Photosynthetic carbon assimilation in the coccolithophorid Emiliania huxleyi (Haptophyta): Evidence for the predominant operation of the C3 cycle and the contribution of beta-carboxylases to the active anaplerotic reaction

The coccolithophorid Emiliania huxleyi (Haptophyta) is a representative and unique marine phytoplankton species that fixes inorganic carbon by photosynthesis and calcification. We examined the initial process of photosynthetic carbon assimilation by analyses of metabolites, enzymes and genes. When the cells were incubated with a radioactive substrate (2.3 mM NaHsup(14)CO3) for 10s under illumination, 70% of the sup(14)C was incorporated into the 80% methanol-soluble fraction. Eighty-five and 15% of sup(14)C in the soluble fraction was incorporated into phosphate esters (P-esters), including the C3 cycle intermediates and a C4 compound, aspartate, respectively. A pulse-chase experiment showed that sup(14)C in P-esters was mainly transferred into lipids, while [sup(14)C]aspartate, [sup(14)C]alanine and [sup(14)C]glutamate levels remained almost constant. These results indicate that the C3 cycle functions as the initial pathway of carbon assimilation and that beta-carboxylation contributes to the production of amino acids in subsequent metabolism. Transcriptional analysis of beta-carboxylases such as pyruvate carboxylase (PYC), phosphoenolpyruvate carboxylase (PEPC) and phosphoenolpyruvate carboxykinase (PEPCK) revealed that PYC and PEPC transcripts were greatly increased under illumination, whereas the PEPCK transcript decreased remarkably. PEPC activity was higher in light-grown cells than in dark-adapted cells. PYC activity was detected in isolated chloroplasts of light-grown cells. According to analysis of their deduced N-terminal sequence, PYC and PEPC are predicted to be located in the chloroplasts and mitochondria, respectively. These results suggest that E. huxleyi possesses unique carbon assimilation mechanisms in which beta-carboxylation by both PYC and PEPC plays important roles in different organelles.