Patterns and regulation of silicon accumulation in Synechococcus spp.

Six clones of the marine cyanobacterium Synechococcus, representing four major clades, were all found to contain significant amounts of silicon in culture. Growth rate was unaffected by silicic acid, Si(OH)4 , concentration between 1 and 120 µM suggesting that Synechococcus lacks an obligate need for silicon (Si). Strains contained two major pools of Si: an aqueous soluble and an aqueous insoluble pool. Soluble pool sizes correspond to estimated intracellular dissolved Si concentrations of 2-24 mM, which would be thermodynamically unstable implying the binding of intracellular soluble Si to organic ligands. The Si content of all clones was inversely related to growth rate and increased with higher [Si(OH)4 ] in the growth medium. Accumulation rates showed a unique bilinear response to increasing [Si(OH)4 ] from 1 to 500 µM with the rate of Si acquisition increasing abruptly between 80 and 100 µM Si(OH)4 . Although these linear responses imply some form of diffusion-mediated transport, Si uptake rates at low Si (~1 µM Si) were inhibited by orthophosphate, suggesting a role of phosphate transporters in Si acquisition. Theoretical calculations imply that observed Si acquisition rates are too rapid to be supported by lipid-solubility diffusion of Si through the plasmalemma; however, facilitated diffusion involving membrane protein channels may suffice. The data are used to construct a working model of the mechanisms governing the Si content and rate of Si acquisition in Synechococcus.

Effects of wetland plants on denitrification rates: a meta-analysis.

Human activity is accelerating changes in biotic communities worldwide. Predicting impacts of these changes on ecosystem services such as denitrification, a process that mitigates the consequences of nitrogen pollution, remains one of the most important challenges facing ecologists. Wetlands especially are valued as important sites of denitrification, and wetland plants are expected to have differing effects on denitrification. We present the results of a meta-analysis, conducted on 419 published estimates of denitrification in wetlands dominated by different plant species. Plants increased denitrification rates by 55% on average. This effect varied significantly among communities as defined by the dominant plant species, but surprisingly did not differ substantially among methods for measuring denitrification or among types of wetlands. We conclude that mechanistically linking functional plant traits to denitrification will be key to predicting the role of wetlands in nitrogen mitigation in a changing world.

Quantitative 3D elemental microtomography of Cyclotella meneghiniana at 400-nm resolution.

X-ray fluorescence tomography promises to map elemental distributions in unstained and unfixed biological specimens in three dimensions at high resolution and sensitivity, offering unparalleled insight in medical, biological, and environmental sciences. X-ray fluorescence tomography of biological specimens has been viewed as impractical-and perhaps even impossible for routine application-due to the large time required for scanning tomography and significant radiation dose delivered to the specimen during the imaging process. Here, we demonstrate submicron resolution X-ray fluorescence tomography of a whole unstained biological specimen, quantifying three-dimensional distributions of the elements Si, P, S, Cl, K, Ca, Mn, Fe, Cu, and Zn in the freshwater diatom Cyclotella meneghiniana with 400-nm resolution, improving the spatial resolution by over an order of magnitude. The resulting maps faithfully reproduce cellular structure revealing unexpected patterns that may elucidate the role of metals in diatom biology and of diatoms in global element cycles. With anticipated improvements in data acquisition and detector sensitivity, such measurements could become routine in the near future.

Luxury iron uptake and storage in pennate diatoms from the equatorial Pacific Ocean.

Iron is a key micronutrient for ocean phytoplankton, and the availability of iron controls primary production and community composition in large regions of the ocean. Pennate diatoms, a phytoplankton group that responds to iron additions in low-iron areas, can have highly variable iron contents, and some groups such as Pseudo-nitzschia, are known to use ferritin to store iron for later use. We quantified and mapped the intracellular accumulation of iron by a natural population of Pseudo-nitzschia from the Fe-limited equatorial Pacific Ocean. A total of 48 h after iron addition, nearly half of the accumulated iron was localized in storage bodies adjacent to chloroplasts believed to represent ferritin. Over the subsequent 48 h, stored iron was distributed to the rest of the cell through subsequent growth and division, partially supporting the iron contents of the daughter cells. This study provides the first quantitative view into the cellular trafficking of iron in a globally relevant phytoplankton group and demonstrates the unique capabilities of synchrotron-based element imaging approaches.

