Novel isotope sensor for spatially-resolved analysis of nutrient exchange in the rhizosphere
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During arbuscular mycorrhiza (AM) symbiosis AM fungi form tree-shaped structures called arbuscules in root cortex cells of host plants. Arbuscules and their host cells are central for reciprocal nutrient exchange between the symbionts.REQUIRED FOR ARBUSCULAR MYCORRHIZATION1(RAM1) encodes a GRAS protein crucial for transcriptionally regulating plant genes needed for arbuscule development and nutrient exchange. Similar to other GRAS proteins, RAM1 likely does not bind to DNA and how RAM1 activates its target promoters remained elusive. Here, we demonstrate that RAM1 interacts with five AM-induced APETALA 2 (AP2) transcription factors of the WRINKLED1-like family called CTTC MOTIF-BINDING TRANSCRIPTION FACTOR1 (CBX1), WRI3, WRI5a, WRI5b, and WRI5c via a C-terminal domain containing the M2/M2a motif. This motif is conserved and enriched in WRI proteins encoded by genomes of AM-competent plants. RAM1 together with any of these WRI proteins activates the promoters of genes required for symbiotic nutrient exchange, namelyRAM2,STUNTED ARBUSCULES (STR),andPHOSPHATE TRANSPORTER 4 (PT4), inNicotiana benthamianaleaves. This activation as well as target promoter induction inLotus japonicushairy roots depends onMYCS(MYCORRHIZA SEQUENCE)-elements andAW-boxes, previously identified as WRI-binding sites. TheWRIgenes are activated in two waves: Transcription ofRAM1,CBX1,andWRI3is coregulated by calcium- and calmodulin-dependent protein kinase-activated CYCLOPS, through theAMCYC-REin their promoter, and DELLA, whileWRI5a,b,andcpromoters containMYCS-elements andAW-boxes and can be activated by RAM1 heterocomplexes with CBX1 or WRI3. We propose that RAM1 provides an activation domain to DNA-binding WRI proteins to activate genes with central roles in AM development and function.
In this paper we demonstrate that several ubiquitous hyporheic exchange mechanisms can be represented simply as a one-dimensional diffusion process, where the diffusivity decays exponentially with depth into the streambed. Based on a meta-analysis of 106 previously published laboratory measurements of hyporheic exchange (capturing a range of bed morphologies, hydraulic conditions, streambed properties, and experimental approaches) we find that the reference diffusivity and mixing length-scale are functions of the permeability Reynolds Number and Schmidt Number. These dimensionless numbers, in turn, can be estimated for a particular stream from the median grain size of the streambed and the stream's depth, slope, and temperature. Application of these results to a seminal study of nitrate removal in 72 headwater streams across the United States, reveals: (a) streams draining urban and agricultural landscapes have a diminished capacity for in-stream and in-bed mixing along with smaller subsurface storage zones compared to streams draining reference landscapes; (b) under steady-state conditions nitrate uptake in the streambed is primarily biologically controlled; and (c) median reaction timescales for nitrate removal in the hyporheic zone are 0.5 and 20 hr for uptake by assimilation and denitrification, respectively. While further research is needed, the simplicity and extensibility of the framework described here should facilitate cross-disciplinary discussions and inform reach-scale studies of pollutant fate and transport and their scale-up to watersheds and beyond.
