The role of the C4 pathway in carbon accumulation and fixation in a marine diatom

Abstract

The role of a C(4) pathway in photosynthetic carbon fixation by marine diatoms is presently debated. Previous labeling studies have shown the transfer of photosynthetically fixed carbon through a C(4) pathway and recent genomic data provide evidence for the existence of key enzymes involved in C(4) metabolism. Nonetheless, the importance of the C(4) pathway in photosynthesis has been questioned and this pathway is seen as redundant to the known CO(2) 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 CO(2) (10 microm), but had little effect on photosynthesis in the C(3) marine Chlorophyte, Chlamydomonas sp. In 3,3-dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate-treated T. weissflogii cells, elevated CO(2) (150 microm) or low O(2) (80-180 microm) restored photosynthesis to the control rate linking PEPCase inhibition with CO(2) supply in this diatom. In C(4) organic carbon-inorganic carbon competition experiments, the (12)C-labeled C(4) products of PEPCase, oxaloacetic acid and its reduced form malic acid suppressed the fixation of (14)C-labeled inorganic carbon by 40% to 50%, but had no effect on O(2) evolution in photosynthesizing diatoms. Oxaloacetic acid-dependent O(2) evolution in T. weissflogii was twice as high in cells acclimated to 10 microm rather than 22 microm CO(2), indicating that the use of C(4) compounds for photosynthesis is regulated over the range of CO(2) concentrations observed in marine surface waters. Short-term (14)C uptake (silicone oil centrifugation) and CO(2) 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 C(4) pathway functions as a CO(2) concentrating mechanism. Together these results demonstrate that the C(4) pathway is important in carbon accumulation and photosynthetic carbon fixation in diatoms at low (atmospheric) CO(2).

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.

Figure 2.

Oxaloacetic acid-dependent O₂ evolution (thick line) and dark O₂ consumption (thin line) in T. weissflogii cells at the C_i compensation point given 1 mm OAA at 120 s.

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.

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).

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 .