Quantification of phosphorus in single cells using synchrotron X-ray fluorescence.

Phosphorus is required for numerous cellular compounds and as a result can serve as a useful proxy for total cell biomass in studies of cell elemental composition. Single-cell analysis by synchrotron X-ray fluorescence (SXRF) enables quantitative and qualitative analyses of cell elemental composition with high elemental sensitivity. Element standards are required to convert measured X-ray fluorescence intensities into element concentrations, but few appropriate standards are available, particularly for the biologically important element P. Empirical P conversion factors derived from other elements contained in certified thin-film standards were used to quantify P in the model diatom Thalassiosira pseudonana, and the measured cell quotas were compared with those measured in bulk by spectrophotometry. The mean cellular P quotas quantified with SXRF for cells on Au, Ni and nylon grids using this approach were not significantly different from each other or from those measured spectrophotometrically. Inter-cell variability typical of cell populations was observed. Additionally, the grid substrates were compared for their suitability to P quantification based on the potential for spectral interferences with P. Nylon grids were found to have the lowest background concentrations and limits of detection for P, while background concentrations in Ni and Au grids were 1.8- and 6.3-fold higher. The advantages and disadvantages of each grid type for elemental analysis of individual phytoplankton cells are discussed.

Lakes as sentinels of climate change.

While there is a general sense that lakes can act as sentinels of climate change, their efficacy has not been thoroughly analyzed. We identified the key response variables within a lake that act as indicators of the effects of climate change on both the lake and the catchment. These variables reflect a wide range of physical, chemical, and biological responses to climate. However, the efficacy of the different indicators is affected by regional response to climate change, characteristics of the catchment, and lake mixing regimes. Thus, particular indicators or combinations of indicators are more effective for different lake types and geographic regions. The extraction of climate signals can be further complicated by the influence of other environmental changes, such as eutrophication or acidification, and the equivalent reverse phenomena, in addition to other land-use influences. In many cases, however, confounding factors can be addressed through analytical tools such as detrending or filtering. Lakes are effective sentinels for climate change because they are sensitive to climate, respond rapidly to change, and integrate information about changes in the catchment.

Effects of Invasive-Plant Management on Nitrogen-Removal Services in Freshwater Tidal Marshes.

Establishing relationships between biodiversity and ecosystem function is an ongoing endeavor in contemporary ecosystem and community ecology, with important practical implications for conservation and the maintenance of ecosystem services. Removal of invasive plant species to conserve native diversity is a common management objective in many ecosystems, including wetlands. However, substantial changes in plant community composition have the potential to alter sediment characteristics and ecosystem services, including permanent removal of nitrogen from these systems via microbial denitrification. A balanced assessment of costs associated with keeping and removing invasive plants is needed to manage simultaneously for biodiversity and pollution targets. We monitored small-scale removals of Phragmites australis over four years to determine their effects on potential denitrification rates relative to three untreated Phragmites sites and adjacent sites dominated by native Typha angustifolia. Sediment ammonium increased following the removal of vegetation from treated sites, likely as a result of decreases in both plant uptake and nitrification. Denitrification potentials were lower in removal sites relative to untreated Phragmites sites, a pattern that persisted at least two years following removal as native plant species began to re-colonize treated sites. These results suggest the potential for a trade-off between invasive-plant management and nitrogen-removal services. A balanced assessment of costs associated with keeping versus removing invasive plants is needed to adequately manage simultaneously for biodiversity and pollution targets.

Microplankton trace element contents: implications for mineral limitation of mesozooplankton in an HNLC area.

