Diel-Regulated Transcriptional Cascades of Microbial Eukaryotes in the North Pacific Subtropical Gyre

Abstract

Open-ocean surface waters host a diverse community of single-celled eukaryotic plankton (protists) consisting of phototrophs, heterotrophs, and mixotrophs. The productivity and biomass of these organisms oscillate over diel cycles, and yet the underlying transcriptional processes are known for few members of the community. Here, we examined a 4-day diel time series of transcriptional abundance profiles for the protist community (0.2–100 μm in cell size) in the North Pacific Subtropical Gyre near Station ALOHA. De novo assembly of poly-A+ selected metatranscriptomes yielded over 30 million contigs with taxonomic and functional annotations assigned to 54 and 25% of translated contigs, respectively. The completeness of the resulting environmental eukaryotic taxonomic bins was assessed, and 48 genera were further evaluated for diel patterns in transcript abundances. These environmental transcriptome bins maintained reproducible temporal partitioning of total gene family abundances, with haptophyte and ochrophyte genera generally showing the greatest diel partitioning of their transcriptomes. The haptophyte Phaeocystis demonstrated the highest proportion of transcript diel periodicity, while most other protists had intermediate levels of periodicity regardless of their trophic status. Dinoflagellates, except for the parasitoid genus Amoebophrya, exhibit the fewest diel oscillations of transcript abundances. Diel-regulated gene families were enriched in key metabolic pathways; photosynthesis, carbon fixation, and fatty acid biosynthesis gene families had peak times concentrated around dawn, while gene families involved in protein turnover (proteasome and protein processing) are most active during the high intensity daylight hours. TCA cycle, oxidative phosphorylation and fatty acid degradation predominantly peaked near dusk. We identified temporal pathway enrichments unique to certain taxa, including assimilatory sulfate reduction at dawn in dictyophytes and signaling pathways at early evening in haptophytes, pointing to possible taxon-specific channels of carbon and nutrients through the microbial community. These results illustrate the synchrony of transcriptional regulation to the diel cycle and how the protist community of the North Pacific Subtropical Gyre structures their transcriptomes to guide the daily flux of matter and energy through the gyre ecosystem.

Marine Microeukaryote Metatranscriptomics: Sample Processing and Bioinformatic Workflow Recommendations for Ecological Applications

“…High frequency sampling may identify metabolic trends that either change on a diel cycle (e.g., energy partitioning), or are unaffected by temporal dynamics (e.g., chronic nutrient stress). In particular, there is significant diel periodicity in metabolic processes carried out by protists in surface waters (Kolody et al, 2019;Becker et al, 2021;Groussman et al, 2021), and time of day should therefore be considered in sampling designs.…”

“…Note that the number of RNA copies per cell may differ by organism, with larger cells generally containing higher RNA concentrations (Marguerat and Bähler, 2012). Transcript pools furthermore fluctuate over the course of a day, with phytoplankton groups under different diel transcriptional regulation (Groussman et al, 2021). These factors may contribute to the copies L -1 estimate among taxa not fully scaling with absolute cell densities.…”

“…Broadly, these approaches can be used to find similarities in gene expression profiles across sample types and/ or relate transcript levels to environmental conditions. Ordination plots have been utilized to show time-dependent patterns in microeukaryote gene expression (Groussman et al, 2021), taxon-specific microeukaryote functional profiles (Hu et al, 2018), and differences in surface and deep dinoflagellate metatranscriptome profiles (Cohen et al, 2021). The input data for ordinations may be normalized read counts associated with annotated or unannotated transcripts at the community level (e.g., community-wide TPM), or normalized at a given taxonomic level of interest (e.g., haptophytes).…”

“…When applied to marine systems, it may offer information on the biogeochemical and ecological roles of community members across ecosystems and environmental conditions. Marine metatranscriptomic studies to date have been conducted across spatial and temporal scales (e.g., Sun et al, 2020;Becker et al, 2021;Cohen et al, 2021;Coesel et al, 2021;Groussman et al, 2021;Harke et al, 2021;Muratore et al, 2022), and in concert with experimental incubations/microcosms in which key biological, chemical and/or physical parameters are manipulated (e.g., Alexander et al, 2015b;Bertrand et al, 2015;Lampe et al, 2018). Such metatranscriptomic investigations into marine protists have expanded our understanding of their nutrient physiology (Alexander et al, 2015a;Pearson et al, 2015;Lampe et al, 2018;Caputi et al, 2019;Kolody et al, 2019), nutritional modes (Hu et al, 2018;Lambert et al, 2021), coastal bloom dynamics (Gong et al, 2017;Ji et al, 2018;Metegnier et al, 2020), and contributions to ocean biogeochemistry (Carradec et al, 2018;Cohen et al, 2021).…”

“…Marine metatranscriptomic initiatives have greatly benefited from the expansion of reference sequence libraries derived from laboratory isolates, such as the Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP) (Keeling et al, 2014;Caron et al, 2017). The MMETSP database is composed of over 678 protistan transcriptomes from 405 unique strains (Krinos et al, 2021) and is widely used in current marine omic workflows (e.g., Lampe et al, 2018;Kolody et al, 2019;Groussman et al, 2021;Krinos et al, 2022). Oceanographic field expeditions such as Tara Oceans (Carradec et al, 2018) and bioGEOTRACES (Biller et al, 2018) are leveraging this database and other recently generated eukaryotic transcriptomes and genomes to uncover the taxonomic and functional roles of protists across ocean basins (Carradec et al, 2018;Alexander et al, 2021;Weissman et al, 2021;Blaxter et al, 2022;Delmont et al, 2022).…”