Complementary environmental analysis and functional characterization of lower glycolysis-gluconeogenesis in the diatom plastid.

Organic carbon fixed in chloroplasts through the Calvin-Benson-Bassham Cycle can be diverted toward different metabolic fates, including cytoplasmic and mitochondrial respiration, gluconeogenesis, and synthesis of diverse plastid metabolites via the pyruvate hub. In plants, pyruvate is principally produced via cytoplasmic glycolysis, although a plastid-targeted lower glycolytic pathway is known to exist in non-photosynthetic tissue. Here, we characterized a lower plastid glycolysis-gluconeogenesis pathway enabling the direct interconversion of glyceraldehyde-3-phosphate and phospho-enol-pyruvate in diatoms, ecologically important marine algae distantly related to plants. We show that two reversible enzymes required to complete diatom plastid glycolysis-gluconeogenesis, Enolase and bis-phosphoglycerate mutase (PGAM), originated through duplications of mitochondria-targeted respiratory isoforms. Through CRISPR-Cas9 mutagenesis, integrative 'omic analyses, and measured kinetics of expressed enzymes in the diatom Phaeodactylum tricornutum, we present evidence that this pathway diverts plastid glyceraldehyde-3-phosphate into the pyruvate hub, and may also function in the gluconeogenic direction. Considering experimental data, we show that this pathway has different roles dependent in particular on day length and environmental temperature, and show that the cpEnolase and cpPGAM genes are expressed at elevated levels in high-latitude oceans where diatoms are abundant. Our data provide evolutionary, meta-genomic, and functional insights into a poorly understood yet evolutionarily recurrent plastid metabolic pathway.

Contribution of genome-scale metabolic modelling to niche theory.

Standard niche modelling is based on probabilistic inference from organismal occurrence data but does not benefit yet from genome-scale descriptions of these organisms. This study overcomes this shortcoming by proposing a new conceptual niche that resumes the whole metabolic capabilities of an organism. The so-called metabolic niche resumes well-known traits such as nutrient needs and their dependencies for survival. Despite the computational challenge, its implementation allows the detection of traits and the formal comparison of niches of different organisms, emphasising that the presence-absence of functional genes is not enough to approximate the phenotype. Further statistical exploration of an organism's niche sheds light on genes essential for the metabolic niche and their role in understanding various biological experiments, such as transcriptomics, paving the way for incorporating better genome-scale description in ecological studies.

Evolution of the acceptor side of photosystem I: ferredoxin, flavodoxin, and ferredoxin-NADP+ oxidoreductase.

The development of oxygenic photosynthesis by primordial cyanobacteria ~2.7 billion years ago led to major changes in the components and organization of photosynthetic electron transport to cope with the challenges of an oxygen-enriched atmosphere. We review herein, following the seminal contributions as reported by Jaganathan et al. (Functional genomics and evolution of photosynthetic systems, vol 33, advances in photosynthesis and respiration, Springer, Dordrecht, 2012), how these changes affected carriers and enzymes at the acceptor side of photosystem I (PSI): the electron shuttle ferredoxin (Fd), its isofunctional counterpart flavodoxin (Fld), their redox partner ferredoxin-NADP+ reductase (FNR), and the primary PSI acceptors F x and F A/F B. Protection of the [4Fe-4S] centers of these proteins from oxidative damage was achieved by strengthening binding between the F A/F B polypeptide and the reaction center core containing F x, therefore impairing O2 access to the clusters. Immobilization of F A/F B in the PSI complex led in turn to the recruitment of new soluble electron shuttles. This function was fulfilled by oxygen-insensitive [2Fe-2S] Fd, in which the reactive sulfide atoms of the cluster are shielded from solvent by the polypeptide backbone, and in some algae and cyanobacteria by Fld, which employs a flavin as prosthetic group and is tolerant to oxidants and iron limitation. Tight membrane binding of FNR allowed solid-state electron transfer from PSI bridged by Fd/Fld. Fine tuning of FNR catalytic mechanism led to formidable increases in turnover rates compared with FNRs acting in heterotrophic pathways, favoring Fd/Fld reduction instead of oxygen reduction.

