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Living cells orchestrate enzyme activities to produce myriads of biopolymers but cell-biological understanding of such processes is scarce. Starch, a plant biopolymer forming discrete, semi-crystalline granules within plastids, plays a central role in glucose storage, which is fundamental to life. Combining complementary imaging techniques and Arabidopsis genetics we reveal that, in chloroplasts, multiple starch granules initiate in stromal pockets between thylakoid membranes. These initials coalesce, then grow anisotropically to form lenticular granules. The major starch polymer, amylopectin, is synthesized at the granule surface, while the minor amylose component is deposited internally. The non-enzymatic domain of STARCH SYNTHASE 4, which controls the protein’s localization, is required for anisotropic growth. These results present us with a conceptual framework for understanding the biosynthesis of this key nutrient. Starch is the major form of energy storage in plant cells and forms discrete, semi-crystalline granules within plastids. Here the authors use electron tomography and nanoSIMS to show that Arabidopsis starch granules initiate in stromal pockets between thylakoid membranes that coalesce before growing anisotropically.

Reef-building corals form essential, mutualistic endosymbiotic associations with photosynthetic Symbiodinium dinoflagellates, providing their animal host partner with photosynthetically derived nutrients that allow the coral to thrive in oligotrophic waters. However, little is known about the dynamics of these nutritional interactions at the (sub)cellular level. Here, we visualize with submicrometer spatial resolution the carbon and nitrogen fluxes in the intact coral-dinoflagellate association from the reef coral Pocillopora damicornis by combining nanoscale secondary ion mass spectrometry (NanoSIMS) and transmission electron microscopy with pulse-chase isotopic labeling using [ 13 C]bicarbonate and [ 15 N]nitrate. This allows us to observe that (i) through light-driven photosynthesis, dinoflagellates rapidly assimilate inorganic bicarbonate and nitrate, temporarily storing carbon within lipid droplets and starch granules for remobilization in nighttime, along with carbon and nitrogen incorporation into other subcellular compartments for dinoflagellate growth and maintenance, (ii) carbon-containing photosynthates are translocated to all four coral tissue layers, where they accumulate after only 15 min in coral lipid droplets from the oral gastroderm and within 6 h in glycogen granules from the oral epiderm, and (iii) the translocation of nitrogen-containing photosynthates is delayed by 3 h. IMPORTANCE   Our results provide detailed in situ subcellular visualization of the fate of photosynthesis-derived carbon and nitrogen in the coral-dinoflagellate endosymbiosis. We directly demonstrate that lipid droplets and glycogen granules in the coral tissue are sinks for translocated carbon photosynthates by dinoflagellates and confirm their key role in the trophic interactions within the coral-dinoflagellate association. Our results provide detailed in situ subcellular visualization of the fate of photosynthesis-derived carbon and nitrogen in the coral-dinoflagellate endosymbiosis. We directly demonstrate that lipid droplets and glycogen granules in the coral tissue are sinks for translocated carbon photosynthates by dinoflagellates and confirm their key role in the trophic interactions within the coral-dinoflagellate association.

Background The development of nanoscale secondary ion mass spectrometry (NanoSIMS) has revolutionized the study of biological tissues by enabling, e.g., the visualization and quantification of metabolic processes at subcellular length scales. However, the associated sample preparation methods all result in some degree of tissue morphology distortion and loss of soluble compounds. To overcome these limitations an entirely cryogenic sample preparation and imaging workflow is required. Results Here, we report the development of a CryoNanoSIMS instrument that can perform isotope imaging of both positive and negative secondary ions from flat block-face surfaces of vitrified biological tissues with a mass- and image resolution comparable to that of a conventional NanoSIMS. This capability is illustrated with nitrogen isotope as well as trace element mapping of freshwater hydrozoan Green Hydra tissue following uptake of 15 N-enriched ammonium. Conclusion With a cryo-workflow that includes vitrification by high pressure freezing, cryo-planing of the sample surface, and cryo-SEM imaging, the CryoNanoSIMS enables correlative ultrastructure and isotopic or elemental imaging of biological tissues in their most pristine post-mortem state. This opens new horizons in the study of fundamental processes at the tissue- and (sub)cellular level. Teaser CryoNanoSIMS: subcellular mapping of chemical and isotopic compositions of biological tissues in their most pristine post-mortem state.