The anaerobic oxidation of methane (AOM) is a significant worldwide microbial process in anoxic lake and ocean sediments, responsible for sequestering up to 80% of this greenhouse gas. Often considered a metabolism on the edge of thermodynamic probability, the impact of ANaerobic MEthane-oxidizing ‘ANME’ archaea on carbon and nutrient cycling in sediment ecosystems is far reaching. They not only serve as a sink for methane coupled to diverse electron acceptors, but also catalyze the transformation of many important nutrients including nitrogen, phosphate, and iron. While information about the potential mechanisms supporting metabolism in AOM is now available, remarkably little has been learned about their nutritional requirements, their dependencies on bacterial partners, and consequentially their ultimate impact on nutrient transformation and bioavailability within sedimentary ecosystems, and beyond. Further, the role of viruses within sediment ecosystems represents an essential but vastly understudied aspect of the transformation of carbon and nutrients by AOM. Viruses are now widely appreciated as central players in biogeochemical cycles across diverse ecosystems. These nanoscale predators have been shown to enhance the turnover of essential nutrients, thus stimulating microbial growth and environmental viruses themselves may constitute an important reservoir of nitrogen and phosphorous. Little is known about the role of viruses in methane-impacted sedimentary ecosystems, however prior genomic and microscopy evidence suggests that AOM consortia are susceptible to phage infection. The overarching scientific goal of this multi-disciplinary research proposal is to build on these recent discoveries and expand our understanding of interactions and fundamental activities involved in cycling of carbon and nutrients by syntrophic methanotrophic archaeal-bacterial consortia and associated viruses in anoxic sedimentary environments. Our three specific objectives are to 1) Quantify energy and nutrient exchange (e.g. N, P, Fe and vitamins) within AOM consortia and between ANME-bacterial partners; 2) Identify virus-host interactions associated with AOM and assess C and N transfer through viruses in methane-impacted sediment ecosystems; 3) Model energy and nutrient exchange in AOM consortia and viral-host interactions (i.e. viral activity), and their environmental distribution patterns. Our experimental emphasis cuts across scales that are important for understanding microbial and viral interactions and activities within their habitats, as well as community wide biogeochemical transformations. These research goals will be accomplished through the application of novel molecular techniques targeting DNA, RNA, proteins, and metabolites combined with a unique multi-modal analytical imaging pipeline. We will then model the ecophysiological capabilities of diverse sediment-hosted methanotrophic consortia to develop a more comprehensive understanding of the energetic and nutritional interactions between different AOM partner couplings that occur in sediments.
The subtropical North Atlantic Ocean (STNA) has been experiencing a steady decline in both Chlorophyll a (the primary photosynthetic pigment in phytoplankton used to identify abundance in the ocean) and net primary production (the net amount carbon fixed in the surface ocean by phytoplankton after losses to respiration). This decline is spread across the entire STNA and has been ongoing since the beginning of the satellite ocean color record in 1997. Previous work has attributed these changes to decreased nutrient exchange between nutrient-poor surface waters and nutrient-rich deep waters resulting from increased stratification driven by surface warming due to climate change. Our results from an analysis of satellite data, Argo float data, and historical cruise data show significant inconsistencies with this hypothesis. We observe a trend of increased stratification for half the STNA basin but decreased stratification in the other half. Where we see increased stratification, we also see changes in nitrate that would result in an increased delivery of nitrate to surface waters if vertical transport across the stratified layers was the dominant source of nutrients. Our working hypothesis is that changes in circulation in the subtropical gyre have resulted in an altered mix of source waters with distinct nutrient characteristics. The mechanism behind the decline in primary production in the STNA remains unclear. We will use the ECCO-Darwin data-assimilative global ocean biogeochemistry model to further investigate the mechanisms behind the observed changes in production and nutrients in the STNA.
Wheat plants (Triticum aestivum) were grown for 43 days in a micro-porous tube nutrient delivery system. Roots were unable to penetrate the microporous tube, but grew on the surface and maintained capillary contact with the nutrient solution on the inside of the tube through the 5-microns pores of the porous tube. Water potential in the system was controlled at -0.4, -0.8, and -3.0 kPa by adjusting the applied pressure (hydrostatic head) to the nutrient solution flowing through the microporous tubes. A relatively small decrease in applied water potential from -0.4 to -3.0 kPa resulted in a 34% reduction of shoot growth but only a moderate reduction in the mid-day leaf water potential from - 1.3 to - 1.7 MPa. Carbon dioxide assimilation decreased and water use efficiency increased with the more negative applied water Potentials, while intercellular C02 concentration remained constant. The physiological responses observed in this study in response to small constant differences in applied water potentials were much greater than expected from either the applied water potential or the observed plant water potential. Even though the micro-porous tube may not represent natural conditions and could possibly introduce morphological and physiological artifacts , it enables a high degree of control of water potential that facilitates the investigation of many aspects of water relations not practical with other experimental systems.