Mesozooplankton production in high-nutrient low-chlorophyll regions of the ocean may be reduced if the trace element concentrations in their food are insufficient to meet growth and metabolic demands. We used elemental microanalysis (SXRF) of single-celled plankton to determine their trace metal contents during a series of semi-Lagrangian drift studies in an HNLC upwelling region, the Costa Rica Dome (CRD). Cells from the surface mixed layer had lower Fe:S but higher Zn:S and Ni:S than those from the subsurface chlorophyll maximum at 22-30 m. Diatom Fe:S values were typically 3-fold higher than those in flagellated cells. The ratios of Zn:C in flagellates and diatoms were generally similar to each other, and to co-occurring mesozooplankton. Estimated Fe:C ratios in flagellates were lower than those in co-occurring mesozooplankton, sometimes by more than 3-fold. In contrast, Fe:C in diatoms was typically similar to that in zooplankton. RNA:DNA ratios in the CRD were low compared with other regions, and were related to total autotrophic biomass and weakly to the discrepancy between Zn:C in flagellated cells and mesozooplankton tissues. Mesozooplankton may have been affected by the trace element content of their food, even though trace metal limitation of phytoplankton was modest at best.

Factors affecting Fe and Zn contents of mesozooplankton from the Costa Rica Dome.

Mineral limitation of mesozooplankton production is possible in waters with low trace metal availability. As a step toward estimating mesozooplankton Fe and Zn requirements under such conditions, we measured tissue concentrations of major and trace nutrient elements within size-fractioned zooplankton samples collected in and around the Costa Rica Upwelling Dome, a region where phytoplankton growth may be co-limited by Zn and Fe. The geometric mean C, N, P contents were 27, 5.6 and 0.21 mmol gdw-1, respectively. The values for Fe and Zn were 1230 and 498 nmol gdw-1, respectively, which are low compared with previous measurements. Migrant zooplankton caused C and P contents of the 2-5 mm fraction to increase at night relative to the day while the Fe and Zn contents decreased. Fe content increased with size while Zn content decreased with size. Fe content was strongly correlated to concentrations of two lithogenic tracers, Al and Ti. We estimate minimum Fe:C ratios in large migrant and resident mixed layer zooplankton to be 15 and 60 µmol mol-1, respectively. The ratio of Zn:C ranged from 11 µmol mol-1 for the 0.2-0.5 mm size fraction to 33 µmol mol-1 for the 2-5 mm size fraction.

The trace metal composition of marine phytoplankton.

Trace metals are required for numerous processes in phytoplankton and can influence the growth and structure of natural phytoplankton communities. The metal contents of phytoplankton reflect biochemical demands as well as environmental availability and influence the distribution of metals in the ocean. Metal quotas of natural populations can be assessed from analyses of individual cells or bulk particle assemblages or inferred from ratios of dissolved metals and macronutrients in the water column. Here, we review the available data from these approaches for temperate, equatorial, and Antarctic waters in the Pacific and Atlantic Oceans. The data show a generalized metal abundance ranking of Fe≈Zn>Mn≈Ni≈Cu≫Co≈Cd; however, there are notable differences between taxa and regions that inform our understanding of ocean metal biogeochemistry. Differences in the quotas estimated by the various techniques also provide information on metal behavior. Therefore, valuable information is lost when a single metal stoichiometry is assumed for all phytoplankton.

Zernike phase contrast in scanning microscopy with X-rays.

Scanning X-ray microscopy focuses radiation to a small spot and probes the sample by raster scanning. It allows information to be obtained from secondary signals such as X-ray fluorescence, which yields an elemental mapping of the sample not available in full-field imaging. The analysis and interpretation from these secondary signals can be considerably enhanced if these data are coupled with structural information from transmission imaging. However, absorption often is negligible and phase contrast has not been easily available. Originally introduced with visible light, Zernike phase contrast(1) is a well-established technique in full-field X-ray microscopes for visualization of weakly absorbing samples(2-7). On the basis of reciprocity, we demonstrate the implementation of Zernike phase contrast in scanning X-ray microscopy, revealing structural detail simultaneously with hard-X-ray trace-element measurements. The method is straightforward to implement without significant influence on the resolution of the fluorescence images and delivers complementary information. We show images of biological specimens that clearly demonstrate the advantage of correlating morphology with elemental information.

