Mónica V. Orellana

Spontaneous assembly of marine dissolved organic matter into polymer gels

A large pool of organic carbon resides in the worlds oceans in the form of dissolved organic matter (DOM),. DOM is operationally defined as the fraction of organic matter that passes through a filter with a given pore size (which can range from less than 0.1 μm to 0.46 μm). This fraction has a longer oceanic residence time — and is generally less biodegradable — than particulate organic matter (POM). Processes transforming DOM into POM are therefore crucial for our understanding of the cycling of organic material in the oceans. The aggregation of marine colloids, which constitute 10–40% of DOM,,, is thought to be an important step in the transformation of DOM into POM. It has been suggested that colloids, as well as transparent exopolymer particles and large aggregates (‘marine snow’) can be viewed as polymer gels. Whether free DOM polymers can indeed spontaneously assemble to form polymer gels has, however, not yet been shown. Here we present experimental observations that demonstrate that marine polymer gels can assemble from free DOM polymers, and that their formation mechanism, physical characteristics and mineralization can be understood in terms of polymer gel theory. The principles and methods of polymer gel physics thus have the potential to provide profound new insights into the processes controlling the exchange between the DOM and POM pools and the cycling of marine organic matter.

Marine microgels as a source of cloud condensation nuclei in the high Arctic

Marine microgels play an important role in regulating ocean basin-scale biogeochemical dynamics. In this paper, we demonstrate that, in the high Arctic, marine gels with unique physicochemical characteristics originate in the organic material produced by ice algae and/or phytoplankton in the surface water. The polymers in this dissolved organic pool assembled faster and with higher microgel yields than at other latitudes. The reversible phase transitions shown by these Arctic marine gels, as a function of pH, dimethylsulfide, and dimethylsulfoniopropionate concentrations, stimulate the gels to attain sizes below 1 μm in diameter. These marine gels were identified with an antibody probe specific toward material from the surface waters, sized, and quantified in airborne aerosol, fog, and cloud water, strongly suggesting that they dominate the available cloud condensation nuclei number population in the high Arctic (north of 80°N) during the summer season. Knowledge about emergent properties of marine gels provides important new insights into the processes controlling cloud formation and radiative forcing, and links the biology at the ocean surface with cloud properties and climate over the central Arctic Ocean and, probably, all oceans.

FLAVODOXIN EXPRESSION AS AN INDICATOR OF IRON LIMITATION IN MARINE DIATOMS1

We have prevously shown that a marine chlorophyte expressed flavodoxin under iron limitation but not under other nutrient stress conditions. Here we use polyclonal antiserum raised against the diatom Phaeodactylum tricornutum Bohlin to show that a similar response is observed in this species. Using our antibody, western blotting techniques, and standard colorimetric detection (4‐chloro‐1‐naphthol), we can detect at least a 25–50‐fold increase in flavodoxin in iron‐depleted compared to iron‐replete cells. In iron‐limited batch cultures of P. tricornulum, flavodoxin accumulation was inversely proportional to growth rate and was not detectable in cultures containing initially more than 750 nm of iron. We demonstrated that the accumulation of flavodoxin under iron stress is widespread among marine diatoms and that it may be possible to use the presence or absence of flavodoxin in natural marine diatom assemblages to detect iron limitation. However, our polyclonal antisera appears to be specific for diatoms and did not cross‐react with Synechococcus sp., Micromonas pusilla (Butcher) Manton et Parke, Dunaliella tertiolecta Butcher, Chlorella sp., Emiliania huxleii (Lohm.) Hay et Parke, or Isochrysis galbana Parke. A reverse bioassay experiment was conducted with natural phytoplankton assemblages containing mainly diatoms from Long Island Sound and in shelf waters near Cape Hatteras, two areas not suspected to be iron‐limited. Although flavodoxin was not detected in situ in these areas, natural populations of diatoms driven into iron limitation expres.sed flavodoxin. Flavodoxin was detected in mats of the diatom Rhizosolenia castracanei Cleve collected from the Equatorial Pacific during a JGOFS cruise in 1992, consistent with the hypothesis that iron may be limiting in this high‐nutrient, low‐chlorophyll region.

