5. Uptake and release of CO2 by T. weissflogii (solid line) and Chlamydomonas sp. (dashed line) during alternating periods of light and dark (bar on x axis) and following the addition (in the light) of 20 mM MPA in 1% SDS (arrows) at constant pH (7.5) recorded in a membrane inlet mass spectrometer. Note that in the presence of the carbonic anhydrase inhibitor acetazolamide, the production of CO2 by the cells in these concentrated suspensions may lead to CO2 concentrations that are higher than that at equilibrium with .

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS AND DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Phytoplankton Cultures

Axenic cultures of the marine diatom Thalassiosira weissflogii (CCMP 1336) and the marine chlorophyte Chlamydomonas sp. (CCMP 222) were maintained in air-equilibrated synthetic ocean water (Aquil; Price et al., 1988, 1989) in continuous light (200 µmol photon m^–2 s^–1) at 18°C. Stock cultures were maintained in glass tubes and experimental cultures were grown in acid-soaked (1 N HCl) clear polycarbonate bottles. Cell growth was monitored by measuring in vivo fluorescence and microscope cell counts. Cells for all experiments were harvested in mid-exponential growth phase (µ = 1 d^–1).

PEPCase Inhibition Experiments

The inhibition of whole cell photosynthesis by DCDP, a specific, competitive (with PEP) inhibitor of PEPCase (Jenkins et al., 1987), was measured in T. weissflogii and Chlamydomonas sp. Photosynthesis was measured as oxygen evolution using an oxygen electrode cell (Hansatech, King's Lynn, UK) with 400 µmol photon m^–2 s^–1 photosynthetically active radiation at 22°C. For PEPCase inhibition treatments, DCDP was added to a final concentration of 750 µM from a 10-mM stock solution in 10 mM HCl. Cells were concentrated to 2 x 10⁶ (T. weissflogii) and 5.6 x 10⁶ (Chlamydomonas) cells mL^–1 in seawater culture media with 2 mM bicine (pH 7.4), 15 to 30 µM CO₂, and 300 to 400 µM O₂. Photosynthesis rates of DCDP-inhibited cells with excess CO₂ (150 µM CO₂, 300–400 µM O₂) and low O₂ (80–180 µM O₂, 15–30 µM CO₂) were also measured. DCDP inhibition experiments were also tried at pH 8 to maintain lower CO₂, but DCDP was not taken up above pH 7.4.

C₄ Acid-Dependent Oxygen Evolution Experiments

The stimulation of O₂ evolution by C₄ compounds was studied in T. weissflogii cells brought to the C_i compensation point (photosynthesis is equal to respiration) in an oxygen electrode. Cells were concentrated to 10⁶ cells mL^–1 in buffer (25 mM HEPES, 350 mM sorbitol, pH 7) and incubated in the light until nearly all inorganic carbon was consumed and the concentration of O₂ remained constant. Based on the photosynthesis-C_i relationship and respiration rates of this diatom, the total C_i at the compensation point was estimated to be <2 µM. Once the cells were brought to the C_i compensation point, 1 mM OAA was added. Maximum OAA-dependent O₂ evolution rates were compared with maximum rates in cells resuspended in buffer with 1 mM C_i. Cells were grown as described above and for at least two transfers (9–10 generations) in media bubbled with either air (10 µM CO₂) or air containing 700 µmol mol^–1 CO₂ (22 µM CO₂).

C₄-C_i Competition Experiments

The inhibition of inorganic carbon fixation by OAA or its reduced form, malic acid, was measured in photosynthesizing T. weissflogii cells concentrated to 2.5 x 10⁵ cells mL^–1 in buffer (25 mM HEPES, 350 mM sorbitol, pH 7.5) and incubated in the light (400 µmol photon m^–2 s^–1) at 22°C with 1.2 mM ¹⁴C-C_i. C₄ organic acids were added from freshly prepared stock solutions of the free acids (50 mM) to give final concentrations in the incubations of 2 mM OAA or malic acid. For the malic acid experiments, incubations were begun with the simultaneous addition of ¹⁴C-C_i (pH 9.5) and C₄ acid dissolved in HEPES buffer (pH 7.5) to cells that had been photosynthesizing in the light for 5 min. In the OAA experiment, photosynthesizing cells were incubated with 2 mM OAA for 30 s prior to the addition of ¹⁴C-C_i. At various times during the 5-min incubations, 1-mL subsamples were transferred to 2 mL methanol plus 50 µL 6 N HCl. The liquid was evaporated and the residues resuspended in a small volume (0.2–0.3 mL) of ultra-pure water. The acid-stable (organic) ¹⁴C content of extracts was quantified by liquid scintillation counting. Carbon fixation rates were estimated from the slopes of the ¹⁴C fixation curves and the specific activity of the radiocarbon. The spontaneous decarboxylation rate of oxaloacetic acid in the HEPES/sorbitol buffer was sufficiently slow (<1 nmol min^–1 over 5 min) so as not to be a significant source of CO₂. The effects of OAA and malic acid on photosynthetic O₂ evolution in T. weissflogii were also measured.

