Expression and Inhibition of the Carboxylating and Decarboxylating Enzymes in the Photosynthetic C4 Pathway of Marine Diatoms

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Abstract
There is evidence that the CO(2)-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 pCO(2), but did not detect such regulation in Phaeodactylum tricornutum grown under similar conditions. Transcripts encoding phosphoenolpyruvate carboxykinase did not appear to be regulated by pCO(2) in either diatom. In T. pseudonana and T. weissflogii, net CO(2) 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 KHCO(3). In T. pseudonana, T. weissflogii, and P. tricornutum, rates of net O(2) evolution were reduced by an average of 67%, 55%, and 62%, respectively, in the presence of 20 microm quercetin, also an inhibitor of PEPC. Quercetin promoted net CO(2) 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 CO(2) 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 O(2) evolution was reduced by 90% in T. pseudonana. In T. weissflogii and P. tricornutum, 5 mm 3-MPA totally inhibited net CO(2) fixation and O(2) evolution. Neither quercetin nor 3-MPA had a significant inhibitory effect on photosynthetic O(2) evolution or CO(2) uptake in the marine chlorophyte isolates Chlamydomonas sp. or Dunaliella tertiolecta. Our evidence supports the idea that C(4)-based CO(2)-concentrating mechanisms are generally distributed in diatoms. This conclusion is discussed within the context of the evolutionary success of diatoms.

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