John Cliff

Exploring the transfer of recent plant photosynthates to soil microbes: mycorrhizal pathway vs direct root exudation

Plants rapidly release photoassimilated carbon (C) to the soil via direct root exudation and associated mycorrhizal fungi, with both pathways promoting plant nutrient availability. This study aimed to explore these pathways from the roots vascular bundle to soil microbial communities. Using nanoscale secondary ion mass spectrometry (NanoSIMS) imaging and 13C-phospho- and neutral lipid fatty acids, we traced in-situ flows of recently photoassimilated C of 13CO2-exposed wheat (Triticum aestivum) through arbuscular mycorrhiza (AM) into root- and hyphae-associated soil microbial communities. Intraradical hyphae of AM fungi were significantly 13C-enriched compared to other root-cortex areas after 8 h of labelling. Immature fine root areas close to the root tip, where AM features were absent, showed signs of passive C loss and co-location of photoassimilates with nitrogen taken up from the soil solution. A significant and exclusively fresh proportion of 13C-photosynthates was delivered through the AM pathway and was utilised by different microbial groups compared to C directly released by roots. Our results indicate that a major release of recent photosynthates into soil leave plant roots via AM intraradical hyphae already upstream of passive root exudations. AM fungi may act as a rapid hub for translocating fresh plant C to soil microbes.

In situ mapping of nutrient uptake in the rhizosphere using nanoscale secondary ion mass spectrometry.

Plant roots and microorganisms interact and compete for nutrients within the rhizosphere, which is considered one of the most biologically complex systems on Earth. Unraveling the nitrogen (N) cycle is key to understanding and managing nutrient flows in terrestrial ecosystems, yet to date it has proved impossible to analyze and image N transfer in situ within such a complex system at a scale relevant to soil-microbe-plant interactions. Linking the physical heterogeneity of soil to biological processes marks a current frontier in plant and soil sciences. Here we present a new and widely applicable approach that allows imaging of the spatial and temporal dynamics of the stable isotope 15N assimilated within the rhizosphere. This approach allows visualization and measurement of nutrient resource capture between competing plant cells and microorganisms. For confirmation we show the correlative use of nanoscale secondary ion mass spectrometry, and transmission electron microscopy, to image differential partitioning of 15NH4+ between plant roots and native soil microbial communities at the submicron scale. It is shown that 15N compounds can be detected and imaged in situ in individual microorganisms in the soil matrix and intracellularly within the root. Nanoscale secondary ion mass spectrometry has potential to allow the study of assimilatory processes at the submicron level in a wide range of applications involving plants, microorganisms, and animals.

Pathways for Neoarchean pyrite formation constrained by mass-independent sulfur isotopes

It is generally thought that the sulfate reduction metabolism is ancient and would have been established well before the Neoarchean. It is puzzling, therefore, that the sulfur isotope record of the Neoarchean is characterized by a signal of atmospheric mass-independent chemistry rather than a strong overprint by sulfate reducers. Here, we present a study of the four sulfur isotopes obtained using secondary ion MS that seeks to reconcile a number of features seen in the Neoarchean sulfur isotope record. We suggest that Neoarchean ocean basins had two coexisting, significantly sized sulfur pools and that the pathways forming pyrite precursors played an important role in establishing how the isotopic characteristics of each of these pools was transferred to the sedimentary rock record. One of these pools is suggested to be a soluble (sulfate) pool, and the other pool (atmospherically derived elemental sulfur) is suggested to be largely insoluble and unreactive until it reacts with hydrogen sulfide. We suggest that the relative contributions of these pools to the formation of pyrite depend on both the accumulation of the insoluble pool and the rate of sulfide production in the pyrite-forming environments. We also suggest that the existence of a significant nonsulfate pool of reactive sulfur has masked isotopic evidence for the widespread activity of sulfate reducers in the rock record.

Large sulfur isotope fractionations associated with Neoarchean microbial sulfate reduction

