Elsevier

Protist

Volume 163, Issue 2, March 2012, Pages 232-251
Protist

Original Paper
Coupled Effects of Light and Nitrogen Source on the Urea Cycle and Nitrogen Metabolism over a Diel Cycle in the Marine Diatom Thalassiosira pseudonana

https://doi.org/10.1016/j.protis.2011.07.008Get rights and content

Diatoms are photoautotrophic organisms capable of growing on a variety of inorganic and organic nitrogen sources. Discovery of a complete urea cycle in diatoms was surprising, as this pathway commonly functions in heterotrophic organisms to rid cells of waste nitrogen. To determine how the urea cycle is integrated into cellular nitrogen metabolism and energy management, the centric diatom Thalassiosira pseudonana was maintained in semi-continuous batch cultures on nitrate, ammonium, or urea as the sole nitrogen source, under a 16: 8 light: dark cycle and at light intensities that were low, saturating, or high for growth. Steady-state transcript levels were determined for genes encoding enzymes linked to the urea cycle, urea hydrolysis, glutamine synthesis, pyrimidine synthesis, photorespiration, and energy storage. Transcript abundances were significantly affected by nitrogen source, light intensity and a diel cycle. The impact of N source on differential transcript accumulation was most apparent under the highest light intensity. Models of cellular metabolism under high light were developed based on changes in transcript abundance and predicted enzyme localizations. We hypothesize that the urea cycle is integrated into nitrogen metabolism through its connection to glutamine and in the eventual production of urea. These findings have important implications for nitrogen flow in the cell over diel cycles at surface ocean irradiances.

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

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