Synechococcus nitrogen gene loss in iron-limited ocean regions.

Synechococcus are the most abundant cyanobacteria in high latitude regions and are responsible for an estimated 17% of annual marine net primary productivity. Despite their biogeochemical importance, Synechococcus populations have been unevenly sampled across the ocean, with most studies focused on low-latitude strains. In particular, the near absence of Synechococcus genomes from high-latitude, High Nutrient Low Chlorophyll (HNLC) regions leaves a gap in our knowledge of picocyanobacterial adaptations to iron limitation and their influence on carbon, nitrogen, and iron cycles. We examined Synechococcus populations from the subarctic North Pacific, a well-characterized HNLC region, with quantitative metagenomics. Assembly with short and long reads produced two near complete Synechococcus metagenome-assembled genomes (MAGs). Quantitative metagenome-derived abundances of these populations matched well with flow cytometry counts, and the Synechococcus MAGs were estimated to comprise >99% of the Synechococcus at Station P. Whereas the Station P Synechococcus MAGs contained multiple genes for adaptation to iron limitation, both genomes lacked genes for uptake and assimilation of nitrate and nitrite, suggesting a dependence on ammonium, urea, and other forms of recycled nitrogen leading to reduced iron requirements. A global analysis of Synechococcus nitrate reductase abundance in the TARA Oceans dataset found nitrate assimilation genes are also lower in other HNLC regions. We propose that nitrate and nitrite assimilation gene loss in Synechococcus may represent an adaptation to severe iron limitation in high-latitude regions where ammonium availability is higher. Our findings have implications for models that quantify the contribution of cyanobacteria to primary production and subsequent carbon export.

Widespread use of proton-pumping rhodopsin in Antarctic phytoplankton.

Photosynthetic carbon (C) fixation by phytoplankton in the Southern Ocean (SO) plays a critical role in regulating air-sea exchange of carbon dioxide and thus global climate. In the SO, photosynthesis (PS) is often constrained by low iron, low temperatures, and low but highly variable light intensities. Recently, proton-pumping rhodopsins (PPRs) were identified in marine phytoplankton, providing an alternate iron-free, light-driven source of cellular energy. These proteins pump protons across cellular membranes through light absorption by the chromophore retinal, and the resulting pH energy gradient can then be used for active membrane transport or for synthesis of adenosine triphosphate. Here, we show that PPR is pervasive in Antarctic phytoplankton, especially in iron-limited regions. In a model SO diatom, we found that it was localized to the vacuolar membrane, making the vacuole a putative alternative phototrophic organelle for light-driven production of cellular energy. Unlike photosynthetic C fixation, which decreases substantially at colder temperatures, the proton transport activity of PPR was unaffected by decreasing temperature. Cellular PPR levels in cultured SO diatoms increased with decreasing iron concentrations and energy production from PPR photochemistry could substantially augment that of PS, especially under high light intensities, where PS is often photoinhibited. PPR gene expression and high retinal concentrations in phytoplankton in SO waters support its widespread use in polar environments. PPRs are an important adaptation of SO phytoplankton to growth and survival in their cold, iron-limited, and variable light environment.

Diminished carbon and nitrate assimilation drive changes in diatom elemental stoichiometry independent of silicification in an iron-limited assemblage.

In the California Current Ecosystem, upwelled water low in dissolved iron (Fe) can limit phytoplankton growth, altering the elemental stoichiometry of the particulate matter and dissolved macronutrients. Iron-limited diatoms can increase biogenic silica (bSi) content >2-fold relative to that of particulate organic carbon (C) and nitrogen (N), which has implications for carbon export efficiency given the ballasted nature of the silica-based diatom cell wall. Understanding the molecular and physiological drivers of this altered cellular stoichiometry would foster a predictive understanding of how low Fe affects diatom carbon export. In an artificial upwelling experiment, water from 96 m depth was incubated shipboard and left untreated or amended with dissolved Fe or the Fe-binding siderophore desferrioxamine-B (+DFB) to induce Fe-limitation. After 120 h, diatoms dominated the communities in all treatments and displayed hallmark signatures of Fe-limitation in the +DFB treatment, including elevated particulate Si:C and Si:N ratios. Single-cell, taxon-resolved measurements revealed no increase in bSi content during Fe-limitation despite higher transcript abundance of silicon transporters and silicanin-1. Based on these findings we posit that the observed increase in bSi relative to C and N was primarily due to reductions in C fixation and N assimilation, driven by lower transcript expression of key Fe-dependent genes.