Dissecting ancient microbial sulfur cycling Before the rise of oxygen, life on Earth depended on the marine sulfur cycle. The fractionation of different sulfur isotopes provides clues to which biogeochemical cycles were active long ago (see the Perspective by Ueno). Zhelezinskaia et al. found negative isotope anomalies in Archean rocks from Brazil and posit that metabolic fluxes from sulfate-reducing microorganisms influenced the global sulfur cycle, including sulfur in the atmosphere. In contrast, Paris et al. found positive isotope anomalies in Archean sediments from South Africa, implying that the marine sulfate pool was more disconnected from atmospheric sulfur. As an analog for the Archean ocean, Crowe et al. measured sulfur isotope signatures in modern Lake Matano, Indonesia, and suggest that low seawater sulfate concentrations restricted early microbial activity. Science, this issue p. 703, p. 742, p. 739; see also p. 735 Sulfate-reducing bacteria in Earth’s earliest oceans likely influenced the global carbon cycle. [Also see Perspective by Ueno] The minor extent of sulfur isotope fractionation preserved in many Neoarchean sedimentary successions suggests that sulfate-reducing microorganisms played an insignificant role in ancient marine environments, despite evidence that these organisms evolved much earlier. We present bulk, microdrilled, and ion probe sulfur isotope data from carbonate-associated pyrite in the ~2.5-billion-year-old Batatal Formation of Brazil, revealing large mass-dependent fractionations (approaching 50 per mil) associated with microbial sulfate reduction, as well as consistently negative Δ33S values (~ –2 per mil) indicative of atmospheric photochemical reactions. Persistent 33S depletion through ~60 meters of shallow marine carbonate implies long-term stability of seawater sulfate abundance and isotope composition. In contrast, a negative Δ33S excursion in lower Batatal strata indicates a response time of ~40,000 to 150,000 years, suggesting Neoarchean sulfate concentrations between ~1 and 10 μM.

Visualising gold inside tumour cells following treatment with an antitumour gold(I) complex.

Gold(I) phosphine complexes, such as [Au(d2pype)(2)]Cl, (1, where d2pype is 1,2-bis(di-2-pyridyl phosphinoethane)), belong to a class of promising chemotherapeutic candidates that have been shown to be selectively toxic to tumourigenic cells, and may act via uptake into tumour cell mitochondria. For a more holistic understanding of their mechanism of action, a deeper knowledge of their subcellular distribution is required, but to date this has been limited by a lack of suitable imaging techniques. In this study the subcellular distribution of gold was visualised in situ in human breast cancer cells treated with 1, using nano-scale secondary ion mass spectrometry. NanoSIMS ion maps of (12)C(14)N(-), (31)P(-), (34)S(-) and (197)Au(-) allowed, for the first time, visualisation of cellular morphology simultaneously with subcellular distribution of gold. Energy filtered transmission electron microscopy (EFTEM) element maps for gold were also obtained, allowing for observation of nuclear and mitochondrial morphology with excellent spatial resolution, and gold element maps comparable to the data obtained with NanoSIMS. Following 2 h treatment with 1, the subcellular distribution of gold was associated with sulfur-rich regions in the nucleus and cytoplasm, supporting the growing evidence for the the mechanism of action of Au(I) compounds based on inhibition of thiol-containing protein families, such as the thioredoxin system. The combination of NanoSIMS and EFTEM has broader applicability for studying the subcellular distribution of other types of metal-based drugs.

Nanoscale analysis of pyritized microfossils reveals differential heterotrophic consumption in the ∼1.9-Ga Gunflint chert

The 1.88-Ga Gunflint biota is one of the most famous Precambrian microfossil lagerstätten and provides a key record of the biosphere at a time of changing oceanic redox structure and chemistry. Here, we report on pyritized replicas of the iconic autotrophic Gunflintia–Huroniospora microfossil assemblage from the Schreiber Locality, Canada, that help capture a view through multiple trophic levels in a Paleoproterozoic ecosystem. Nanoscale analysis of pyritic Gunflintia (sheaths) and Huroniospora (cysts) reveals differing relic carbon and nitrogen distributions caused by contrasting spectra of decay and pyritization between taxa, reflecting in part their primary organic compositions. In situ sulfur isotope measurements from individual microfossils (δ34SV-CDT +6.7‰ to +21.5‰) show that pyritization was mediated by sulfate-reducing microbes within sediment pore waters whose sulfate ion concentrations rapidly became depleted, owing to occlusion of pore space by coeval silicification. Three-dimensional nanotomography reveals additional pyritized biomaterial, including hollow, cellular epibionts and extracellular polymeric substances, showing a preference for attachment to Gunflintia over Huroniospora and interpreted as components of a saprophytic heterotrophic, decomposing community. This work also extends the record of remarkable biological preservation in pyrite back to the Paleoproterozoic and provides criteria to assess the authenticity of even older pyritized microstructures that may represent some of the earliest evidence for life on our planet.

