Original PaperCoupled Effects of Light and Nitrogen Source on the Urea Cycle and Nitrogen Metabolism over a Diel Cycle in the Marine Diatom Thalassiosira pseudonana
Introduction
Diatoms are a key group of marine phytoplankton that are dominant in nutrient-rich coastal regions, and contribute an estimated 40% of total oceanic primary productivity (Nelson et al. 1995). Diatom blooms commonly occur in regions where nitrogen (N) source is variable and they possess a suite of N-related transporters and enzymes (e.g., Allen, 2005, Armbrust et al., 2004, Hildebrand, 2005, Hildebrand and Dahlin, 2000) and utilize a variety of inorganic (e.g., nitrate, NO3-; ammonium, NH4+) and organic (e.g., urea; amino acids) N sources for growth. Diatoms exhibit their fastest growth rates on reduced forms of N such as NH4+ or urea (Dortch, 1990, Dortch et al., 1991, Peers et al., 2000, Syrett, 1981), in part due to the low energetic costs associated with assimilation of these forms (Hildebrand 2005).
Analysis of whole genome sequences of marine diatoms is providing new insights into mechanisms underlying the biogeochemical roles of these organisms. One of the more surprising outcomes was identification of genes required for a complete urea cycle in Thalassiosira pseudonana (Armbrust et al. 2004). This pathway was subsequently identified in the genomes of the diatoms Phaeodactylum tricornutum (Bowler et al. 2008) and Fragilariopsis cylindrus (http://genome.jgi-psf.org/Fracy1/Fracy1.home.html), and there is now evidence for a complete cycle in additional members of the chromaveolates (e.g., Emiliania huxleyii; http://genome.jgi-psf.org/Emihu1/Emihu1.home.html) (Allen et al. 2011). Recent studies suggest that plants also possess the enzymes necessary for a complete urea cycle; however, localization of key enzymes differs from that of heterotrophs and diatoms (Gaufichon et al., 2010, Taylor et al., 2010).
In heterotrophs, the initial and rate-limiting step in the urea cycle occurs within mitochondria. Carbamoyl phosphate synthetase (CPS) catalyzes a 2-step ligation of 2 molecules of ATP, bicarbonate and NH4+ to form carbamoyl phosphate (P) (Beevers and Storey, 1976, Tatibana and Shigesad, 1972). One of two isoforms of CPS (CPSI or CPSIII) is used in the heterotrophic urea cycle, depending on the organism. A third isoform, CPSII, is involved in pyrimidine synthesis in the cytosol and utilizes glutamine. In higher plants, a plastid-localized CPSII serves a dual-role in pyridimine synthesis and the production of carbamoyl-P (Slocum 2005). CPSIII and CPSII use the amide group from glutamine as the primary N donor, whereas CPSI requires NH4+ as its primary substrate (Holden et al., 1998, Hong et al., 1994). In diatoms, two novel forms of CPS have been identified: unCPS utilizes NH4+ in the mitochondria as part of the urea cycle and pgCPS2 utilizes glutamine in the cytosol (Allen et al. 2011).
Detection of the urea cycle, including a mitochondria-targeted unCPS, in diatoms was unexpected as this pathway commonly functions in heterotrophic organisms to rid cells of waste NH4+, which diatoms can use as a sole N source for growth. Several hypotheses may explain the functional role of the urea cycle in diatoms, including temporary energy storage through formation of creatine-P, recycling of N through the production of amino acids, including arginine required for polyamine synthesis, the formation of a urea by-product, or as a sink for the photorespiration-generated NH4+ (Allen et al. 2006, 2011; Armbrust et al., 2004, Bowler et al., 2008, Vardi et al., 2008). Detection of all urea cycle enzymes in T. pseudonana under a variety of laboratory conditions (Nunn et al. 2009), as well as the presence of many diatom- urea cycle transcripts in a field metatranscriptome, suggest that this pathway plays a central role in diatom metabolism (Marchetti et al. submitted). Furthermore, changes in urea cycle intermediates in P. tricornutum in response to N availability have implicated the urea cycle in C and N redistribution via the tricarboxylic acid cycle (TCA) and the glutamine synthetase/glutamate synthase (GS-GOGAT) pathway (Allen et al. 2011).
