Phosphorus and nitrogen starvation reveal life-cycle specific responses in the metabolome of Emiliania huxleyi (Haptophyta)

The coccolithophore Emiliania huxleyi is a microalga with biogeochemical and biotechnological relevance, due to its high abundance in the ocean and its ability to form intricate calcium carbonate structures. Depletion of macronutrients in oceanic waters is very common and will likely enhance with advancing climate change. We present the first comprehensive metabolome study analyzing the effect of phosphorus (P) and nitrogen (N) starvation on the diploid and haploid life-cycle stage, applying various metabolome analysis methods to gain new insights in intracellular mechanisms to cope with nutrient starvation. P-starvation led to an accumulation of many generic and especially N-rich metabolites, including lipids, osmolytes, and pigments. This suggests that P-starvation primarily arrests cell-cycling due to lacking P for nucleic acid synthesis, but that enzymatic functionality is widely preserved. Also, the de-epoxidation ratio of the xanthophyll cycle was upregulated in the diploid stage under P-starvation, indicating increased nonphotochemical quenching, a response typically observed under high light stress. In contrast, N-starvation resulted in a decrease of most central metabolites, also P-containing ones, especially in the diploid stage, indicating that most enzymatic functionality ceased. The two investigated nutrient starvation conditions caused significantly different responses, contrary to previous assumptions derived from transcriptomic studies. Data highlight that instantaneous biochemical flux is a more dominant driver of the metabolome than the transcriptomically rearranged pathway patterns. Due to the fundamental nature of the observed responses it may be speculated that microalgae with similar nutrient requirements can cope better with P-starvation than with N-starvation

Effects of CO2 and their modulation by light in the life-cycle stages of the coccolithophore Emiliania huxleyi

The effects of ocean acidification on the life-cycle stages of the coccolithophore Emiliania huxleyi and their modulation by light were examined. Calcifying diploid and noncalcifying haploid cells (Roscoff culture collection strains 1216 and 1217) were acclimated to present-day and elevated CO2 partial pressures (PCO2; 38.5 vs. 101.3 Pa, i.e., 380 vs. 1000 µatm) under low and high light (50 vs. 300 µmol photons m-2 s-1). Growth rates as well as cellular quotas and production rates of C and N were measured. Sources of inorganic C for biomass buildup were determined using a 14C disequilibrium assay. Photosynthetic O2 evolution was measured as a function of dissolved inorganic C and light by means of membrane-inlet mass spectrometry. The diploid stage responded to elevated PCO2 by shunting resources from the production of particulate inorganic C toward organic C yet keeping the production of total particulate C constant. As the effect of ocean acidification was stronger under low light, the diploid stage might be less affected by increased acidity when energy availability is high. The haploid stage maintained elemental composition and production rates under elevated PCO2. Although both life-cycle stages involve different ways of dealing with elevated PCO2, the responses were generally modulated by energy availability, being typically most pronounced under low light. Additionally, PCO2 responses resembled those induced by high irradiances, indicating that ocean acidification affects the interplay between energy-generating processes (photosynthetic light reactions) and processes competing for energy (biomass buildup and calcification). A conceptual model is put forward explaining why the magnitude of single responses is determined by energy availability

Physiological effects of ocean acidification on the coccolithophore Emiliania huxleyi and their modulation by light

To investigate the modulation of OA-responses by light intensity, cells of E. huxleyi strain RCC 1216 were acclimated to ambient and high pCO2 (380 vs. 1000 µatm) under limiting and saturating light intensities (50 vs. 300 µmol photons m-2 s-1). Growth rates and cellular quotas of POC and PIC were measured. Photosynthetic O2 evolution was measured as a function of light and of [DIC]

Characterization of the life-cycle stages of the coccolithophore Emiliania huxleyi and their responses to Ocean Acidification

