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

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 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 muM), 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 CO2 (150 muM) or low O-2 (80-180 muM) restored photosynthesis to the control rate linking PEPCase inhibition with CO2 supply in this diatom. In C-4 organic carbon-inorganic carbon competition experiments, the C-12-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 muM rather than 22 muM CO2, indicating that the use of C-4 compounds for photosynthesis is regulated over the range of CO2 concentrations observed in marine surface waters. Short-term C-14 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 C-4 pathway functions as a CO2 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) CO2.