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Evolution and metabolic significance of the urea cycle in photosynthetic diatoms

Abstract

Diatoms dominate the biomass of phytoplankton in nutrient-rich conditions and form the basis of some of the world’s most productive marine food webs1,2,3,4. The diatom nuclear genome contains genes with bacterial and plastid origins as well as genes of the secondary endosymbiotic host (the exosymbiont5)1,6,7,8,9,10, yet little is known about the relative contribution of each gene group to diatom metabolism. Here we show that the exosymbiont-derived ornithine-urea cycle, which is similar to that of metazoans but is absent in green algae and plants, facilitates rapid recovery from prolonged nitrogen limitation. RNA-interference-mediated knockdown of a mitochondrial carbamoyl phosphate synthase impairs the response of nitrogen-limited diatoms to nitrogen addition. Metabolomic analyses indicate that intermediates in the ornithine-urea cycle are particularly depleted and that both the tricarboxylic acid cycle and the glutamine synthetase/glutamate synthase cycles are linked directly with the ornithine-urea cycle. Several other depleted metabolites are generated from ornithine-urea cycle intermediates by the products of genes laterally acquired from bacteria. This metabolic coupling of bacterial- and exosymbiont-derived proteins seems to be fundamental to diatom physiology because the compounds affected include the major diatom osmolyte proline12 and the precursors for long-chain polyamines required for silica precipitation during cell wall formation11. So far, the ornithine-urea cycle is only known for its essential role in the removal of fixed nitrogen in metazoans. In diatoms, this cycle serves as a distribution and repackaging hub for inorganic carbon and nitrogen and contributes significantly to the metabolic response of diatoms to episodic nitrogen availability. The diatom ornithine-urea cycle therefore represents a key pathway for anaplerotic carbon fixation into nitrogenous compounds that are essential for diatom growth and for the contribution of diatoms to marine productivity.

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Figure 1: Carbamoyl phosphate synthase phylogeny and divergence timing.
Figure 2: Transcript levels of genes encoding components of the urea cycle and the nitrate assimilation pathway in wild-type P. tricornutum cells.
Figure 3: Growth characteristics and metabolite abundance in wild-type and unCPS RNAi P. tricornutum lines.
Figure 4: Conceptual overview of the roles of unCPS and the diatom urea cycle on the basis of metabolite data from wild-type and RNAi lines.

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Acknowledgements

We thank A. Meichenin and C. Lichtlé for assistance with electron microscropy, J. C. Thomas for CPS purification and activity experiments, A. Falciatore for advice on RNAi constructs and A. Main for screening and evaluation of RNAi lines. This study was supported by the National Science Foundation (NSF-OCE-0722374, NSF-OCE-0727997, NSF-MCB-1024913) and JCVI internal funding to A.E.A., the European Commission Diatomics project and Agence Nationale de la Recherche (France) (C.B.) and the Czech Science Foundation (206/08/1423) (M.O.).

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Contributions

A.E.A. and C.B. designed the study. A.E.A. performed CPS localization, confocal microscopy, protein purification, activity, overexpression and other laboratory experiments. A.E.A. and C.L.D. designed nitrogen-recovery experiments and the physiological characterization of the RNAi as well as wild-type experiments, which were performed by H.Z and C.L.D.. H.Z. generated and screened RNAi lines. H.Z. and H.H. performed long-term and short-term nitrogen-recovery and related qPCR experiments. D.A.J. ran qPCR reactions and assisted with analyses of qPCR data. M.O., A.H. and A.E.A. generated and analysed phylogenetic and molecular clock data. A.N-N. and A.R.F. performed metabolite profiling of samples collected from RNAi and wild-type cultures. J.P.M., C.L.D. and A.E.A. analysed qPCR, metabolite and western blot data in detail. A.E.A. wrote the paper with assistance from C.L.D., A.R.F., C.B. and M.O. All the authors discussed the results and commented on the manuscript.

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Correspondence to Andrew E. Allen.

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Allen, A., Dupont, C., Oborník, M. et al. Evolution and metabolic significance of the urea cycle in photosynthetic diatoms. Nature 473, 203–207 (2011). https://doi.org/10.1038/nature10074

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