Anthropogenic carbon dioxide emissions cause a chemical phenomenon known as Ocean Acidification (OA). The associated changes in seawater chemistry are believed to have significant impact especially on coccolithophores, unicellular calcifying primary producers that take an outstanding role in the regulation of the marine carbon pumps. This thesis investigated the calcifying diploid and the non-calcifying haploid life-cycle stages of the globally dominant coccolithophore Emiliania huxleyi, and their responses to OA. Emphasis was put on investigating the role of energy-availability (i.e., irradiance) in the manifestation of OA-responses. A suite of methods was applied to resolve the effects on the phenomenological level (growth, elemental quotas and production), the physiological level (photosynthesis, carbon acquisition) and the level of gene expression (transcriptomics). In publication I, haploid and diploid cells were compared using microarray-based transcriptome profiling to assess stage-specific gene expression. The study identified genes related to distinct cell-biological traits, such as calcification in the diplont as well as flagellae and lipid respiration in the haplont. It further revealed that the diploid stage needs to make more regulatory efforts to epigenetically administrate its double amount of DNA, and therefore strongly controls its gene expression on the basis of transcription. The haplont in turn, possessing only a single sized genome, does not require these administrative efforts and seems to drive a more unrestricted gene expression. The proteome is apparently regulated on the basis of rapid turnover, i.e., post-translational. The haploid and diploid genomes may therefore be regarded as cellular ‘operating systems’ that streamline the life-cycle stages to occupy distinct ecological niches. Publication II investigated the responses of the life-cycle stages to OA under limiting and saturating light intensities. Growth rates as well as quotas and production rates of carbon (C) and nitrogen (N) were measured. In addition, inorganic C acquisition and photosynthesis were determined with a 14C-tracer technique and mass spectrometrybased gas-flux measurements. Under OA, the diploid stage shunted resources from calcification towards biomass production, yet keeping the production of total particulate carbon constant. In the haploid stage, elemental composition and production rates were more or less unaffected although major physiological acclimations were evident, pointing towards efforts to maintain homeostasis. Apparently, both life-cycle stages pursue distinct strategies to deal with OA. As a general pattern, OA-responses were strongly modulated by energy availability and typically most pronounced under low light. A concept explaining the energy-dependence of responses was proposed. In publication III, microarray-based transcriptome profiling was used to screen for cellular processes that underlie the observed phenomenological and physiological responses observed in the life-cycle stages (publication II). In the diplont, the increased biomass production under OA seems to be caused by production of glycoconjugates and lipids. 8 The lowered calcification may be attributed to impaired signal-transduction and iontransport mechanisms. The haplont utilized genes and metabolic pathways distinct from the diploid stage, reflecting the stage-specific usage of certain portions of the genome. With respect to functionality and energy-dependence, however, the transcriptomic OAresponses resembled those of the diplont. In both stages, signal transduction and ionhomeostasis were equally OA-sensitive under all light intensities. The effects on carbon metabolism and photophysiology, however, were clearly modulated by light availability. These interactive effects can be explained with the influence of both OA and light on the cellular ‘redox hub’, a major sensory system controlling the network of metabolic sources and sinks of reductive energy. In the general discussion, the newly gained views on the life-cycle stages are synthesized and biogeochemical implications of light-dependent OA-effects on coccolithophore calcification are considered. Furthermore, emerging physiological response patterns are identified to develop unifying concepts that can explain the energy-dependence of physiological effects. Finally, the critical role of redox regulation in the responses to changing environmental parameters is argued and research perspectives are given how to further resolve effects of the changing environment on marine phytoplankton

Charakterisierung der Lebensabschnittsphasen der Coccolithophoride Emiliania huxleyi und ihrer Reaktionen gegenüber der Ozeanversauerung

Anthropogenic carbon dioxide emissions cause a chemical phenomenon known as Ocean Acidification (OA). The associated changes in seawater chemistry are believed to have significant impact especially on coccolithophores, unicellular calcifying primary producers that take an outstanding role in the regulation of the marine carbon pumps. This thesis investigated the calcifying diploid and the non-calcifying haploid life-cycle stages of the globally dominant coccolithophore Emiliania huxleyi, and their responses to OA. Emphasis was put on investigating the role of energy-availability (i.e., irradiance) in the manifestation of OA-responses. A suite of methods was applied to resolve the effects on the phenomenological level (growth, elemental quotas and production), the physiological level (photosynthesis, carbon acquisition) and the level of gene expression (transcriptomics). In publication I, haploid and diploid cells were compared using microarray-based transcriptome profiling to assess stage-specific gene expression. The study identified genes related to distinct cell-biological traits, such as calcification in the diplont as well as flagellae and lipid respiration in the haplont. It further revealed that the diploid stage needs to make more regulatory efforts to epigenetically administrate its double amount of DNA, and therefore strongly controls its gene expression on the basis of transcription. The haplont in turn, possessing only a single sized genome, does not require these administrative efforts and seems to drive a more unrestricted gene expression. The proteome is apparently regulated on the basis of rapid turnover, i.e., post-translational. The haploid and diploid genomes may therefore be regarded as cellular operating systems that streamline the life-cycle stages to occupy distinct ecological niches. Publication II investigated the responses of the life-cycle stages to OA under limiting and saturating light intensities. Growth rates as well as quotas and production rates of carbon (C) and nitrogen (N) were measured. In addition, inorganic C acquisition and photosynthesis were determined with a 14C-tracer technique and mass spectrometry-based gas-flux measurements. Under OA, the diploid stage shunted resources from calcification towards biomass production, yet keeping the production of total particulate carbon constant. In the haploid stage, elemental composition and production rates were more or less unaffected although major physiological acclimations were evident, pointing towards efforts to maintain homeostasis. Apparently, both life-cycle stages pursue distinct strategies to deal with OA. As a general pattern, OA-responses were strongly modulated by energy availability and typically most pronounced under low light. A concept explaining the energy-dependence of responses was proposed. In publication III, microarray-based transcriptome profiling was used to screen for cellular processes that underlie the observed phenomenological and physiological responses observed in the life-cycle stages (publication II). In the diplont, the increased biomass production under OA seems to be caused by production of glycoconjugates and lipids. The lowered calcification may be attributed to impaired signal-transduction and ion-transport mechanisms. The haplont utilized genes and metabolic pathways distinct from the diploid stage, reflecting the stage-specific usage of certain portions of the genome. With respect to functionality and energy-dependence, however, the transcriptomic OA-responses resembled those of the diplont. In both stages, signal transduction and ion-homeostasis were equally OA-sensitive under all light intensities. The effects on carbon metabolism and photophysiology, however, were clearly modulated by light availability. These interactive effects can be explained with the influence of both OA and light on the cellular redox hub , a major sensory system controlling the network of metabolic sources and sinks of reductive energy. In the general discussion, the newly gained views on the life-cycle stages are synthesized and biogeochemical implications of light-dependent OA-effects on coccolithophore calcification are considered. Furthermore, emerging physiological response patterns are identified to develop unifying concepts that can explain the energy-dependence of physiological effects. Finally, the critical role of redox regulation in the responses to changing environmental parameters is argued and research perspectives are given how to further resolve effects of the changing environment on marine phytoplankton