From nets to networks: tools for deciphering phytoplankton metabolic interactions within communities and their global significance.

Our oceans are populated with a wide diversity of planktonic organisms that form complex dynamic communities at the base of marine trophic networks. Within such communities are phytoplankton, unicellular photosynthetic taxa that provide an estimated half of global primary production and support biogeochemical cycles, along with other essential ecosystem services. One of the major challenges for microbial ecologists has been to try to make sense of this complexity. While phytoplankton distributions can be well explained by abiotic factors such as temperature and nutrient availability, there is increasing evidence that their ecological roles are tightly linked to their metabolic interactions with other plankton members through complex mechanisms (e.g. competition and symbiosis). Therefore, unravelling phytoplankton metabolic interactions is the key for inferring their dependency on, or antagonism with, other taxa and better integrating them into the context of carbon and nutrient fluxes in marine trophic networks. In this review, we attempt to summarize the current knowledge brought by ecophysiology, organismal imaging, in silico predictions and co-occurrence networks using 'omics data, highlighting successful combinations of approaches that may be helpful for future investigations of phytoplankton metabolic interactions within their complex communities.This article is part of the theme issue 'Connected interactions: enriching food web research by spatial and social interactions'.

Better together? Lessons on sociality from Trichodesmium.

The N2-fixing cyanobacterium Trichodesmium is an important player in the oceanic nitrogen and carbon cycles. Trichodesmium occurs both as single trichomes and as colonies containing hundreds of trichomes. In this review, we explore the benefits and disadvantages of colony formation, considering physical, chemical, and biological effects from nanometer to kilometer scale. Showing that all major life challenges are affected by colony formation, we claim that Trichodesmium's ecological success is tightly linked to its colonial lifestyle. Microbial interactions in the microbiome, chemical gradients within the colony, interactions with particles, and elevated mobility in the water column shape a highly dynamic microenvironment. We postulate that these dynamics are key to the resilience of Trichodesmium and other colony formers in our changing environment.

High Growth Rate of Diatoms Explained by Reduced Carbon Requirement and Low Energy Cost of Silica Deposition.

The rapid growth of diatoms makes them one of the most pervasive and productive types of plankton in the world's ocean, but the physiological basis for their high growth rates remains poorly understood. Here, we evaluate the factors that elevate diatom growth rates, relative to other plankton, using a steady-state metabolic flux model that computes the photosynthetic C source from intracellular light attenuation and the carbon cost of growth from empirical cell C quotas, across a wide range of cell sizes. For both diatoms and other phytoplankton, growth rates decline with increased cell volume, consistent with observations, because the C cost of division increases with size faster than photosynthesis. However, the model predicts overall higher growth rates for diatoms due to reduced C requirements and the low energetic cost of Si deposition. The C savings from the silica frustule are supported by metatranscriptomic data from Tara Oceans, which show that the abundance of transcripts for cytoskeleton components in diatoms is lower than in other phytoplankton. Our results highlight the importance of understanding the origins of phylogenetic differences in cellular C quotas, and suggest that the evolution of silica frustules may play a critical role in the global dominance of marine diatoms. IMPORTANCE This study addresses a longstanding issue regarding diatoms, namely, their fast growth. Diatoms, which broadly are phytoplankton with silica frustules, are the world's most productive microorganisms and dominate in polar and upwelling regions. Their dominance is largely supported by their high growth rate, but the physiological reasoning behind that characteristic has been obscure. In this study, we combine a quantitative model and metatranscriptomic approaches and show that diatoms' low carbon requirements and low energy costs for silica frustule production are the key factors supporting their fast growth. Our study suggests that the effective use of energy-efficient silica as a cellular structure, instead of carbon, enables diatoms to be the most productive organisms in the global ocean.

A robust approach to estimate relative phytoplankton cell abundances from metagenomes.