Different iron storage strategies among bloom-forming diatoms.

Diatoms are prominent eukaryotic phytoplankton despite being limited by the micronutrient iron in vast expanses of the ocean. As iron inputs are often sporadic, diatoms have evolved mechanisms such as the ability to store iron that enable them to bloom when iron is resupplied and then persist when low iron levels are reinstated. Two iron storage mechanisms have been previously described: the protein ferritin and vacuolar storage. To investigate the ecological role of these mechanisms among diatoms, iron addition and removal incubations were conducted using natural phytoplankton communities from varying iron environments. We show that among the predominant diatoms, Pseudo-nitzschia were favored by iron removal and displayed unique ferritin expression consistent with a long-term storage function. Meanwhile, Chaetoceros and Thalassiosira gene expression aligned with vacuolar storage mechanisms. Pseudo-nitzschia also showed exceptionally high iron storage under steady-state high and low iron conditions, as well as following iron resupply to iron-limited cells. We propose that bloom-forming diatoms use different iron storage mechanisms and that ferritin utilization may provide an advantage in areas of prolonged iron limitation with pulsed iron inputs. As iron distributions and availability change, this speculated ferritin-linked advantage may result in shifts in diatom community composition that can alter marine ecosystems and biogeochemical cycles.

Transcriptomic and proteomic responses of the oceanic diatom Pseudo-nitzschia granii to iron limitation.

Diatoms are a highly successful group of photosynthetic protists that often thrive under adverse environmental conditions. Members of the genus Pseudo-nitzschia are ecologically important diatoms which are able to subsist during periods of chronic iron limitation and form dense blooms following iron fertilization events. The cellular strategies within diatoms that orchestrate these physiological responses to variable iron concentrations remain largely uncharacterized. Using a combined transcriptomic and proteomic approach, we explore the exceptional ability of a diatom isolated from the iron-limited Northeast Pacific Ocean to reorganize its intracellular processes as a function of iron. We compared the molecular responses of Pseudo-nitzschia granii observed under iron-replete and iron-limited growth conditions to those of other model diatoms. Iron-coordinated molecular responses demonstrated some agreement between gene expression and protein abundance, including iron-starvation-induced-proteins, a putative iron transport system and components of photosynthesis and the Calvin cycle. Pseudo-nitzschia granii distinctly differentially expresses genes encoding proteins involved in iron-independent photosynthetic electron transport, urea acquisition and vitamin synthesis. We show that P. granii is unique among studied diatoms in its physiology stemming from distinct cellular responses, which may underlie its ability to subsist in low iron regions and rapidly bloom to outcompete other diatom taxa following iron enrichment.

Development of a molecular-based index for assessing iron status in bloom-forming pennate diatoms.

Iron availability limits primary productivity in large areas of the world's oceans. Ascertaining the iron status of phytoplankton is essential for understanding the factors regulating their growth and ecology. We developed an incubation-independent, molecular-based approach to assess the iron nutritional status of specific members of the diatom community, initially focusing on the ecologically important pennate diatom Pseudo-nitzschia. Through a comparative transcriptomic approach, we identified two genes that track the iron status of Pseudo-nitzschia with high fidelity. The first gene, ferritin (FTN), encodes for the highly specialized iron storage protein induced under iron-replete conditions. The second gene, ISIP2a, encodes an iron-concentrating protein induced under iron-limiting conditions. In the oceanic diatom Pseudo-nitzschia granii (Hasle) Hasle, transcript abundance of these genes directly relates to changes in iron availability, with increased FTN transcript abundance under iron-replete conditions and increased ISIP2a transcript abundance under iron-limiting conditions. The resulting ISIP2a:FTN transcript ratio reflects the iron status of cells, where a high ratio indicates iron limitation. Field samples collected from iron grow-out microcosm experiments conducted in low iron waters of the Gulf of Alaska and variable iron waters in the California upwelling zone verify the validity of our proposed Pseudo-nitzschia Iron Limitation Index, which can be used to ascertain in situ iron status and further developed for other ecologically important diatoms.