Silicone Oil Centrifugation

Short-term ¹⁴C carbon accumulation experiments were conducted using the silicone oil centrifugation technique (Badger et al., 1980; Tortell et al., 2000). Cells were resuspended in C_i-free 50 mM HEPES buffer with seawater salts (pH = 8). Concentrated cells (3.5 x 10⁶ for T. weissflogii and 9 x 10⁶ for Chlamydomonas sp. in 200 µL) were layered on top of silicon oil in 0.6-mL microcentrifuge tubes. Illumination was provided from the side at a photon flux density of 500 µmol m^–2 s^–1 using a tungsten-halogen bulb from a slide projector. To start the incubation 1 mM NaH¹⁴CO₃ was added and mixed with the pipette. Incubations (duration 10 s) were terminated by centrifugation through the silicon oil layer into a basic cell extraction and inorganic carbon trapping solution (6 N NaOH with 10% methanol). Other trapping solutions (all at pH 8) included 50% TriReagent (Sigma, St. Louis), 2% SDS and 8 mM MPA (Toronto Research Chemicals), and 50% Percol (to provide density at low ionic strength) with 1% SDS and 20 mM MPA. The resulting pellet was immediately frozen in liquid nitrogen. The base of the centrifuge tube was cut off and retained quantified using liquid scintillation counting. The ¹⁴C counts (corrected for quench) for the unacidified samples were used to determine the total accumulated carbon (inorganic and organic) and those for the acidified samples were used to determine the amount of organic carbon fixed.

Membrane Inlet Mass Spectrometry

The short-term accumulation and release of carbon was followed using a membrane inlet system attached to a Prisma QMS-200 (Pfeiffer) quadrapole mass spectrometer with closed ion source recording at mass/charge (m/z) ratios of 40 and 44. The membrane inlet system was modified from a water-jacketed DW/2 oxygen electrode chamber (Hansatech Instruments) in which the electrode base plate was replaced by a stainless steel base plate with a gas port drilled through the center. The standard Teflon membrane (thickness 12.5 µm) supplied with the DW/2 system was used. Illumination was provided by a tungsten projector bulb at 300 µmol m^–2 s^–1. Temperature was maintained at 20°C. The mass spectrometer was calibrated for CO₂ using buffer equilibrated with 100 and 750 µmol mol^–1 CO₂. Cells were concentrated to 9.4 x 10⁶ (T. weissflogii) and 3.6 x 10⁶ (Chlamydomonas) cells mL^–1 in buffer (25 mM HEPES, 350 mM sorbitol, pH 7.5) with 100 mM acetazolamide to suppress extracellular carbonic anhydrase activity and an initial C_i concentration of 100 µM. Release of CO₂ was measured in cells transferred to the dark and in cells treated with 1% SDS plus 20 mM MPA (pH = 7.5), an inhibitor of the C₄ PEPCKase.

	ACKNOWLEDGMENTS

 
We thank Katsura Izui (Kyoto University) for kindly providing the DCDP.

Received February 19, 2004; returned for revision April 27, 2004; accepted April 29, 2004.

	FOOTNOTES

 
¹ This work was supported by the NSF-EMSI program through the Center for Environmental Bioinorganic Chemistry (CEBIC) and by a Hatch/McIntyre-Stennis grant through the New Jersey Agricultural Experiment Station.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.041319.

^* Corresponding author; e-mail reinfelder{at}envsci.rutgers.edu; fax 732–932–8644.

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