Uncovering framboidal pyrite biogenicity using nano-scale CNorg mapping

Framboidal pyrite has been used as a paleo-redox proxy and a biomarker in ancient sediments, but the interpretation of pyrite framboids can be controversial, especially where later overgrowths have obscured primary textures. Here we show how nano-scale chemical mapping of organic carbon and nitrogen (CN org ) can detect relict framboids within Precambrian pyrite grains and determine their formation mechanism. Pyrite grains associated with an Ediacaran fossil Lagerstatte from Newfoundland (ca. 560 Ma) hold significance for our understanding of taphonomy and redox history of the earliest macrofossil assemblages. They show distinct chemical zoning with respect to CN org . Relict framboids are revealed as spheroidal zones within larger pyrite grains, whereby pure pyrite microcrystals are enclosed by a mesh-like matrix of pyrite possessing elevated CN org , replicating observations from framboids growing within modern biofilms. Subsequent pyrite overgrowths also incorporated CN org from biofilms, with concentric CN org zoning showing that the availability of CN org progressively decreased during later pyrite growth. Multiple framboids are commonly cemented together by these overgrowths to form larger grains, with relict framboids only detectable in CN org maps. In situ sulfur isotope data (δ 34 S = ∼−24‰ to −15‰) show that the source of sulfur for the pyrite was also biologically mediated, most likely via a sulfate-reducing microbial metabolism within the biofilms. Relict framboids have significantly smaller diameters than the pyrite grains that enclose them, suggesting that the use of framboid diameters to infer water column paleo-redox conditions should be approached with caution. This work shows that pyrite framboids have formed within organic biofilms for at least 560 m.y., and provides a novel methodology that could readily be extended to search for such biomarkers in older rocks and potentially on other planets.

Improved particle location and isotopic screening measurements of sub-micron sized particles by Secondary Ion Mass Spectrometry

There are a number of applications within cosmochemistry, environmental studies, nuclear safeguards and nuclear forensic analyses that require capabilities for the location and isotopic measurement of sub-micron to micron-sized particles. This task can be divided into two sub-tasks: the first problem is to find the particle of interest in a matrix of other materials and the second is to perform accurate and precise isotopic measurements of the individual particles. This paper describes results obtained on real and standard samples using a newly developed Automated Particle Measurement (APM) software, for both Small Geometry (SG) and Large Geometry (LG) Secondary Ion Mass Spectrometry (SIMS) instruments. The speed and quality of screening measurements, in particular on the LG-SIMS, are far better than previously available. This paper mainly focuses on the analyses of uranium particles for safeguards verification purposes, but the described method can also be used for other applications.

High-resolution secondary ion mass spectrometry analysis of carbon dynamics in mycorrhizas formed by an obligately myco-heterotrophic orchid.

Mycorrhiza formation represents a significant carbon (C) acquisition alternative for orchid species, particularly those that remain achlorophyllous through all life stages. As it is known that orchid mycorrhizas facilitate nutrient transfer (most notably of C), it has not been resolved if C transfer occurs only after lysis of mycorrhizal structures (fungal pelotons) or also across the mycorrhizal interface of pre-lysed pelotons. We used high-resolution secondary ion mass spectrometry (nanoSIMS) and labelling with enriched (13) CO2 to trace C transfers, at subcellular scale, across mycorrhizal interfaces formed by Rhizanthella gardneri, an achlorphyllous orchid. Carbon was successfully traced in to the fungal portion of orchid mycorrhizas. However, we did not detect C movement across intact mycorrhizal interfaces up to 216 h post (13) CO2 labelling. Our findings provide support for the hypothesis that C transfer from the mycorrhizal fungus to orchid, at least for R. gardneri, likely occurs after lysis of the fungal peloton.

Application of nanoscale secondary ion mass spectrometry to plant cell research

Imaging resource flow in soil-plant systems remains central to understanding plant development and interactions with the environment. Typically, subcellular resolution is required to fully elucidate the compartmentation, behavior, and mode of action of organic compounds and mineral elements within plants. For many situations this has been limited by the poor spatial resolution of imaging techniques and the inability to undertake studies in situ. Here we demonstrate the potential of Nanoscale Secondary Ion Mass Spectrometry (NanoSIMS), which is capable of the quantitative high-resolution spatial imaging of stable isotopes (e.g. 12C, 13C, 14N, 15N, 16O, 18O, 31P, 34S) within intact plant-microbial-soil systems. We present examples showing how the approach can be used to investigate competition for 15N-labeled nitrogen compounds between plant roots and soil microorganisms living in the rhizosphere and the spatial imaging of 31P in roots. We conclude that NanoSIMS has great potential to elucidate the flow of isotopically-labeled compounds in complex media (e.g. soil) and opens up countless new opportunities for studying plant responses to abiotic stress (e.g. 18O3, elevated 13CO2), signal exchange, nutrient flow and plant-microbial interactions.