Marine diatom proteorhodopsins and their potential role in coping with low iron availability.

Proteorhodopsins (PR) are retinal-binding membrane proteins that function as light-driven proton pumps to generate energy for metabolism and growth. Recently PR-like genes have been identified in some marine eukaryotic protists, including diatoms, dinoflagellates, haptophytes and cryptophytes. These rhodopsins are homologous to green-light-absorbing, ATP-generating PRs present within bacteria. Here we show that in the oceanic diatom Pseudo-nitzschia granii, PR-like gene and protein expressions increase appreciably under iron limitation. In a survey of available transcriptomes, PR-like genes in diatoms are generally found in isolates from marine habitats where seasonal to chronic growth limitation by the micronutrient iron is prevalent, yet similar biogeographical patterns are not apparent in other phytoplankton taxa. We propose that rhodopsin-based phototrophy could account for a proportion of energy synthesis in marine eukaryotic photoautotrophs, especially when photosynthesis is compromised by low iron availability. This alternative ATP-generating pathway could have significant effects on plankton community structure and global ocean carbon cycling.

Comparative metatranscriptomics identifies molecular bases for the physiological responses of phytoplankton to varying iron availability.

In vast expanses of the oceans, growth of large phytoplankton such as diatoms is limited by iron availability. Diatoms respond almost immediately to the delivery of iron and rapidly compose the majority of phytoplankton biomass. The molecular bases underlying the subsistence of diatoms in iron-poor waters and the plankton community dynamics that follow iron resupply remain largely unknown. Here we use comparative metatranscriptomics to identify changes in gene expression associated with iron-stimulated growth of diatoms and other eukaryotic plankton. A microcosm iron-enrichment experiment using mixed-layer waters from the northeastern Pacific Ocean resulted in increased proportions of diatom transcripts and reduced proportions of transcripts from most other taxa within 98 h after iron addition. Hundreds of diatom genes were differentially expressed in the iron-enriched community compared with the iron-limited community; transcripts of diatom genes required for synthesis of photosynthesis and chlorophyll components, nitrate assimilation and the urea cycle, and synthesis of carbohydrate storage compounds were significantly overrepresented. Transcripts of genes encoding rhodopsins in eukaryotic phytoplankton were significantly underrepresented following iron enrichment, suggesting rhodopsins help cells cope with low-iron conditions. Oceanic diatoms appear to display a distinctive transcriptional response to iron enrichment that allows chemical reduction of available nitrogen and carbon sources along with a continued dependence on iron-free photosynthetic proteins rather than substituting for iron-containing functional equivalents present within their gene repertoire. This ability of diatoms to divert their newly acquired iron toward nitrate assimilation may underlie why diatoms consistently dominate iron enrichments in high-nitrate, low-chlorophyll regions.

Ferritin is used for iron storage in bloom-forming marine pennate diatoms.

