Sebastian D. Rokitta

Transcriptome analyses reveal differential gene expression patterns between the life-cycle stages of Emiliania Huxleyi (haptophyta) and reflect specialization to different ecological niches

Coccolithophores, especially the abundant, cosmopolitan species Emiliania huxleyi (Lohmann) W. W. Hay et H. P. Mohler, are one of the main driving forces of the oceanic carbonate pump and contribute significantly to global carbon cycling, due to their ability to calcify. A recent study indicates that termination of diploid blooms by viral infection induces life‐cycle transition, and speculation has arisen about the role of the haploid, noncalcifying stage in coccolithophore ecology. To explore gene expression patterns in both life‐cycle stages, haploid and diploid cells of E. huxleyi (RCC 1217 and RCC 1216) were acclimated to limiting and saturating photon flux densities. Transcriptome analyses were performed to assess differential genomic expression related to different ploidy levels and acclimation light intensities. Analyses indicated that life‐cycle stages exhibit different properties of regulating genome expression (e.g., pronounced gene activation and gene silencing in the diploid stage), proteome maintenance (e.g., increased turnover of proteins in the haploid stage), as well as metabolic processing (e.g., pronounced primary metabolism and motility in the haploid stage and calcification in the diploid stage). Furthermore, higher abundances of transcripts related to endocytotic and digestive machinery were observed in the diploid stage. A qualitative feeding experiment indicated that both life‐cycle stages are capable of particle uptake (0.5 μm diameter) in late‐stationary growth phase. Results showed that the two life‐cycle stages represent functionally distinct entities that are evolutionarily shaped to thrive in the environment they typically inhabit.

Ocean acidification affects redox-balance and ion-homeostasis in the life-cycle stages of Emiliania huxleyi.

Ocean Acidification (OA) has been shown to affect photosynthesis and calcification in the coccolithophore Emiliania huxleyi, a cosmopolitan calcifier that significantly contributes to the regulation of the biological carbon pumps. Its non-calcifying, haploid life-cycle stage was found to be relatively unaffected by OA with respect to biomass production. Deeper insights into physiological key processes and their dependence on environmental factors are lacking, but are required to understand and possibly estimate the dynamics of carbon cycling in present and future oceans. Therefore, calcifying diploid and non-calcifying haploid cells were acclimated to present and future CO2 partial pressures (pCO2; 38.5 Pa vs. 101.3 Pa CO2) under low and high light (50 vs. 300 µmol photons m−2 s−1). Comparative microarray-based transcriptome profiling was used to screen for the underlying cellular processes and allowed to follow up interpretations derived from physiological data. In the diplont, the observed increases in biomass production under OA are likely caused by stimulated production of glycoconjugates and lipids. The observed lowered calcification under OA can be attributed to impaired signal-transduction and ion-transport. The haplont utilizes distinct genes and metabolic pathways, reflecting the stage-specific usage of certain portions of the genome. With respect to functionality and energy-dependence, however, the transcriptomic OA-responses resemble those of the diplont. In both life-cycle stages, OA affects the cellular redox-state as a master regulator and thereby causes a metabolic shift from oxidative towards reductive pathways, which involves a reconstellation of carbon flux networks within and across compartments. Whereas signal transduction and ion-homeostasis appear equally OA-sensitive under both light intensities, the effects on carbon metabolism and light physiology are clearly modulated by light availability. These interactive effects can be attributed to the influence of OA and light on the redox equilibria of NAD and NADP, which function as major sensors for energization and stress. This generic mode of action of OA may therefore provoke similar cell-physiological responses in other protists.

Acidification, not carbonation, is the major regulator of carbon fluxes in the coccolithophore Emiliania huxleyi.

Summary A combined increase in seawater [CO2] and [H+] was recently shown to induce a shift from photosynthetic HCO3 − to CO2 uptake in Emiliania huxleyi. This shift occurred within minutes, whereas acclimation to ocean acidification (OA) did not affect the carbon source. To identify the driver of this shift, we exposed low‐ and high‐light acclimated E. huxleyi to a matrix of two levels of dissolved inorganic carbon (1400, 2800 μmol kg−1) and pH (8.15, 7.85) and directly measured cellular O2, CO2 and HCO3 − fluxes under these conditions. Exposure to increased [CO2] had little effect on the photosynthetic fluxes, whereas increased [H+] led to a significant decline in HCO3 − uptake. Low‐light acclimated cells overcompensated for the inhibition of HCO3 − uptake by increasing CO2 uptake. High‐light acclimated cells, relying on higher proportions of HCO3 − uptake, could not increase CO2 uptake and photosynthetic O2 evolution consequently became carbon‐limited. These regulations indicate that OA responses in photosynthesis are caused by [H+] rather than by [CO2]. The impaired HCO3 − uptake also provides a mechanistic explanation for lowered calcification under OA. Moreover, it explains the OA‐dependent decrease in photosynthesis observed in high‐light grown phytoplankton.

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

BackgroundGlobal 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.ResultsThe 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.ConclusionsThe 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.

P- and N-Depletion Trigger Similar Cellular Responses to Promote Senescence in Eukaryotic Phytoplankton

Global change will affect multiple physico-chemical parameters of the oceans, amongst them also the abundances of macronutrients like phosphorus and nitrogen that are critical for phytoplankton growth. Here we assessed the transcriptomic responses to phosphorus (P) depletion in the haploid and diploid life-cycle stage of the coccolithophore Emiliania huxleyi (RCC1217/1216) and compared the results with an existing dataset on nitrogen (N) depletion. The responses to the two depletion scenarios within one particular life-cycle stage were more similar at the transcriptome level than the responses of the two stages towards only one particular depletion scenario, emphasizing the tripartite nature of the coccolithophore genome. When cells senesced in both scenarios, they applied functionally similar programs to shut down cell cycling, re-adjust biochemical pathways and increase metabolic turnover to efficiently recycle elements. Those genes that exclusively responded to either P- or N-depletion modulated the general response to enhance scavenging, uptake and attempted storage of the limiting nutrient. The metabolic adjustments during senescence involved conserved and ancient pathways (e.g., proline oxidation or the glycolytic bypass) that prolong survival on the one hand, but on the other hand give rise to toxic messengers (e.g., reactive oxygen species or methylglyoxal). Continued senescence thus promotes various processes that lead to cell death, which can be delayed only for a limited time. As a consequence, the interplay of the involved processes determines how long cells can endure severe nutrient depletion before they lyse and provide their constituent nutrients to the more viable competitors in their environment. These responses to nutrient depletion are observable in other phytoplankton, but it appears that E. huxleyi´s outstanding endurance under nutrient deficiency is due to its versatile high-affinity uptake systems and an efficient, NAD-independent malate oxidation that is absent from most other taxa.