Matt R. Kilburn

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.

Two coexisting sulfur metabolisms in a ca. 3400 Ma sandstone

A sandstone from the ca. 3400 Ma Strelley Pool Formation of Western Australia contains pristine micron-sized pyrite intimately associated with organic material coating framework quartz grains. A synsedimentary to early diagenetic origin for this pyrite is indicated by its occurrence in black, bedded sandstone at the base of the formation, and in reworked black clasts higher up in the formation. High-resolution multiple sulfur isotope analysis (32S, 33S, 34S) using secondary ion mass spectrometry (NanoSIMS and large-radius ion microprobe) reveals δ34SVCDT (Vienna Canyon Diablo troilite) values between ∼–12‰ and +6‰, and Δ33S values between –1.65‰ and +1.43‰, from pyrite grains within a single thin section. A large spread of δ34S values over only 5–10 μm, together with the spatial association of pyrite with carbon and nitrogen, indicates biological processing of sulfur. The presence of both +Δ33S and –Δ33S signals overprinted by significant mass-dependent δ34S fractionation in this pyrite population indicates for the first time that both microbial sulfate reduction of aqueous sulfate (–Δ33S) and microbial disproportionation of elemental sulfur (+Δ33S) were co-occurring in an open-marine, sedimentary hosted ecosystem in the Paleoarchean.

Use of NanoSIMS in the search for early life on Earth: ambient inclusion trails in a c. 3400 Ma sandstone

Ambient inclusion trails (AIT) are enigmatic microtubular structures created by the migration of mineral crystals through a lithified substrate. The decomposition of organic material has been suggested as the driving force for the crystal migration, but has yet to be rigorously tested. AIT may hold potential as a biosignature for investigating early life on Earth if the associated organic material can be shown to be biological. This paper attempts to test the formation mechanism and biogenicity of AIT from the c. 3400 Ma Strelley Pool sandstone of Western Australia using NanoSIMS technology. In doing so, we demonstrate the unique ability of the NanoSIMS to combine sub-micron scale imaging with in situ chemical and isotopic data, thereby enhancing our ability to evaluate the biogenicity criteria for Archaean microstructures. Enrichments of a suite of major elements (C, N, P, S) and trace elements (Co, Fe, Ni, Zn), often associated with biological processes, are found within several AIT in this sandstone. C and N enrichments are most common along AIT margins, and correlate with depletions of Si, O, Ca and Mg, indicating that this material is indeed organic in nature. δ13C values of this carbonaceous material average −26‰. Petrographic observations show that some of the AIT occur in the centre of detrital sandstone grains where they were sealed from later fluid flow and therefore preserve primary Archaean (bio)geochemistry. In contrast, AIT found around the outer edges of sandstone grains may contain more recent organic material introduced by later fluid migration and are an unreliable biosignature. Using the petrographic and geochemical data a multi-stage model for AIT formation and subsequent diagenetic modification is proposed. The possible sources of the primary organic material are discussed and we conclude that the data are consistent with a biological origin for these AIT. An abiogenic origin is more difficult to sustain but cannot yet be completely excluded for AIT in general.

Competition between plant and bacterial cells at the microscale regulates the dynamics of nitrogen acquisition in wheat (Triticum aestivum)

The ability of plants to compete effectively for nitrogen (N) resources is critical to plant survival. However, controversy surrounds the importance of organic and inorganic sources of N in plant nutrition because of our poor ability to visualize and understand processes happening at the root–microbial–soil interface. Using high-resolution nano-scale secondary ion mass spectrometry stable isotope imaging (NanoSIMS-SII), we quantified the fate of 15N over both space and time within the rhizosphere. We pulse-labelled the soil surrounding wheat (Triticum aestivum) roots with either or 15N-glutamate and traced the movement of 15N over 24 h. Imaging revealed that glutamate was rapidly depleted from the rhizosphere and that most 15N was captured by rhizobacteria, leading to very high 15N microbial enrichment. After microbial capture, approximately half of the 15N-glutamate was rapidly mineralized, leading to the excretion of , which became available for plant capture. Roots proved to be poor competitors for 15N-glutamate and took up N mainly as . Spatial mapping of 15N revealed differential patterns of 15N uptake within bacteria and the rapid uptake and redistribution of 15N within roots. In conclusion, we demonstrate the rapid cycling and transformation of N at the soil–root interface and that wheat capture of organic N is low in comparison to inorganic N under the conditions tested.

NanoSIMS multi-element imaging reveals internalisation and nucleolar targeting for a highly-charged polynuclear platinum compound

Simultaneous multi-element imaging using NanoSIMS (nano-scale secondary ion mass spectrometry), exploiting the novel combination of (195)Pt and (15)N in platinum-am(m)ine antitumour drugs, provides information on the internalisation and subcellular localisation of both metal and ligands, and allows identification of ligand exchange.

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.

Going nano: A new step toward understanding the processes governing freshwater ooid formation

Marine and freshwater ooids were historically thought to form by purely physicochemical processes in turbulent environments. Recently, organomineralization has been identified as a key process for the initiation of freshwater ooid cortex formation, but the exact biochemical mechanism(s) involved and subsequent contribution to the development of the growing cortex remain unknown. Here, we show that photosynthetic microbes not only enhance early carbonate precipitation around the ooid nucleus but also control the formation of the entire cortex in freshwater ooids from Lake Geneva, Switzerland. Microbial extracellular polymeric substances are first permineralized as amorphous magnesium silicates ( am Mg-Si) before being calcified. An ∼5‰–6‰ depletion of 13 C in ooid cortices compared to both bulk values and carbonate nuclei supports this photosynthetic microbial mechanism and argues against contributions from sulfate-reducing bacteria or methanogens. These data have significant implications for paleoenvironmental studies since photosynthetic microbes now provide an alternative to turbulent hydrodynamic conditions in the formation of freshwater ooids.

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.