Transcriptomic responses of Emiliania huxleyi to Ocean Acidification

To investigate effects of ocean acidification (OA) and their energy-dependent modulation in Emiliania huxleyi, diploid cells (RCC 1216) were acclimated to present and future CO2 partial pressures (pCO2; 380 vs. 1000 µatm) under low and high light (50 vs. 300 µmol photons m-2 s-1). Microarray-based transcriptome profiling was used to screen for cellular processes that underlie the physiological responses observed earlier (Rokitta & Rost 2012). OA was shown to influence fluxes of organic metabolites within and across compartments, and their partitioning between oxidative (e.g. glycolysis) and reductive pathways (e.g. pentose phosphate pathway), which is the likely cause for increased POC production. Furthermore, altered signal-transduction (e.g. phosphatidylinositolphosphate- and sphingosine-based signals) and membrane-potentials (e.g. by altered active ion transport) seem to be a major cause of impaired calcification under OA. While OA influenced signal-transduction and ion homeostasis independent of the light level, the effects of OA on the carbon metabolism were prominently modulated by energy availability. This interdependence of carbon metabolism and light physiology likely derives from their reliance on the redox equilibria of NAD+ and NADP+, which are cellular sensors for energy state and stress level. Due to the fundamental role of the affected processes, responses to OA are likely to occur similarly in other marine protists

Seawater carbonate chemistry and effects of CO2 and their modulation by light in the life-cycle stages of the coccolithophore Emiliania huxleyi strains RCC 1216 and 1217 during experiments, 2012

The effects of ocean acidification on the life-cycle stages of the coccolithophore Emiliania huxleyi and their by light were examined. Calcifying diploid and noncalcifying haploid cells (Roscoff culture collection 1216 and 1217) were acclimated to present-day and elevated CO2 partial pressures (PCO2; 38.5 vs. 101.3 Pa, ., 380 vs. 1000 matm) under low and high light (50 vs. 300 mmol photons m-2 s-1). Growth rates as well as quotas and production rates of C and N were measured. Sources of inorganic C for biomass buildup were using a 14C disequilibrium assay. Photosynthetic O2 evolution was measured as a function of dissolved inorganic C and light by means of membrane-inlet mass spectrometry. The diploid stage responded to elevated PCO2 by shunting resources from the production of particulate inorganic C toward organic C yet keeping the production of total particulate C constant. As the effect of ocean acidification was stronger under low light, the diploid stage might be less affected by increased acidity when energy availability is high. The haploid stage maintained elemental composition and production rates under elevated PCO2. Although both life-cycle stages involve different ways of dealing with elevated PCO2, the responses were generally modulated by energy availability, being typically most pronounced under low light. Additionally, PCO2 responses resembled those induced by high irradiances, indicating that ocean acidification affects the interplay between energy-generating processes (photosynthetic light reactions) and processes competing for energy (biomass buildup and calcification). A conceptual model is put forward explaining why the magnitude of single responses is determined by energy availability

Emiliania huxleyi endures N-limitation with an efficient metabolic budgeting and effective ATP synthesis