Primary productivity in 30-40% of the world's oceans is limited by availability of the micronutrient iron. Regions with chronically low iron concentrations are sporadically pulsed with new iron inputs by way of dust or lateral advection from continental margins. Addition of iron to surface waters in these areas induces massive phytoplankton blooms dominated primarily by pennate diatoms. Here we provide evidence that the bloom-forming pennate diatoms Pseudo-nitzschia and Fragilariopsis use the iron-concentrating protein, ferritin, to safely store iron. Ferritin has not been reported previously in any member of the Stramenopiles, a diverse eukaryotic lineage that includes unicellular algae, macroalgae and plant parasites. Phylogenetic analyses suggest that ferritin may have arisen in this small subset of diatoms through a lateral gene transfer. The crystal structure and functional assays of recombinant ferritin derived from Pseudo-nitzschia multiseries reveal a maxi-ferritin that exhibits ferroxidase activity and binds iron. The protein is predicted to be targeted to the chloroplast to control the distribution and storage of iron for proper functioning of the photosynthetic machinery. Abundance of Pseudo-nitzschia ferritin transcripts is regulated by iron nutritional status, and is closely tied to the loss and recovery of photosynthetic competence. Enhanced iron storage with ferritin allows the oceanic diatom Pseudo-nitzschia granii to undergo several more cell divisions in the absence of iron than the comparably sized, oceanic centric diatom Thalassiosira oceanica. Ferritin in pennate diatoms probably contributes to their success in chronically low-iron regions that receive intermittent iron inputs, and provides an explanation for the importance of these organisms in regulating oceanic CO(2) over geological timescales.

IDENTIFICATION AND ASSESSMENT OF DOMOIC ACID PRODUCTION IN OCEANIC PSEUDO-NITZSCHIA (BACILLARIOPHYCEAE) FROM IRON-LIMITED WATERS IN THE NORTHEAST SUBARCTIC PACIFIC(1).

We identified and investigated the potential toxicity of oceanic Pseudo-nitzschia species from Ocean Station Papa (OSP), located in a high-nitrate, low-chlorophyll (HNLC) region of the northeast (NE) subarctic Pacific Ocean. Despite their relatively low abundances in the indigenous phytoplankton assemblage, Pseudo-nitzschia species richness is high. The morphometric characteristics of five oceanic Pseudo-nitzschia isolates from at least four species are described using SEM and TEM. The species identified are Pseudo-nitzschia dolorosa Lundholm et Moestrup, P. granii Hasle, P. heimii Manguin, and P. cf. turgidula (Hust.) Hasle. Additional support for the taxonomic classifications based on frustule morphology is provided through the sequencing of the internal transcribed spacer 1 (ITS1) rDNA. Pseudo-nitzschia species identification was also assessed by the construction of ITS1 clone libraries and using automated ribosomal intergenic spacer analysis (ARISA) for environmental samples collected during the Subarctic Ecosystem Response to Iron Enrichment Study (SERIES), conducted in close proximity to OSP in July of 2002. Based on ITS1 sequences, the presence of P. granii, P. heimii, P. cf. turgidula, and at least five other putative, unidentified Pseudo-nitzschia ITS1 variants was confirmed within iron-enriched phytoplankton assemblages at OSP. None of the oceanic isolates produced detectable levels of particulate domoic acid (DA) when in prolonged stationary phase due to silicic acid starvation. The lack of detectable concentrations of DA suggests that either these strains produce very little or no toxin, or that the physiological conditions required to promote particulate DA production were not met and thus differ from their coastal, toxigenic congeners.

The decline and fate of an iron-induced subarctic phytoplankton bloom.

Iron supply has a key role in stimulating phytoplankton blooms in high-nitrate low-chlorophyll oceanic waters. However, the fate of the carbon fixed by these blooms, and how efficiently it is exported into the ocean's interior, remains largely unknown. Here we report on the decline and fate of an iron-stimulated diatom bloom in the Gulf of Alaska. The bloom terminated on day 18, following the depletion of iron and then silicic acid, after which mixed-layer particulate organic carbon (POC) concentrations declined over six days. Increased particulate silica export via sinking diatoms was recorded in sediment traps at depths between 50 and 125 m from day 21, yet increased POC export was not evident until day 24. Only a small proportion of the mixed-layer POC was intercepted by the traps, with more than half of the mixed-layer POC deficit attributable to bacterial remineralization and mesozooplankton grazing. The depletion of silicic acid and the inefficient transfer of iron-increased POC below the permanent thermocline have major implications both for the biogeochemical interpretation of times of greater iron supply in the geological past, and also for proposed geo-engineering schemes to increase oceanic carbon sequestration.