Wheat plants (Triticum aestivum) were grown for 43 days in a micro-porous tube nutrient delivery system. Roots were unable to penetrate the microporous tube, but grew on the surface and maintained capillary contact with the nutrient solution on the inside of the tube through the 5-micron pores of the porous tube. Water potential in the system was controlled at -0.4, -0.8, and -3.0 kPa by adjusting the applied pressure (hydrostatic head) to the nutrient solution flowing through the microporous tubes. A relatively small decrease in applied water potential from -0.4 to -3.0 kPa resulted in a 34% reduction of shoot growth but only a moderate reduction in the midday leaf water potential from -1.3 to -1.7 MPa. Carbon dioxide assimilation decreased and water use efficiency increased with the more negative applied water potentials, while intercellular CO2 concentration remained constant. This was associated with a decrease in stomatal conductance to water vapor from 1.90 to 0.98 mol/(sq m sec) and a decrease in total apparent hydraulic conductance from 47 to 12 (micro)mol/(sec MPa). Although the applied water potentials were in the -0.4 to -3.0 kPa range, the actual water potential perceived by the plant roots appeared to be in the range of -0.26 to -0.38 MPa as estimated by the leaf water potential of bagged plants. The amount of K, Ca, Mg, Zn, Cu, and B accumulated with each unit of transpired water increased as the applied water potential became less negative. The increase in accumulation ranged from 1.4-fold for K to 2.2-fold for B. The physiological responses observed in this study in response to small constant differences in applied water potentials were much greater than expected from either the applied water potential or the observed plant water potential. Even though the micro-porous tube may not represent natural conditions and could possibly introduce morphological and physiological artifacts, it enables a high degree of control of water potential that facilitates the investigation of many aspects of water relations not practical with other experimental systems.
Microalgae are increasingly recognized for their potential in wastewater treatment and the sustainable production of feedstock for fuel, feed, food, and other bioproducts. Like conventional agricultural systems, algal cultivation involves complex microbial communities. However, despite their pivotal role in cultivation outcomes, especially at the commodity-scale, the critical interactions between microalgae and their microbiomes are often overlooked. Here we synthesize current knowledge on the taxonomic diversity, ecological roles, and biotechnological potential of algal microbiomes, with a focus on their interactions with algal hosts through nutrient exchange, growth modulation, pathogen defense, and environmental conditioning. We also examine how environmental factors such as nutrient availability, salinity, and temperature influence these interactions. Advances in microbiome engineering, including synthetic biology and ecological approaches, offer opportunities to enhance beneficial algal-microbiome interactions, thereby improving growth, resilience, and yield. These advancements could lead to more sustainable and economically viable microalgae cultivation, with far-reaching implications for environmental management and biotechnological innovation. By addressing key economic and environmental barriers, microbiome engineering holds transformative potential to revolutionize large-scale algae cultivation and provide sustainable solutions to global challenges.
Nutrient exchange forms the basis of the ancient symbiotic relationship that occurs between most land plants and arbuscular mycorrhizal (AM) fungi. Plants provide carbon (C) to AM fungi and fungi provide the plant with nutrients such as nitrogen (N) and phosphorous (P). Nutrient addition can alter this symbiotic coupling in key ways, such as reducing AM fungal root colonization and changing the AM fungal community composition. However, environmental parameters that differentiate ecosystems and drive plant distribution patterns (e.g., pH, moisture), are also known to impact AM fungal communities. Identifying the relative contribution of environmental factors impacting AM fungal distribution patterns is important for predicting biogeochemical cycling patterns and plant-microbe relationships across ecosystems. Here, to evaluate the relative impacts of local environmental conditions and long-term nutrient addition on AM fungal abundance and composition across grasslands, we studied experimental plots amended for 10 years with N, P, or N and P fertilizer in different grassland ecosystem types, including tallgrass prairie, montane, shortgrass prairie, and desert grasslands. Contrary to our hypothesis, we found ecosystem type, not nutrient treatment, was the main driver of AM fungal root colonization, diversity, and community composition, even when accounting for site-specific nutrient limitations. We identified several important environmental drivers of grassland ecosystem AM fungal distribution patterns, including aridity, mean annual temperature, root moisture, and soil pH. This work provides empirical evidence for niche partitioning strategies of AM fungal functional guilds and emphasizes the importance of long-term, large scale research projects to provide ecologically relevant context to nutrient addition studies.
Omics-based studies have evolved over the past two decades to provide rich datasets from which deeper system-level understanding can be unraveled. Omics technologies (e.g., genomics, transcriptomics, proteomics, and metabolomics) can provide specific molecular insights into plant and microbe fitness, disease states, stress, cell signaling/cell-cell communication, and nutrient exchange, and these molecular observations can be correlated with system phenotype and function. The overall health of plant-microbe systems and their surrounding environments can be assessed, and a greater understanding of processes related to nutrient exchange/cycling, plant disease, and ecosystem homeostasis can now be achieved via the latest approaches to plant-microbe omics analysis.