Modeling the effect of temperature on bioaccumulation of metals by a marine bioindicator organism, Mytilus edulis.

To determine whether regional or seasonal variability in water temperatures might affect the bioaccumulation of metals by marine invertebrates, we used a biokinetic/bioenergetic approach to model metal bioaccumulation (Ag, Am, Cd, Co, Se, and Zn) from dietary and dissolved sources by blue mussels, Mytilus edulis, acclimated at 2 and 12 degrees C. Accumulation of metal from the aqueous phase was not affected by temperature. However, Ag, Am, and Zn from diet were respectively accumulated up to 5.7-fold, 5.3-fold, and 2-fold more effectively at 2 degrees C than at 12 degrees C, largely because these three metals were assimilated from food more effectively at the lower temperature. In contrast, bioaccumulation of Cd, Co, Se from diet was not substantially affected by temperature even though efflux constants for these metals were up to 4-fold lower at 2 degrees C than at 12 degrees C. Total bioaccumulation of Ag, Am, and Zn was up to 1.7-3.6-fold higher at 2 degrees C than at 12 degrees C, with the largest differences predicted for high food conditions. Temperature-related variability in bioaccumulation of metals should be considered when interpreting patterns in metal tissue concentrations and when adapting management strategies developed for temperate seas to polar areas.

Exploring ocean biogeochemistry by single-cell microprobe analysis of protist elemental composition.

The biogeochemical cycles of many elements in the ocean are linked by their simultaneous incorporation into protists. In order to understand these elemental interactions and their implications for global biogeochemical cycles, accurate measures of cellular element stoichiometries are needed. Bulk analysis of size-fractionated particulate material obscures the unique biogeochemical roles of different functional groups such as diatoms, calcifying protists, and diazotrophs. Elemental analysis of individual protist cells can be performed using electron, proton, and synchrotron X-ray microprobes. Here we review the capabilities and limitations of each approach and the application of these advanced techniques to cells collected from natural communities. Particular attention is paid to recent studies of plankton biogeochemistry in low-iron waters of the Southern Ocean. Single-cell analyses have revealed significant inter-taxa differences in phosphorus, iron, and nickel quotas. Differences in the response of autotrophs and heterotrophs to iron fertilization were also observed. Two-dimensional sub-cellular mapping indicates the importance of iron to photosynthetic machinery and of zinc to nuclear organelles. Observed changes in diatom silicification and cytoplasm content following iron fertilization modify our understanding of the relationship between iron availability and silicification. These examples demonstrate the advantages of studying ocean biogeochemistry at the level of individual cells.

Quantifying trace elements in individual aquatic protist cells with a synchrotron X-ray fluorescence microprobe.

The study of trace metal cycling by aquatic protists is limited by current analytical techniques. Standard "bulk" element analysis techniques that rely on physical separations to concentrate cells for analysis cannot separate cells from co-occurring detrital material or other cells of differing taxonomy or trophic function. Here we demonstrate the ability of a synchrotron-based X-ray fluorescence (SXRF) microprobe to quantify the elements Si, Mn, Fe, Ni, and Zn in individual aquatic protist cells. This technique distinguishes between different types of cells in an assemblage and between cells and other particulate matter. Under typical operating conditions, the minimum detection limits are 7.0 x 10(-16) mol microm(-2) for Si and between 5.0 x 10(-20) and 3.9 x 10(-19) mol microm(-2) for Mn, Fe, Ni, and Zn; this sensitivity is sufficient to detect these elements in cells from even the most pristine waters as demonstrated in phytoplankton cells collected from remote areas of the Southern Ocean. Replicate analyses of single cells produced variations of <5% for Si, Mn, Fe, and Zn and <10% for Ni. Comparative analyses of cultured phytoplankton cells generally show no significant differences in cellular metal concentrations measured with SXRF and standard bulk techniques (spectrophotometry and graphite furnace atomic absorption spectrometry). SXRF also produces two-dimensional maps of element distributions in cells, thereby providing information not available with other analytical approaches. This technique enables the accurate and precise measurement of trace metals in individual aquatic protists collected from natural environments.