Quantifying inorganic nitrogen assimilation by synechococcus using bulk and single-cell mass spectrometry: A comparative study

Copyright © 2018 Giardina, Cheong, Marjo, Clode, Guagliardo, Pickford, Pernice, Seymour and Raina. Microorganisms drive most of the major biogeochemical cycles in the ocean, but the rates at which individual species assimilate and transform key elements is generally poorly quantified. One of these important elements is nitrogen, with its availability limiting primary production across a large proportion of the ocean. Nitrogen uptake by marine microbes is typically quantified using bulk-scale approaches, such as Elemental Analyzer-Isotope Ratio Mass Spectrometry (EA-IRMS), which averages uptake over entire communities, masking microbial heterogeneity. However, more recent techniques, such as secondary ion mass spectrometry (SIMS), allow for elucidation of assimilation rates at the scale at which they occur: the single-cell level. Here, we combine and compare the application of bulk (EA-IRMS) and single-cell approaches (NanoSIMS and Time-of-Flight-SIMS) for quantifying the assimilation of inorganic nitrogen by the ubiquitous marine primary producer Synechococcus. We aimed to contrast the advantages and disadvantages of these techniques and showcase their complementarity. Our results show that the average assimilation of 15N by Synechococcus differed based on the technique used: values derived from EA-IRMS were consistently higher than those derived from SIMS, likely due to a combination of previously reported systematic depletion as well as differences in sample preparation. However, single-cell approaches offered additional layers of information, whereby NanoSIMS allowed for the quantification of the metabolic heterogeneity among individual cells and ToF-SIMS enabled identification of nitrogen assimilation into peptides. We suggest that this coupling of stable isotope-based approaches has great potential to elucidate the metabolic capacity and heterogeneity of microbial cells in natural environments

Soil microbial responses to biochars varying in particle size, surface and pore properties

Biochars are known for their heterogeneity, especially in pore and surface structure associated with pyrolysis processes and sources of feedstocks. The surface area of biochar is likely to be an important determinant of the extent of soil microbial attachment, whereas the porous structure of biochar is expected to provide protection for soil microorganisms. Potential interactions between biochars from different sources and with different particle sizes were investigated in relation to soil microbial properties in a short-term incubation study. Three particle size (sieved) fractions (0.5–1.0, 1.0–2.0 and 2.0–4.0 mm) from three woody biochars produced from jarrah wood, jarrah and wandoo wood and Australian wattle branches, respectively, were incubated in soil at 25 °C for 56 d. Observation by scanning electron microscopy (SEM) and characterisation of pore and surface area showed that all three woody biochars provided potential habitats for soil microorganisms due to their high porosity and surface areas. The biochars were structurally heterogeneous, varying in porosity and surface structure both within and between the biochar sources. After the 56-d incubation, hyphal colonisation was observed on biochar surfaces and in larger biochar pores. Soil clumping occurred on biochar particles, cementing and covering exposed biochar pores. This may have altered surface area and pore availability for microbial colonisation. Transient changes in soil microbial biomass, without a consistent trend, were observed among biochars during the 56-d incubation

Sediment characteristics influence the fertilisation success of the corals Acropora tenuis and Acropora millepora

Elevated suspended sediment concentrations (SSCs) often impact coral fertilisation success, but sediment composition can influence effect thresholds, which is problematic for accurately predicting risk. Here, we derived concentration–response thresholds and cause-effect pathways for SSCs comprising a range of realistic mineral and organic compositions on coral fertilisation success. Effect concentration thresholds (EC10: 10% fertilisation inhibition) varied markedly, with fertilisation highly sensitive to inshore organic-clay rich sediments and bentonite clay at 40 mg L−1). The effect thresholds for relevant sediment types were combined with in situ turbidity data from locations near dredging operations to assess the risks posed by dredging to coral fertilisation at these locations

Trait convergence in photosynthetic nutrient-use efficiency along a 2-million year dune chronosequence in a global biodiversity hotspot