Phytoplankton account for >45% of global primary production, and have an enormous impact on aquatic food webs and on the entire Earth System. Their members are found among prokaryotes (cyanobacteria) and multiple eukaryotic lineages containing chloroplasts. Genetic surveys of phytoplankton communities generally consist of PCR amplification of bacterial (16S), nuclear (18S) and/or chloroplastic (16S) rRNA marker genes from DNA extracted from environmental samples. However, our appreciation of phytoplankton abundance or biomass is limited by PCR-amplification biases, rRNA gene copy number variations across taxa, and the fact that rRNA genes do not provide insights into metabolic traits such as photosynthesis. Here, we targeted the photosynthetic gene psbO from metagenomes to circumvent these limitations: the method is PCR-free, and the gene is universally and exclusively present in photosynthetic prokaryotes and eukaryotes, mainly in one copy per genome. We applied and validated this new strategy with the size-fractionated marine samples collected by Tara Oceans, and showed improved correlations with flow cytometry and microscopy than when based on rRNA genes. Furthermore, we revealed unexpected features of the ecology of these ecosystems, such as the high abundance of picocyanobacterial aggregates and symbionts in the ocean, and the decrease in relative abundance of phototrophs towards the larger size classes of marine dinoflagellates. To facilitate the incorporation of psbO in molecular-based surveys, we compiled a curated database of >18,000 unique sequences. Overall, psbO appears to be a promising new gene marker for molecular-based evaluations of entire phytoplankton communities.

Convergent evolution and horizontal gene transfer in Arctic Ocean microalgae.

Microbial communities in the world ocean are affected strongly by oceanic circulation, creating characteristic marine biomes. The high connectivity of most of the ocean makes it difficult to disentangle selective retention of colonizing genotypes (with traits suited to biome specific conditions) from evolutionary selection, which would act on founder genotypes over time. The Arctic Ocean is exceptional with limited exchange with other oceans and ice covered since the last ice age. To test whether Arctic microalgal lineages evolved apart from algae in the global ocean, we sequenced four lineages of microalgae isolated from Arctic waters and sea ice. Here we show convergent evolution and highlight geographically limited HGT as an ecological adaptive force in the form of PFAM complements and horizontal acquisition of key adaptive genes. Notably, ice-binding proteins were acquired and horizontally transferred among Arctic strains. A comparison with Tara Oceans metagenomes and metatranscriptomes confirmed mostly Arctic distributions of these IBPs. The phylogeny of Arctic-specific genes indicated that these events were independent of bacterial-sourced HGTs in Antarctic Southern Ocean microalgae.

Heterotrophic bacterial diazotrophs are more abundant than their cyanobacterial counterparts in metagenomes covering most of the sunlit ocean.

Biological nitrogen fixation contributes significantly to marine primary productivity. The current view depicts few cyanobacterial diazotrophs as the main marine nitrogen fixers. Here, we used 891 Tara Oceans metagenomes derived from surface waters of five oceans and two seas to generate a manually curated genomic database corresponding to free-living, filamentous, colony-forming, particle-attached, and symbiotic bacterial and archaeal populations. The database provides the genomic content of eight cyanobacterial diazotrophs including a newly discovered population related to known heterocystous symbionts of diatoms, as well as 40 heterotrophic bacterial diazotrophs that considerably expand the known diversity of abundant marine nitrogen fixers. These 48 populations encapsulate 92% of metagenomic signal for known nifH genes in the sunlit ocean, suggesting that the genomic characterization of the most abundant marine diazotrophs may be nearing completion. Newly identified heterotrophic bacterial diazotrophs are widespread, express their nifH genes in situ, and also occur in large planktonic size fractions where they might form aggregates that provide the low-oxygen microenvironments required for nitrogen fixation. Critically, we found heterotrophic bacterial diazotrophs to be more abundant than cyanobacterial diazotrophs in most metagenomes from the open oceans and seas, emphasizing the importance of a wide range of heterotrophic populations in the marine nitrogen balance.

Carbon Dioxide Concentration Mechanisms in Natural Populations of Marine Diatoms: Insights From Tara Oceans.