The N required for entry into the urea cycle may be generated from NH4+ or glutamine via the GS-GOGAT pathway (Zehr and Falkowski 1988). In T. pseudonana, three isoforms of GS have been identified: GSI, GSII and GSIII (Armbrust et al., 2004, Takabayashi et al., 2005). Transcriptional data is currently available for the genes encoding GSII and GSIII (Brown et al., 2009, Parker and Armbrust, 2005, Takabayashi et al., 2005). GSII acts within the plastid where it utilizes the NH4+ derived from reduction of NO3− (Brown et al. 2009). GSIII was hypothesized to function in the cytosol to assimilate the NH4+ taken up directly by the cells in T. pseudonana (Brown et al. 2009), although direct evidence for this localization is lacking. A combination of in silico analyses and confocal microscopy using GFP labeling with P. tricornutum suggests that GSIII is targeted to the mitochondria in diatoms (Siaut et al. 2007).
To date, several studies have provided the framework for utilizing transcript abundances as a tool for elucidating the complex interactions between N metabolism, N assimilation and changing irradiance in diatoms (e.g., Granum et al., 2009, Hildebrand, 2005, Hildebrand and Dahlin, 2000, Kang et al., 2009, Kroth et al., 2008, Mock et al., 2008, Parker and Armbrust, 2005, Parker et al., 2004). Previous work has identified transcriptional regulation in a variety of organisms for the genes examined in this study: from bacteria to complex metazoans (e.g., CPSIII, Pierard et al. 1980; CPSII, Denis-Duphil 1989; URE [urease], reviewed in Mobley et al. 1995; GSII, Takabayashi et al. 2005; GDCT, Parker et al. 2004, Parker and Armbrust 2005; CK [creatine kinase], Jaynes et al. 1986). Post-transcriptional modification may also affect downstream processes (e.g., Poulsen et al. 2006); however, changes in transcript abundance provide a first snapshot of the cell's response to its environment.
Our experimental design incorporated multi-way ANOVAs to explicitly test interactions between N source and light on cell physiology and gene expression. In silico analysis and quantitative reverse transcriptase PCR were used to identify connections between the urea cycle and other pathways integral to cell metabolism. A model of N flow in T. pseudonana was developed from this data that includes protein localization and suggests that the urea cycle plays a critical role in both N metabolism and energy balance in the cell.
Section snippets
Growth Rate Comparisons
Growth rates were significantly different based on the interaction of N source and light intensity (two-way ANOVA, p < 0.05; Table 1; Supplementary Table S1). The 50 μmol photons m-2 s-1 irradiance was defined as low light (LL), the 190 μmol photons m-2 s-1 irradiance as saturating light (SL) and the 400 μmol photons m-2 s-1 as high light (HL) for growth. The Fv /Fm also varied significantly based on the interaction of N source and light intensity in both the light and the dark (p < 0.05; Table 1;
Discussion
Our results demonstrate that light intensity and N source differentially affect diurnal transcript accumulation of genes involved in the key metabolic pathways of the urea cycle (unCPS); urea hydrolysis (URE); N assimilation (GSI; GSII; GSIII); pyrimidine biosynthesis (pgCPSII); photorespiration (GDCT); and the formation of energy-storage compound creatine-P (CK). With few exceptions, the highest light treatment yielded the most transcripts for all genes and conditions surveyed; under high
Methods
Culture conditions: Axenic cultures of Thalassiosira pseudonana (Hustedt) Hasle et Heimdal (Provasoli- Guillard National Center for Culture of Marine Phytoplankton, CCMP 1335) were maintained in semi-continuous batch cultures (Brand et al. 1981) on a 16: 8 light: dark cycle at 13 °C in artificial seawater amended with f/2 concentrations of silicate, phosphate, vitamins and trace metals (Berges et al. 2001). Cultures were acclimated to growth at 3 light intensities: 50 μmol photons m-2 s-1 (LL),
Acknowledgements
The authors would like to thank Julie Koester for her insightful comments on this manuscript, helpful discussions and assistance with statistical applications. We would also like to acknowledge Chris Berthiaume for his assistance with the RNAseq transcriptome data, Dave Schruth for help with the R software package and Claire Ellis for her assistance with gene sequencing efforts. We also appreciate feedback from two anonymous reviewers.
This work is supported by a Gordon and Betty Moore
References (77)
Beyond sequence homology: Redundant ammonium transporters in a marine diatom are not functionally equivalent
J Phycol
(2005)- et al.
An ecological and evolutionary context for integrated nitrogen metabolism and related signaling pathways in marine diatoms
Curr Opin Plant Biol
(2006) - et al.