Genome-wide diel growth state transitions in the diatom Thalassiosira pseudonana

Marine diatoms are important primary producers that thrive in diverse and dynamic environments. They do so, in theory, by sensing changing conditions and adapting their physiology accordingly. Using the model species Thalassiosira pseudonana, we conducted a detailed physiological and transcriptomic survey to measure the recurrent transcriptional changes that characterize typical diatom growth in batch culture. Roughly 40% of the transcriptome varied significantly and recurrently, reflecting large, reproducible cell-state transitions between four principal states: (i) “dawn,” following 12 h of darkness; (ii) “dusk,” following 12 h of light; (iii) exponential growth and nutrient repletion; and (iv) stationary phase and nutrient depletion. Increases in expression of thousands of genes at the end of the reoccurring dark periods (dawn), including those involved in photosynthesis (e.g., ribulose-1,5-bisphosphate carboxylase oxygenase genes rbcS and rbcL), imply large-scale anticipatory circadian mechanisms at the level of gene regulation. Repeated shifts in the transcript levels of hundreds of genes encoding sensory, signaling, and regulatory functions accompanied the four cell-state transitions, providing a preliminary map of the highly coordinated gene regulatory program under varying conditions. Several putative light sensing and signaling proteins were associated with recurrent diel transitions, suggesting that these genes may be involved in light-sensitive and circadian regulation of cell state. These results begin to explain, in comprehensive detail, how the diatom gene regulatory program operates under varying environmental conditions. Detailed knowledge of this dynamic molecular process will be invaluable for new hypothesis generation and the interpretation of genetic, environmental, and metatranscriptomic data from field studies.

Marine biopolymer self-assembly: implications for carbon cycling in the ocean

Dissolved organic matter is the largest reservoir of reduced carbon in the ocean and is primarily composed of small biopolymers. It is a critical substrate for the microbial community and plays a pivotal role in global carbon cycling.

OPTIMIZATION OF AN IMMUNOFLUORESCENT ASSAY OF THE INTERNAL ENZYME RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE (RUBISCO) IN SINGLE PHYTOPLANKTON CELLS1

Immunochemical probes are widely used to identih different species and to quantify and understand the role that different antigens play within cells. We optimized a single‐cell immunofluorescent assay for the carbon fixation enzyme ribulose‐1,5‐bisphosphate carboxylase (Rubisco) in order to quantify the enzyme by flow cytometry in phytoplankton cells. The criteria for optimization of the immunofluorescent assay for Rubisco in single cells included maximization of Rubisco immunogenicity, minimization of Rubisco diffusion out of the cells, minimization of cell breakage, and maximization of the cell labeling. Several fixatives (cross‐linkers and denaturing) and permeabilizing agents were tested on 26 species of phytoplankton. The only fixative / permeabilizing agent that fulfilled the criteria established for the assay was 96% ethanol. Phytoplankton cells collected from the field needed further treatment with a strong oxidant to permeabilize ethanolfixed cells and thus allow the antibody probe to access the Rubisco antigen. This study should have a general applicability to the study of other soluble photosynthetic antigens in single phytoplankton cells.