Efficient nutrient recycling underpins the ecological success of cnidarian-algal symbioses in oligotrophic waters. In these symbioses, nitrogen limitation restricts the growth of algal endosymbionts in hospite and stimulates their release of photosynthates to the cnidarian host. However, the mechanisms controlling nitrogen availability and their role in symbiosis regulation remain poorly understood. Here, we studied the metabolic regulation of symbiotic nitrogen cycling in the sea anemone Aiptasia by experimentally altering labile carbon availability in a series of experiments. Combining 13 C and 15 N stable isotope labeling experiments with physiological analyses and NanoSIMS imaging, we show that the competition for environmental ammonium between the host and its algal symbionts is regulated by labile carbon availability. Light regimes optimal for algal photosynthesis increase carbon availability in the holobiont and stimulate nitrogen assimilation in the host metabolism. Consequently, algal symbiont densities are lowest under optimal environmental conditions and increase toward the lower and upper light tolerance limits of the symbiosis. This metabolic regulation promotes efficient carbon recycling in a stable symbiosis across a wide range of environmental conditions. Yet, the dependence on resource competition may favor parasitic interactions, explaining the instability of the cnidarian-algal symbiosis as environmental conditions in the Anthropocene shift towards its tolerance limits. Photosymbioses enable efficient nutrient recycling between heterotrophic and phototrophic organisms. This study shows that nutrient cycling in a cnidarian-algal symbiosis is regulated through resource competition between symbiotic partners. Mutualistic interactions can therefore emerge from mutual exploitation in nutrient–exchange symbioses.

Sponges are the oldest known extant animal-microbe symbiosis. These ubiquitous benthic animals play an important role in marine ecosystems in the cycling of dissolved organic matter (DOM), the largest source of organic matter on Earth. The conventional view on DOM cycling through microbial processing has been challenged by the interaction between this efficient filter-feeding host and its diverse and abundant microbiome. Here we quantify, for the first time, the role of host cells and microbial symbionts in sponge heterotrophy. We combined stable isotope probing and nanoscale secondary ion mass spectrometry to compare the processing of different sources of DOM (glucose, amino acids, algal-produced) and particulate organic matter (POM) by a high-microbial abundance (HMA) and low-microbial abundance (LMA) sponge with single-cell resolution. Contrary to common notion, we found that both microbial symbionts and host choanocyte (i.e. filter) cells and were active in DOM uptake. Although all DOM sources were assimilated by both sponges, higher microbial biomass in the HMA sponge corresponded to an increased capacity to process a greater variety of dissolved compounds. Nevertheless, in situ feeding data demonstrated that DOM was the primary carbon source for both the LMA and HMA sponge, accounting for ~90% of their heterotrophic diets. Microbes accounted for the majority (65–87%) of DOM assimilated by the HMA sponge (and ~60% of its total heterotrophic diet) but <5% in the LMA sponge. We propose that the evolutionary success of sponges is due to their different strategies to exploit the vast reservoir of DOM in the ocean.

Correlative light and electron microscopy allows localization of specific molecules at the ultrastructural level in biological tissue but does not provide information about metabolic turnover or the distribution of labile molecules, such as micronutrients. We present a method to directly correlate (immuno)fluorescent microscopy, (immuno)TEM imaging and NanoSIMS isotopic mapping of the same tissue section, with nanometer-scale spatial precision. The process involves chemical fixation of the tissue, cryo sectioning, thawing, and air-drying under a thin film of polyvinyl alcohol. It permits to effectively retain labile compounds and strongly increases NanoSIMS sensitivity for 13 C-enrichment. The method is illustrated here with correlated distribution maps of a carbonic anhydrase enzyme isotype, β-tubulin proteins, and 13 C- and 15 N-labeled labile micronutrients (and their anabolic derivates) within the tissue of a reef-building symbiotic coral. This broadly applicable workflow expands the wealth of information that can be obtained from multi-modal, sub-cellular observation of biological tissue. Loussert-Fonta et al. have developed a method to directly correlate fluorescent microscopy, (immuno)TEM imaging and NanoSIMS isotopic mapping of the same tissue section, with nanometer-scale spatial precision. They illustrate the technique by imaging tissue from a symbiotic coral, Stylophora pistillata .