Marine diatoms, the most successful photoautotrophs in the ocean, efficiently sequester a significant part of atmospheric CO2 to the ocean interior through their participation in the biological carbon pump. However, it is poorly understood how marine diatoms fix such a considerable amount of CO2, which is vital information toward modeling their response to future CO2 levels. The Tara Oceans expeditions generated molecular data coupled with in situ biogeochemical measurements across the main ocean regions, and thus provides a framework to compare diatom genetic and transcriptional flexibility under natural CO2 variability. The current study investigates the interlink between the environmental variability of CO2 and other physicochemical parameters with the gene and transcript copy numbers of five key enzymes of diatom CO2 concentration mechanisms (CCMs): Rubisco activase and carbonic anhydrase (CA) as part of the physical pathway, together with phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, and malic enzyme as part of the potential C4 biochemical pathway. Toward this aim, we mined >200 metagenomes and >220 metatranscriptomes generated from samples of the surface layer of 66 globally distributed sampling sites and corresponding to the four main size fractions in which diatoms can be found: 0.8-5 µm, 5-20 µm, 20-180 µm, and 180-2,000 µm. Our analyses revealed that the transcripts for the enzymes of the putative C4 biochemical CCM did not in general display co-occurring profiles. The transcripts for CAs were the most abundant, with an order of magnitude higher values than the other enzymes, thus implying the importance of physical CCMs in diatom natural communities. Among the different classes of this enzyme, the most prevalent was the recently characterized iota class. Consequently, very little information is available from natural diatom assemblages about the distribution of this class. Biogeographic distributions for all the enzymes show different abundance hotspots according to the size fraction, pointing to the influence of cell size and aggregation in CCMs. Environmental correlations showed a complex pattern of responses to CO2 levels, total phytoplankton biomass, temperature, and nutrient concentrations. In conclusion, we propose that biophysical CCMs are prevalent in natural diatom communities.

Global drivers of eukaryotic plankton biogeography in the sunlit ocean.

Eukaryotic plankton are a core component of marine ecosystems with exceptional taxonomic and ecological diversity, yet how their ecology interacts with the environment to drive global distribution patterns is poorly understood. In this work, we use Tara Oceans metabarcoding data, which cover all major ocean basins, combined with a probabilistic model of taxon co-occurrence to compare the biogeography of 70 major groups of eukaryotic plankton. We uncover two main axes of biogeographic variation. First, more-diverse groups display clearer biogeographic patterns. Second, large-bodied consumers are structured by oceanic basins, mostly through the main current systems, whereas small-bodied phototrophs are structured by latitude and follow local environmental conditions. Our study highlights notable differences in biogeographies across plankton groups and investigates their determinants at the global scale.

Complex Response of the Chlorarachniophyte Bigelowiella natans to Iron Availability.

The productivity of the ocean is largely dependent on iron availability, and marine phytoplankton have evolved sophisticated mechanisms to cope with chronically low iron levels in vast regions of the open ocean. By analyzing the metabarcoding data generated from the Tara Oceans expedition, we determined how the global distribution of the model marine chlorarachniophyte Bigelowiella natans varies across regions with different iron concentrations. We performed a comprehensive proteomics analysis of the molecular mechanisms underpinning the adaptation of B. natans to iron scarcity and report on the temporal response of cells to iron enrichment. Our results highlight the role of phytotransferrin in iron homeostasis and indicate the involvement of CREG1 protein in the response to iron availability. Analysis of the Tara Oceans metagenomes and metatranscriptomes also points to a similar role for CREG1, which is found to be widely distributed among marine plankton but to show a strong bias in gene and transcript abundance toward iron-deficient regions. Our analyses allowed us to define a new subfamily of the CobW domain-containing COG0523 putative metal chaperones which are involved in iron metabolism and are restricted to only a few phytoplankton lineages in addition to B. natans At the physiological level, we elucidated the mechanisms allowing a fast recovery of PSII photochemistry after resupply of iron. Collectively, our study demonstrates that B. natans is well adapted to dynamically respond to a changing iron environment and suggests that CREG1 and COG0523 are important components of iron homeostasis in B. natans and other phytoplankton.IMPORTANCE Despite low iron availability in the ocean, marine phytoplankton require considerable amounts of iron for their growth and proliferation. While there is a constantly growing knowledge of iron uptake and its role in the cellular processes of the most abundant marine photosynthetic groups, there are still largely overlooked branches of the eukaryotic tree of life, such as the chlorarachniophytes. In the present work, we focused on the model chlorarachniophyte Bigelowiella natans, integrating physiological and proteomic analyses in culture conditions with the mining of omics data generated by the Tara Oceans expedition. We provide unique insight into the complex responses of B. natans to iron availability, including novel links to iron metabolism conserved in other phytoplankton lineages.