Biological functions of asparagine synthetase in plants
Plant Sci
(2010) - et al.
Carbamoyl phosphate synthetase: A tunnel runs through it
Curr Opin Struc Biol
(1998) - et al.
Carbamyl phosphate synthetase III, an evolutionary intermediate in the transition between glutamine-dependent and ammonia-dependent carbamyl phosphate synthetases
J Mol Biol
(1994) - et al.
Protein targeting to the chloroplasts of photosynthetic eukaryotes: Getting there is half the fun
Biochim Biophys Acta
(2005) - et al.
Assumption-free analysis of quantitative real-time polymerase chain reaction (pcr) data
Neurosci Lett
(2003) - et al.
Molecular toolbox for studying diatom biology in Phaeodactylum tricornutum
Gene
(2007) Genes, enzymes and regulation of arginine biosynthesis in plants
Plant Physiol Biochem
(2005)- et al.
Control of pyrimidine biosynthesis in mammalian tissues .5. Regulation of glutamine-dependent carbamyl phosphate synthetase - activation by 5-phosphoribosyl 1-pyrophosphate and inhibition by uridine triphosphate
J Biochem-Tokyo
(1972)
Evolution and metabolic significance of the urea cycle in photosynthetic diatoms
Nature
The genome of the diatom Thalassiosira pseudonana: Ecology, evolution, and metabolism
Science
Glutamate synthetase in developing cotyledons of Pisum sativum
Plant Physiol
Improved prediction of signal peptides: SignalP 3.0
J Mol Biol
Laboratory and field responses of algal nitrate reductase to diel periodicity in irradiance, nitrate exhaustion, and the presence of ammonium
Mar Ecol Prog Ser
Evolution of an artificial seawater medium: Improvements in enriched seawater, artificial water over the last two decades
J Phycol
Iron starvation and culture age activate metacaspases and programmed cell death in the marine diatom Thalassiosira pseudonana
Eukaryot Cell
The Phaeodactylum genome reveals the evolutionary history of diatom genomes
Nature
Impact of osmolytes on buoyancy of marine phytoplankton
Mar Biol
A method for the rapid and precise determination of acclimated phytoplankton reproduction rates
J Plankton Res
Unraveling the regulation of nitrogen assimilation in the marine diatom Thalassiosira pseudonana (Bacillariophyceae): Diurnal variations in transcript levels for five genes involved in nitrogen assimilation
J Phycol
The glutamine commute: take the N line and transfer to the A
J Cell Biol
Computational method to predict mitochondrially imported proteins and their targeting sequences
Eur J Biochem
Pyrimidine biosynthesis in Saccharomyces cerevisiae: the ura2 cluster gene, its multifunctional enzyme product, and other structural or regulatory genes involved in de novo UMP synthesis
Biochem Cell Biol
The interaction between ammonium and nitrate uptake in phytoplankton
Mar Ecol Prog Ser
Short-term interaction between nitrate and ammonium uptake in Thalassiosira pseudonana: Effect of preconditioning, nitrogen source and growth rate
Mar Biol
Chitin in diatoms and its association with the cell wall
Eukaryot Cell
MUSCLE: A multiple sequence alignment method with reduced time and space complexity
BMC Bioinformatics
Mitochondrial activities of phosphagen kinases are not widely distributed in the invertebrates
Biol Bull
ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites
Protein Sci
Characterization of urease activity in three marine phytoplankton species, Aureococcus anophagefferens, Prorocentrum minimum, and Thalassiosira weissflogii
Mar Biol
Expressing creatine kinase in transgenic tobacco - a first step towards introducing an energy buffering system in plants
Transgen Res
Molecular evolution of glutamine synthetase ii: Phylogenetic evidence of a non-endosymbiotic gene transfer event early in plant evolution
BMC Evol Biol
Primary carbon and nitrogen metabolic gene expression in the diatom Thalassiosira pseudonana (Bacillariophyceae): Diel periodicity and effects of inorganic carbon and nitrogen
J Phycol
Protein targeting into complex diatom plastids: Functional characterisation of a specific targeting motif
Plant Mol Biol
Cloning and functional characterization of ammonium transporters from the marine diatom Cylindrotheca fusiformis (Bacillariophyceae)
J Phycol
Nitrate transporter genes from the diatom Cylindrotheca fusiformis (Bacillariophyceae): mRNA levels controlled by nitrogen source and by the cell cycle
J Phycol
WoLF PSORT: protein localization predictor
Nucleic Acids Res
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