Journal of Experimental Marine Biology and Ecology
Carbon and nitrogen fluxes in the marine coccolithophore Emiliania huxleyi grown under different nitrate concentrations
Introduction
Assimilation of C and N consume the largest part of ATP and reductants generated in the cell (Huppe and Turpin, 1994). Although competing for energy, the acquisition and metabolism of C and N must be tightly coupled. This is due to the fact that the boundaries of the C to N ratio are defined by the stoichiometry of key components of the cell machinery, such as amino acids, proteins, nucleic acids and chlorophylls (Turpin, 1991 and references therein). The relative size of the different pools, within the above mentioned boundaries, is determined by the N availability relative to C in the environment (Giordano et al., 2001, Palmucci & Giordano, 2009).
Much information on NO3- assimilation is available for model microalgae such as Chlamydomonas reinhardtii (Fernandez and Galvan, 2008 and references therein), but very little is known for ecologically relevant phytoplankton species. In this study, we focused on the widely distributed coccolithophore Emiliania huxleyi (Winter et al., 1994). This organism provides an important contribution to the marine primary production and it is considered to be one of the major producers of calcite in the ocean (Baumann et al., 2004). Very limited data has been published on the NO3- acquisition by E. huxleyi (Page et al., 1999, Riegman et al., 2000). Some data are available for E. huxleyi nitrate reductase (NR), which shows some unique properties compared to the NR proteins of other microalgae (Iwamoto and Shiraiwa, 2003). Native E. huxleyi NR has an overall mass of 514 kD and is composed of six 85 kD homologous subunits (Iwamoto and Shiraiwa, 2003). The Km for NADH and NO3- of purified NR were 40 μM and 104 μM, respectively (Iwamoto and Shiraiwa, 2003). No information is available for E. huxleyi nitrite reductase (NiR), the enzyme responsible for the subsequent reduction of NO2- to NH4+. The NH4+ generated thanks to the catalysis by NiR is incorporated into amino acids via the glutamine synthetase/ glutamate synthase (GS/GOGAT) cycle. Two different GS isoforms of the enzyme, one located in the cytosol (GS1), the other located in the chloroplast (GS2), have been partially characterized in E. huxleyi by Maurin and Le Gal (1997a). Both isoforms are homohexamers with molecular masses of 402 kD for GS1 and 501 kD for GS2, whereas the molecular masses of the subunits of GS1 and GS2 were estimated to be 61 and 70 kD, respectively (Maurin and Le Gal, 1997a). The same authors reported that the Km for hydroxylamine (NH2OH) was approximately 3 mM for both GS isoforms, but GS2 had higher affinity for Gln than GS1. Maurin and Le Gal (1997b) also showed that E. huxleyi total GS activity was stimulated by decreasing NO3- availability and the affinity of GS for NH4+ was higher in N limited cells. In contrast to GS, no data is available in the literature for E. huxleyi GOGAT. The synthesis of amino acids via the GS/GOGAT system typically requires C skeletons from the TCA cycle (Elfiri & Turpin, 1986, Weger & Turpin, 1989).
Almost the entire C contained in macromolecular compounds such as proteins and lipids is fixed by Rubisco. Since Rubisco is characterized by a low affinity for CO2, microalgae cells have to invest a substantial amount of energy to enhance CO2 concentration at the carboxylation site of Rubisco and avoid inorganic carbon (Ci) limitation (Badger et al., 1998). Therefore, cells have developed biophysical and, possibly, biochemical carbon concentrating mechanisms (CCMs) that operate to increase the availability of CO2 for Rubisco (Giordano et al., 2005a and references therein). While it has been shown that the photosynthetic C acquisition of E. huxleyi was regulated in response to light and CO2 (Rost et al., 2003, Rost et al., 2006, Trimborn et al., 2007), the effect of N availability on the CCM has not been studied in this species. The CCMs have been suggested to improve N-use efficiency in microalgae, mainly by increasing the achieved rate of CO2 fixation per unit N in Rubisco (Raven, 1997, Beardall et al., 1998), thereby controlling the cellular elemental ratios, specifically the C to N ratio (Beardall and Giordano, 2002).
In this study, we intend to gain a better understanding of the regulation of intracellular processes that define the C to N ratio in a common phytoplankton cell. With this aim in mind, we investigated the regulation of cellular C and N fluxes and the relative composition of macromolecular pools in response to NO3- availability in E. huxleyi.
Section snippets
Culture conditions
The coccolith-bearing strain B 92/11 (J. C. Green 1990, Plymouth Marine Laboratory) of E. huxleyi was grown in semi continuous cultures in 10 L polycarbonate flasks. Experiments were carried out under a 16:8 h light:dark (LD) cycle at a constant temperature of 15 °C. The applied mean photon flux density was 240 μmol photons m-2 s-1. The culture flasks were aerated with air containing a CO2 partial pressure (pCO2) of 37.5 Pa and placed on a shaker, to keep the cells in suspension. The growth medium
Growth, elemental composition and coccolith morphology
The growth rates were quite similar (1.1 to 1.2 d-1) for the two NO3- treatments. The cell volume, however, was one-forth lower in cells at ambient NO3- (Table 1; Table S1). At ambient NO3-, cells accumulated less organic C and N and the cellular POC content was ca. one-third lower (Table 1; Table S1). Similarly, cells grown at ambient NO3- contained one-third less PON than their high NO3- counterparts (Table. 1; Table S1). Despite changes in POC and PON content, the C to N ratio remained
Discussion
Although C and N quotas and cell volume were lower at ambient than at replete NO3-, growth rates and C to N ratios were similar for the two growth regimes used for this study (Table 1). The constant C to N ratio and the results obtained by FTIR spectroscopy suggest that the abundance of protein relative to the non-nitrogenous pools examined (i.e. carbohydrates and lipids) was not affected by the ambient NO3- treatment, and the lower protein content was simply the consequence of the lower C and
Acknowledgements
We would like to thank Ellen Lichte for technical assistance. The research leading to these results has received funding from the German Research Foundation (DFG) and is part of the project TH 744/2-3. This research was also supported by the Spanish Ministry of Education (Juan de la Cierva programme) cofunded by the European Social Fund and Ministry of Science and Innovation. S. Trimborn and B. Rost acknowledge financial support by the European Research Council under the European Community’s
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