Plant and Cell Physiology Advance Access originally published online on December 24, 2008
Plant and Cell Physiology 2009 50(2):318-329; doi:10.1093/pcp/pcn200
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Photosynthetic Carbon Assimilation in the Coccolithophorid Emiliania huxleyi (Haptophyta): Evidence for the Predominant Operation of the C3 Cycle and the Contribution of β-Carboxylases to the Active Anaplerotic Reaction
Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, 305-8572 Japan
*Corresponding author: E-mail, emilhux{at}biol.tsukuba.ac.jp; Fax, +81-29-853-6614.
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Abstract |
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The coccolithophorid Emiliania huxleyi (Haptophyta) is a representative and unique marine phytoplankton species that fixes inorganic carbon by photosynthesis and calci-fication. 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 NaH14CO3) for 10 s under illumination, 70% of the 14C was incorporated into the 80% methanol-soluble fraction. Eighty-five and 15% of 14C 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 14C in P-esters was mainly transferred into lipids, while [14C]aspartate, [14C]alanine and [14C]glutamate levels remained almost constant. These results indicate that the C3 cycle functions as the initial pathway of carbon assimilation and that β-carboxylation contributes to the production of amino acids in subsequent metabolism. Transcriptional analysis of β-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 assimila-tion mechanisms in which β-carboxylation by both PYC and PEPC plays important roles in different organelles.
Keywords: β-Carboxylation - Coccolithophorid - Emiliania huxleyi (Haptophyta) - Phosphoenolpyruvate carboxylase - Photosynthetic carbon fixation - Pyruvate carboxylase
Abbreviations: BSA, bovine serum albumin; CA, carbonic anhydrase; CCM, carbon-concentrating mechanism; DIC, dissolved inorganic carbons (CO2, HCO3– and CO32–); EST, expressed sequence tag; MA-ESM, Marine Art SF enriched with Erd–Schreiber medium; MDH, malate dehydrogenase; OAA, oxaloacetate; PEP, phosphoenolpyruvate; PEPC, pho-sphoenolpyruvate carboxylase; PEPCK, phosphoenolpyruvate carboxykinase; P-esters, phosphate esters as primary metabolites; 3-PGA, 3-phosphoglycerate; PYC, pyruvate carboxylase; Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase; TLC, thin-layer chromatography.
The nucleotide sequences reported in this paper have been submitted to DDBJ/GenBank/EMBL databanks under accession numbers AB461362 (EhPEPC) and AB461363 (EhPYC).
(Received October 31, 2008; Accepted December 16, 2008)
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