Paleo-diatom composition from Santa Barbara Basin deep-sea sediments: a comparison of 18S-V9 and diat-rbcL metabarcoding vs shotgun metagenomics.

Sedimentary ancient DNA (sedaDNA) analyses are increasingly used to reconstruct marine ecosystems. The majority of marine sedaDNA studies use a metabarcoding approach (extraction and analysis of specific DNA fragments of a defined length), targeting short taxonomic marker genes. Promising examples are 18S-V9 rRNA (~121-130 base pairs, bp) and diat-rbcL (76 bp), targeting eukaryotes and diatoms, respectively. However, it remains unknown how 18S-V9 and diat-rbcL derived compositional profiles compare to metagenomic shotgun data, the preferred method for ancient DNA analyses as amplification biases are minimised. We extracted DNA from five Santa Barbara Basin sediment samples (up to ~11 000 years old) and applied both a metabarcoding (18S-V9 rRNA, diat-rbcL) and a metagenomic shotgun approach to (i) compare eukaryote, especially diatom, composition, and (ii) assess sequence length and database related biases. Eukaryote composition differed considerably between shotgun and metabarcoding data, which was related to differences in read lengths (~112 and ~161 bp, respectively), and overamplification of short reads in metabarcoding data. Diatom composition was influenced by reference bias that was exacerbated in metabarcoding data and characterised by increased representation of Chaetoceros, Thalassiosira and Pseudo-nitzschia. Our results are relevant to sedaDNA studies aiming to accurately characterise paleo-ecosystems from either metabarcoding or metagenomic data.

Global distribution patterns of marine nitrogen-fixers by imaging and molecular methods.

Nitrogen fixation has a critical role in marine primary production, yet our understanding of marine nitrogen-fixers (diazotrophs) is hindered by limited observations. Here, we report a quantitative image analysis pipeline combined with mapping of molecular markers for mining >2,000,000 images and >1300 metagenomes from surface, deep chlorophyll maximum and mesopelagic seawater samples across 6 size fractions (<0.2-2000 µm). We use this approach to characterise the diversity, abundance, biovolume and distribution of symbiotic, colony-forming and particle-associated diazotrophs at a global scale. We show that imaging and PCR-free molecular data are congruent. Sequence reads indicate diazotrophs are detected from the ultrasmall bacterioplankton (<0.2 µm) to mesoplankton (180-2000 µm) communities, while images predict numerous symbiotic and colony-forming diazotrophs (>20 µm). Using imaging and molecular data, we estimate that polyploidy can substantially affect gene abundances of symbiotic versus colony-forming diazotrophs. Our results support the canonical view that larger diazotrophs (>10 µm) dominate the tropical belts, while unicellular cyanobacterial and non-cyanobacterial diazotrophs are globally distributed in surface and mesopelagic layers. We describe co-occurring diazotrophic lineages of different lifestyles and identify high-density regions of diazotrophs in the global ocean. Overall, we provide an update of marine diazotroph biogeographical diversity and present a new bioimaging-bioinformatic workflow.

Phytoplankton in the Tara Ocean.

Photosynthesis evolved in the ocean more than 2 billion years ago and is now performed by a wide range of evolutionarily distinct organisms, including both prokaryotes and eukaryotes. Our appreciation of their abundance, distributions, and contributions to primary production in the ocean has been increasing since they were first discovered in the seventeenth century and has now been enhanced by data emerging from the Tara Oceans project, which performed a comprehensive worldwide sampling of plankton in the upper layers of the ocean between 2009 and 2013. Largely using recent data from Tara Oceans, here we review the geographic distributions of phytoplankton in the global ocean and their diversity, abundance, and standing stock biomass. We also discuss how omics-based information can be incorporated into studies of photosynthesis in the ocean and show the likely importance of mixotrophs and photosymbionts.