The Jurien Bay dune chronosequence in south-western Australia's biodiversity hotspot comprises sites differing in nutrient availability, with phosphorus (P) availability declining strongly with increasing soil age. We have explored the exceptionally high photosynthetic Pause efficiency (PPUE) of Proteaceae in this region, triggering the question what the PPUE of co-occurring species in other families might be along the Jurien Bay chronosequence. We explored how traits associated with PPUE, photosynthetic nitrogen (N)-use efficiency (PNUE) and leaf respiration might converge along the chronosequence, and whether Proteaceae and non-Proteaceae species differ in leaf traits associated with nutrient use. Seven to 10 species were sampled at three sites differing in nutrient availability (ranging from N- to P- limited). Measurements of leaf light-saturated photosynthesis and dark respiration were integrated with measurements of total N and P concentration in both mature and senesced leaves, and leaf mass per unit area (LMA). Contrary to what is known for other chronosequences, rates of photosynthesis and respiration did not decrease with increasing soil age and LMA along the Jurien Bay chronosequence. However, they increased when expressed per unit leaf P. Both N and P were used much more efficiently for photosynthesis on nutrient-poor sites, in both Proteaceae and non-Proteaceae species. Proteaceae had the fastest rate of photosynthesis per unit leaf P, followed by species that preferentially allocate P to mesophyll cells, rather than epidermal cells. Synthesis. Our results show that with declining soil P availability, photosynthetic P-use efficiency of all investigated species from different families increased. Plants growing on the oldest, most nutrient-impoverished soils exhibited similar rates of CO2 exchange as plants growing on more nutrient-rich younger soils, and extraordinarily high photosynthetic P-use efficiency. This indicates convergence in leaf traits related to photosynthetic nutrient use on severely P-impoverished sites.Australian Research Council, Grant/Award Number: CE140100008, DP130100005, DP0985685, DP110101120 and DP0986823; University of Western Australi

First cryo-scanning electron microscopy images and X-ray microanalyses of mucoromycotinian fine root endophytes in vascular plants

Aims. Arbuscule-producing fine root endophytes (FRE) (previously incorrectly Glomus tenue) were recently placed within subphylum Mucoromycotina; the first report of arbuscules outside subphylum Glomeromycotina. Here, we aimed to estimate nutrient concentrations in plant and fungal structures of FRE and to test the utility of cryo-scanning electron microscopy (cryoSEM) for studying these fungi. Methods. To do so, we used replicated cryoSEM and X-ray microanalysis of heavily colonized roots of Trifolium subterraneum. Results. Intercellular hyphae and hyphae in developed arbuscules were consistently very thin; 1.35 ± 0.03 µm and 0.99 ± 0.03 µm in diameter, respectively (mean ± SE). Several intercellular hyphae were often adjacent to each other forming ‘hyphal ropes’. Developed arbuscules showed higher phosphorus concentrations than senesced arbuscules and non-colonized structures. Senesced arbuscules showed greatly elevated concentrations of calcium and magnesium. Conclusions. While uniformly thin hyphae and hyphal ropes are distinct features of FRE, the morphology of fully developed arbuscules, elevated phosphorus in fungal structures, and accumulation of calcium with loss of structural integrity in senesced arbuscules are similar to glomeromycotinian fungi. Thus, we provide evidence that FRE may respond to similar host-plant signals or that the host plant may employ a similar mechanism of association with FRE and AMF

Angiosperm symbioses with non-mycorrhizal fungal partners enhance N acquisition from ancient organic matter in a warming maritime Antarctic

In contrast to the situation in plants inhabiting most of the world’s ecosystems, mycorrhizal fungi are usually absent from roots of the only two native vascular plant species of maritime Antarctica, Deschampsia antarctica and Colobanthus quitensis. Instead, a range of ascomycete fungi, termed dark septate endophytes (DSEs), frequently colonise the roots of these plant species. We demonstrate that colonisation of Antarctic vascular plants by DSEs facilitates not only the acquisition of organic nitrogen as early protein breakdown products, but also as non-proteinaceous D-amino acids and their short peptides, accumulated in slowly-decomposing organic matter, such as moss peat. Our findings suggest that, in a warming maritime Antarctic, this symbiosis has a key role in accelerating the replacement of formerly dominant moss communities by vascular plants, and in increasing the rate at which ancient carbon stores laid down as moss peat over centuries or millennia are returned to the atmosphere as CO2.Additional co-authors: Richard D Bardgett, David W Hopkins and Davey L Jone

Subcellular tracking reveals the location of dimethylsulfoniopropionate in microalgae and visualises its uptake by marine bacteria

Phytoplankton-bacteria interactions drive the surface ocean sulfur cycle and local climatic processes through the production and exchange of a key compound: dimethylsulfoniopropionate (DMSP). Despite their large-scale implications, these interactions remain unquantified at the cellular-scale. Here we use secondary-ion mass spectrometry to provide the first visualization of DMSP at sub-cellular levels, tracking the fate of a stable sulfur isotope (34S) from its incorporation by microalgae as inorganic sulfate to its biosynthesis and exudation as DMSP, and finally its uptake and degradation by bacteria. Our results identify for the first time the storage locations of DMSP in microalgae, with high enrichments present in vacuoles, cytoplasm and chloroplasts. In addition, we quantify DMSP incorporation at the single-cell level, with DMSP-degrading bacteria containing seven times more 34S than the control strain. This study provides an unprecedented methodology to label, retain, and image small diffusible molecules, which can be transposable to other symbiotic systems.This work was supported by ANNiMS (Australian Government, Department of Education, Employment and Workplace Relations), the AMMRF Centre for Microscopy, Characterisation and Analysis (UWA) and by Australian Research Council Grant DE160100636