Expression and inhibition of the carboxylating and decarboxylating enzymes in the photosynthetic C₄ pathway of marine diatoms

There is evidence that the CO₂-concentrating mechanism in the marine diatom Thalassiosira weissflogii operates as a type of single-cell C ₄ photosynthesis. In quantitative-PCR assays, we observed 2- to 4-fold up-regulation of two phosphoenolpyruvate carboxylase (PEPC) gene transcripts in Thalassiosira pseudonana cells adapted to low pCO₂, but did not detect such regulation in Phaeodactylum tricornutum grown under similar conditions. Transcripts encoding phosphoenolpyruvate carboxykinase did not appear to be regulated by pCO₂ in either diatom. In T. pseudonana and T. weissflogii, net CO₂ fixation was blocked by 3, 3-dichloro-2-(dihydroxyphosphinoyl-methyl)-propenoate (a specific inhibitor of PEPC), but was restored by about 50% and 80%, respectively, by addition of millimolar concentrations of KHCO₃. In T. pseudonana, T. weissflogii, and P. tricornutum, rates of net O₂ evolution were reduced by an average of 67%, 55%, and 62%, respectively, in the presence of 20 μM quercetin, also an inhibitor of PEPC. Quercetin promoted net CO₂ leakage from inhibited cells to levels in excess of the equilibrium CO ₂ concentration, suggesting that a fraction of the HCO ₃^- taken up is fated to leak back into the medium as CO₂ when PEPC activity is blocked. In parallel to these experiments, in vitro assays on crude extracts of T. pseudonana demonstrated mean inhibition of 65% of PEPC activity by quercetin. In the presence of 5 mM 3-mercaptopicolinic acid (3-MPA), a classic inhibitor of phosphoenolpyruvate carboxykinase, photosynthetic O₂ evolution was reduced by 90% in T. pseudonana. In T. weissflogii and P. tricornutum, 5 mM 3-MPA totally inhibited net CO₂ fixation and O₂ evolution. Neither quercetin nor 3-MPA had a significant inhibitory effect on photosynthetic O₂ evolution or CO₂ uptake in the marine chlorophyte isolates Chlamydomonas sp. or Dunaliella tertiolecta. Our evidence supports the idea that C₄-based CO₂-concentrating mechanisms are generally distributed in diatoms. This conclusion is discussed within the context of the evolutionary success of diatoms.