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Con Diatoms: Programmed cell death in the Ross Sea Mónica V. Orellana 1,2 Allison Lee 1 , Adrian Lopez Garcia de Lomana 1 , Meredith Jennings 3 , Sarah Bercovici 3 , Alexander Bochdansky 4 , NiHn Baliga 1,5 , and Dennis A. Hansell 1,3 1. InsHtute for Systems Biology, SeaOle WA USA 2. Polar Science CenterAPL, University of Washington, SeaOle WA USA 3. University of Miami 4. Old Dominion University, 5. Department of Microbiology, University of Washington, SeaOle WA USA Abstract FLow Anstra 15µm The Southern Ocean plays a criHcal role for carbon sequestraHon in the oceans. The flux of carbon (C) and nitrogen (N) export to the deep ocean depends on the efficiency of the biological pump transporHng biomass to depth. This study focuses on the impact of programmed cell death (PCD) in the Ross Sea, observed in the late austral summer during TRACERS (TRacing the fate of Algal Carbon Export in the Ross Sea, 2013). Program cell death (PCD) likely modulates the turnover of phytoplankton in the oceans, however very few field data exists recapitulaHng this phenomenon. We present evidence of diatoms undergoing PCD and their proximate causes in the Ross Sea, as well as examine other primary groups of organisms undergoing PCD in the water column. Immunofluorescence coupled with flow cytometry and phylogeneHc analyses of the populaHons undergoing PCD indicate higher occurrences of PCD in diatoms within the eukaryote community at the ocean surface, while prokaryotes undergoing PCD were found at a greater depth. In addiHon, we discuss invesHgaHons conducted to determine the role of PCD in mediaHng carbon export through the producHon of biopolymers as free and assembled polymer gels. DistribuHon of eukaryotes (mainly diatoms) undergoing PCD in the water column. Diatoms dominate the eastern area of the Ross Sea (170 o E) where they are subjected to low iron concentraHons (0.03 nM 0.1 nM) and seasonal depleHon (1,2,3) . (Color scale bar maps for percentage of cells undergoing PCD quanHfied by flow cytometry). Effect of diatoms undergoing PCD. Digital inline holographic microscope (DIHM) images insitu show exopolymeric parHcles (EP) that cause cell aggregaHon and concomitant fast sinking pulses of organic maOer to the deep ocean. EP account for = 20+/- 15μM C Kg -1 . 108m Confocal microscopy shows a variety of diatoms sorted by flow cytometry coupled with immunofluorescence , and indicates that 80 % of the diatoms undergo PCD. Results A. EUKARYOTES : In late summer the majority of the eukaryotes undergoing PCD in the Ross Sea are diatoms, which are the dominant taxa in the eastern area of the Ross Sea (see map). Our metagenomic analysis recapitulated the phylogeneHc tree of eukaryoHc species showing a large diversity of diatoms in the Ross Sea. (Colors correspond to different areas in the Ross Sea, bar size represents species abundance in log scale. % B. PROKARYOTES Prokaryotes undergo PCD at deeper depths than eukaryotes, at the same regions in the Ross Sea. Orellana and Verdugo 2003 Calcium biding stain flow cytometry revealed that 1025% of the dissolved organic maOer (DOM) produced by prokaryotes undergoing PCD assembled as microgels in the water column. Conclusions 1. Planktonic prokaryotes and photosyntheHc eukaryotes play a criHcal role in biogeochemical cycles. 2. PCD is an important mechanism sHmulaHng export producHon in the Ross Sea, however the flow and fate of the exported organic maOer is determined by the taxa that undergoes PCD. 3. Diatoms undergoing PCD in the Ross Sea, sHmulate the release of molecules that aggregate into big (mm size) exopolymer parHcles that rapidly sink into the deep ocean; instead prokaryotes undergoing PCD release molecules that do not aggregate into big parHcles but assemble into colloidal size polymer gels (nm size) and account for 10 to 25% of the DOC. 4. PCD determines parHculate organic carbon and organic nitrogen fluxes and biogeochemical cycling in the Ross Sea. References Coale et al. 2003. Deep Sea Res. 50: 635653; Cocklan et al. 2001. Deep Sea Res. 48: 41274153; Sedwick, et al. 2000. J Geophys Res. Oceans, 105, 1132111336 Turbulent diffusion ProducHvity PCD, Gels Carbon Dynamics 660 GC Sinking CCN Determine the mechanisms by which the biogeochemical transformaHons of nutrients and carbon variables take place in the major water masses located on the Ross Sea shelf. ObjecHves Map: Ross Sea and sampling staHons (1500 samples distributed from 74 o S 80 o S to 160 o E160W and 10 different depths ). Methods: Immunofluorescence coupled with flow cytometry, metagenomics, confocal microscopy, and inline holographic microscopy. 50m 800m Annexin VFITC labeled Sytox blue fluorescence Surface 10m 400m 25m 600m Annexin V FITC labeled Programmed Cell Death Acknowledgements: NSF: 1142049 FITC conjugate Flow cytometric measurements Eukaryotes Prokaryotes Metagenomics show a very diverse populaHon of bacteria. Bacteriodes, Cyanobacteria and Proteobacteria account for the majority of the prokaryotes. Figure showing the framework of the TRACERS study (2013) PCP was measured by flow cytometry. We detected plasma membrane permeability and externalizaHon of phospholipids with Annexin VFITC conjugate. SYTOX blue an impermeable probe that stains the nucleus was used to detect dead cells with compromised plasma membrane. Single cell analysis by flow cytometry, showing cells double stained with Annexin V FITC labeled and Sytox blue. Double posiHve cells are undergoing PCD.