BACKGROUND: Global change will affect patterns of nutrient upwelling in marine environments, potentially becoming even stricter regulators of phytoplankton primary productivity. To better understand phytoplankton nutrient utilization on the subcellular basis, we assessed the transcriptomic responses of the life-cycle stages of the biogeochemically important microalgae Emiliania huxleyi to nitrogen limitation. Cells grown in batch cultures were harvested at 'early' and 'full' nitrogen limitation and were compared with non-limited cells. We applied microarray-based transcriptome profilings, covering ~10.000 known E. huxleyi gene models, and screened for expression patterns that indicate the subcellular responses. RESULTS: The diploid life-cycle stage scavenges nitrogen from external organic sources and -like diatoms- uses the ornithine-urea cycle to rapidly turn over cellular nitrogen. The haploid stage reacts similarly, although nitrogen scavenging is less pronounced and lipid oxidation is more prominent. Generally, polyamines and proline appear to constitute major organic pools that back up cellular nitrogen. Both stages induce a malate:quinone-oxidoreductase that efficiently feeds electrons into the respiratory chain and drives ATP generation with reduced respiratory carbon throughput. CONCLUSIONS: The use of the ornithine-urea cycle to budget the cellular nitrogen in situations of limitation resembles the responses observed earlier in diatoms. This suggests that underlying biochemical mechanisms are conserved among distant clades of marine phototrophic protists. The ornithine-urea cycle and proline oxidation appear to constitute a sensory-regulatory system that monitors and controls cellular nitrogen budgets under limitation. The similarity between the responses of the life-cycle stages, despite the usage of different genes, also indicates a strong functional consistency in the responses to nitrogen-limitation that appears to be owed to biochemical requirements. The malate:quinone-oxidoreductase is a genomic feature that appears to be absent from diatom genomes, and it is likely to strongly contribute to the uniquely high endurance of E. huxleyi under nutrient limitation

Implications of observed inconsistencies in carbonate chemistry measurements for ocean acidification studies

The growing field of ocean acidification research is concerned with the investigation of organisms’ responses to increasing pCO2 values. One important approach in this context is culture work using seawater with adjusted CO2 levels. As aqueous pCO2 is difficult to measure directly in small scale experiments, it is generally calculated from two other measured parameters of the carbonate system (often AT, CT or pH). Unfortunately, the overall uncertainties of measured and subsequently calculated values are often unknown. Especially under high pCO2, this can become a severe problem with respect to the interpretation of physiological and ecological data. In the few datasets from ocean acidification research where all three of these parameters were measured, pCO2 values calculated from AT and CT are typically about 30% lower (i.e. ~300 μatm at a target pCO2 of 1000 μatm) than those calculated from AT and pH or CT and pH. This study presents and discusses these discrepancies as well as likely consequences for the ocean acidification community. Until this problem is solved, one has to consider that calculated parameters of the carbonate system (e.g. pCO2, calcite saturation state) may not be comparable between studies, and that this may have important implications for the interpretation of CO2 perturbation experiments

H+-driven increase in CO2 uptake and decrease in HCO3- uptake explain coccolithophores' acclimation responses to ocean acidification

Recent ocean acidification (OA) studies revealed that seawater [H+] rather than [CO2] or [ inline image] regulate short-term responses in carbon fluxes of Emiliania huxleyi. Here, we investigated whether acclimation to altered carbonate chemistry modulates this regulation pattern and how the carbon supply for calcification is affected by carbonate chemistry. We acclimated E. huxleyi to present-day (ambient [CO2], [ inline image], and pH) and OA conditions (high [CO2], ambient [ inline image], low pH). To differentiate between the CO2 and pH/H+ effects, we also acclimated cells to carbonation (high [CO2] and [ inline image], ambient pH) and acidification (ambient [CO2], low [ inline image], and pH). Under these conditions, growth, production of particulate inorganic and organic carbon, as well as carbon and oxygen fluxes were measured. Under carbonation, photosynthesis and calcification were stimulated due to additional inline image uptake, whereas growth was unaffected. Such stimulatory effects are not apparent after short-term carbonation, indicating that cells adjusted their carbon acquisition during acclimation. Being driven by [ inline image], these regulations can, however, not explain typical OA effects. Under acidification and OA, photosynthesis stayed constant, whereas calcification and growth decreased. Similar to the short-term responses toward high [H+], CO2 uptake significantly increased, but inline image uptake decreased. This antagonistic regulation in CO2 and inline image uptake can explain why photosynthesis, being able to use CO2 and inline image, often benefits from OA, whereas calcification, being mostly dependent on inline image, often decreases. We identified H+ as prime driver of coccolithophores' acclimation responses toward OA. Acidified conditions seem to put metabolic burdens on the cells that result in decreased growth