Oxygen isotope compositions of fossil foraminifera tests are commonly used proxies for ocean paleotemperatures, with reconstructions spanning the last 112 million years. However, the isotopic composition of these calcitic tests can be substantially altered during diagenesis without discernible textural changes. Here, we investigate fluid-mediated isotopic exchange in pristine tests of three modern benthic foraminifera species ( Ammonia sp ., Haynesina germanica , and Amphistegina lessonii ) following immersion into an 18 O-enriched artificial seawater at 90 °C for hours to days. Reacted tests remain texturally pristine but their bulk oxygen isotope compositions reveal rapid and species-dependent isotopic exchange with the water. NanoSIMS imaging reveals the 3-dimensional intra-test distributions of 18 O-enrichment that correlates with test ultra-structure and associated organic matter. Image analysis is used to quantify species level differences in test ultrastructure, which explains the observed species-dependent rates of isotopic exchange. Consequently, even tests considered texturally pristine for paleo-climatic reconstruction purposes may have experienced substantial isotopic exchange; critical paleo-temperature record re-examination is warranted. Paleoclimate reconstructions commonly use oxygen isotope compositions from fossil foraminifera tests as proxies. Here, the authors show that these tests exchange O-isotopes with surrounding fluids, with implications for paleotemperature records.

The density of dinoflagellate microalgae in the tissue of symbiotic corals is an important determinant for health and productivity of the coral animal. Yet, the specific mechanism for their regulation and the consequence for coral nutrition are insufficiently understood due to past methodological limitations to resolve the fine-scale metabolic consequences of fluctuating densities. Here, we characterized the physiological and nutritional consequences of symbiont density variations on the colony and tissue level in Stylophora pistillata from the Red Sea. Alterations in symbiont photophysiology maintained coral productivity and host nutrition across a broad range of symbiont densities. However, we demonstrate that density-dependent nutrient competition between individual symbiont cells, manifested as reduced nitrogen assimilation and cell biomass, probably creates the negative feedback mechanism for symbiont population growth that ultimately defines the steady-state density. Despite fundamental changes in symbiont nitrogen assimilation, we found no density-related metabolic optimum beyond which host nutrient assimilation or tissue biomass declined, indicating that host nutrient demand is sufficiently met across the typically observed range of symbiont densities under ambient conditions.

Corals access inorganic seawater nutrients through their autotrophic endosymbiotic dinoflagellates, but also capture planktonic prey through heterotrophic feeding. Correlating NanoSIMS and TEM imaging, we visualized and quantified the subcellular fate of autotrophic and heterotrophic C and N in the coral Stylophora pistillata using stable isotopes. Six scenarios were compared after 6 h: autotrophic pulse ( 13 C-bicarbonate, 15 N-nitrate) in either unfed or regularly fed corals, and heterotrophic pulse ( 13 C-, 15 N-labelled brine shrimps) in regularly fed corals; each at ambient and elevated temperature. Host assimilation of photosynthates was similar under fed and unfed conditions, but symbionts assimilated 10% more C in fed corals. Photoautotrophic C was primarily channelled into host lipid bodies, whereas heterotrophic C and N were generally co-allocated to the tissue. Food-derived label was detected in some subcellular structures associated with the remobilisation of host lipid stores. While heterotrophic input generally exceeded autotrophic input, it was more negatively affected by elevated temperature. The reduced input from both modes of nutrition at elevated temperature was accompanied by a shift in the partitioning of C and N, benefiting epidermis and symbionts. This study provides a unique view into the nutrient partitioning in corals and highlights the tight connection of nutrient fluxes in symbiotic partners.

Increasing soil salinity causes significant crop losses globally; therefore, understanding plant responses to salt (sodium) stress is of high importance. Plants avoid sodium toxicity through subcellular compartmentation by intricate processes involving a high level of elemental interdependence. Current technologies to visualize sodium, in particular, together with other elements, are either indirect or lack in resolution. Here we used the newly developed cryo nanoscale secondary ion mass spectrometry ion microprobe 1 , which allows high-resolution elemental imaging of cryo-preserved samples and reveals the subcellular distributions of key macronutrients and micronutrients in root meristem cells of Arabidopsis and rice. We found an unexpected, concentration-dependent change in sodium distribution, switching from sodium accumulation in the cell walls at low external sodium concentrations to vacuolar accumulation at stressful concentrations. We conclude that, in root meristems, a key function of the NHX family sodium/proton antiporter SALT OVERLY SENSITIVE 1 (also known as Na + /H + exchanger 7; SOS1/NHX7) is to sequester sodium into vacuoles, rather than extrusion of sodium into the extracellular space. This is corroborated by the use of new genomic, complementing fluorescently tagged SOS1 variants. We show that, in addition to the plasma membrane, SOS1 strongly accumulates at late endosome/prevacuoles as well as vacuoles, supporting a role of SOS1 in vacuolar sodium sequestration. This study demonstrates that cryo nanoscale secondary ion mass spectrometry (CryoNanoSIMS) enables direct multi-elemental imaging at subcellular resolution of macro- and micronutrients or trace elements in plants and may provide insights into the in vivo roles of many transporters.