DIMETHYLSULFONIOPROPIONATE STORAGE IN PHAEOCYSTIS (PRYMNESIOPHYCEAE) SECRETORY VESICLES1

Despite the global importance of dimethylsulfoniopropionate (DMSP)/dimethyl sulfide (DMS) and their role in climate regulation, little is known about the mechanisms of their production and storage in Phaeocystis sp., a major contributor of DMS in polar areas. Phaeocystis secretes polymer microgels, by regulated exocytosis, remaining in condensed phase while stored in secretory vesicles ( Chin et al. 2004 ). In secretory cells, vesicles also store small molecules, which are released during exocytosis. Here, we demonstrated that DMSP and DMS were stored in the secretory vesicles of Phaeocystis antarctica G. Karst. They were trapped within a polyanionic gel matrix, which prevented an accurate measurement of their concentration in the absence of a chelating agent such as EDTA. Understanding the production and the export mechanisms of DMSP and DMS into seawater is important because of the impact the cellular and extracellular pools of these highly relevant biogeochemical metabolites have on the environment. The pool of total DMSP in the presence of Phaeocystis may be underestimated by as much as half. Obtaining accurate budget measurements is the first step toward gaining a better understanding of key issues related to the DMS ocean–air interaction and the effect of phytoplankton DMS production on climate change.

Tracing the source and fate of biopolymers in seawater: application of an immunological technique

To investigate the complex degradation dynamics taking place in the water column, it is necessary to develop probes of high sensitivity and specificity that can identify the source and trace the fate of molecules in the dissolved and particulate pools using unconcentrated seawater. Here we present the application of an immunological probe to perform highly sensitive seawater analysis. We used exopolymers produced by the colonial bloom-forming alga, Phaeocystis (Prymnesiophyte), as a model to test the sensitivity and specificity of an enzyme-linked immunosorbent assay (ELISA) in unconcentrated seawater samples. We tested the ELISA with seawater from the Ross Sea, Gulf of Alaska and the North Water Polynya. We show that ELISA can readily be applied to quantify the concentration of Phaeocystis exopolymers and study their spatial and temporal distribution. With appropriate immunological probes, the ELISA can provide a powerful and highly specific method with nanomolar sensitivity to identify, quantify and trace the dynamics of complex moieties found in seawater.

CYTOCHEMICAL EVIDENCE FOR THE PRESENCE OF ACTIN IN THE NUCLEUS OF THE VOODOO LILY APPENDIX

Immunoflorescence microscopy of sections of the voodoo lily Sauromatum guttatum appendix stained with monoclonal antibodies against α-smooth muscle actin and cytoplasmic actin revealed different staining intensity of different parts of the cell. The anti-cytoplasmic-actin recognized antigens present mainly in the cytoplasm, and the anti-α-smooth muscle-actin recognized more intensively antigens present in the nuclei. A positive staining of the nucleus was also obtained with FITC–phalloidin confirming the presence of actin in its filamenous form in the nucleus. The presence of a nuclear α-smooth muscle-actin-like protein was further confirmed by confocal laser microscopy. On Western blots, the two anti-actins labelled a protein band that comigrated with standard actin at the approximate molecular weight of 43 kDa. Several other proteins interacted with the two antibodies to a different degree. The monoclonal antibodies against β-tubulin subunit stained only the periphery of the cytoplasm and anti-pan cytoplasmic myosin stained the cytoplasm weakly. On a Western blot, anti-β-tubulin subunit primarily recognized a protein band at the appropriate molecular weight of 50 kDa. This is the first cytochemical evidence for the presence of α-smooth muscle-actin-like protein in the plant nucleus.

UV-EXCITED BLUE AUTOFLUORESCENCE OF PSEUDO-NITZSCHIA MULTISERIES (BACILLARIOPHYCEAE)1

A flow cytometer coupled to a scanning monochromator and a fluorescence microscope were used to characterize the fluorescence spectrum of Pseudo‐nitzschia multiseries (Hasle) Hasle, a pennate diatom that produces the neurotoxin domoic acid, a lethal amnesic. In this research, we characterize the fluorescence spectrum of P. multiseries in vivo over the wavelength range of 360 to 850 nm and show that this diatom autofluoresces blue when excited with UV light (350–365 nm). The autofluorescence characterization of Pseudo‐nitzschia may provide new methods for rapid in situ monitoring of diatom populations and reiterates the usefulness of flow cytometry in the analysis and study of marine phytoplankton.