Abstract The presence of Arbuscular Mycorrhizal Fungi (AMF) in vascular land plant roots is one of the most ancient of symbioses supporting nitrogen and phosphorus exchange for photosynthetically derived carbon. Here we provide a multi-scale modeling approach to predict AMF colonization of a worldwide crop from a Recombinant Inbred Line (RIL) population derived from Sorghum bicolor and S. propinquum . The high-throughput phenotyping methods of fungal structures here rely on a Mask Region-based Convolutional Neural Network (Mask R-CNN) in computer vision for pixel-wise fungal structure segmentations and mixed linear models to explore the relations of AMF colonization, root niche, and fungal structure allocation. Models proposed capture over 95% of the variation in AMF colonization as a function of root niche and relative abundance of fungal structures in each plant. Arbuscule allocation is a significant predictor of AMF colonization among sibling plants. Arbuscules and extraradical hyphae implicated in nutrient exchange predict highest AMF colonization in the top root section. Our work demonstrates that deep learning can be used by the community for the high-throughput phenotyping of AMF in plant roots. Mixed linear modeling provides a framework for testing hypotheses about AMF colonization phenotypes as a function of root niche and fungal structure allocations.
Optimizing loblolly pine (Pinus taeda L.) productivity using fertilizers and various site management practices has been a goal of foresters for decades. Nitrogen (N), phosphorus (P), and potassium (K) are the three most operationally applied fertilizers to loblolly pine silviculture and are of primary importance to their total productivity. Fertilizer recommendations for N, P, and K in loblolly pine are primarily made on abiotic factors such as site and soil characteristics, while the biological factors controlling nutrient uptake are typically overlooked in the production and optimization of these stands. Arguably the most important of these biological factors are the diverse ectomycorrhizal fungal (ECM) communities that colonize the fine roots of almost all loblolly pine trees. The mantle formed by ECM fungi on short-root tips presents a barrier for direct apoplastic uptake of N, P, and K from soil solution by pine roots. In well-colonized roots, the tree is dependent on symplastic fungal transport of N, P, and K foraged from the soil by the extraradical hyphal network. This raises the question: Who are we really feeding if the ECM fungi are the ones assimilating most of the tree's total nutritional requirements? Considering multiple species of ECM fungi can inhabit a single root system, many questions remain regarding the drivers of colonization, why some species are more efficient at taking up and exchanging nutrients with their hosts than others, and why certain fertilizers directly affect the morphology of ECM growth. The purposes of this review here are (1) to explore how the most commonly commercially applied macronutrients, N, P, and K, affect the relationship between loblolly pine and ECM communities, and (2) to propose future directions to investigate, preserve, and manipulate these interactions in pine plantations to optimize productivity.
Abstract Interactive microbial communities are ubiquitous, influencing biogeochemical cycles and host health. One widespread interaction is nutrient exchange, or cross-feeding, wherein metabolites are transferred between microbes. Some cross-fed metabolites, such as vitamins, amino acids, and ammonium (NH4+), are communally valuable and impose a cost on the producer. The mechanisms that enforce cross-feeding of communally valuable metabolites are not fully understood. Previously we engineered a cross-feeding coculture between N2-fixing Rhodopseudomonas palustris and fermentative Escherichia coli. Engineered R. palustris excretes essential nitrogen as NH4+ to E. coli, while E. coli excretes essential carbon as fermentation products to R. palustris. Here, we sought to determine whether a reciprocal cross-feeding relationship would evolve spontaneously in cocultures with wild-type R. palustris, which is not known to excrete NH4+. Indeed, we observed the emergence of NH4+ cross-feeding, but driven by adaptation of E. coli alone. A missense mutation in E. coli NtrC, a regulator of nitrogen scavenging, resulted in constitutive activation of an NH4+ transporter. This activity likely allowed E. coli to subsist on the small amount of leaked NH4+ and better reciprocate through elevated excretion of fermentation products from a larger E. coli population. Our results indicate that enhanced nutrient uptake by recipients, rather than increased excretion by producers, is an underappreciated yet possibly prevalent mechanism by which cross-feeding can emerge.
Chitinases degrade chitin, a linear homopolymer of β-1,4-linked N-acetyl-D-glucosamine (GlcNAc) residues found in the cell walls of fungi and the exoskeletons of arthropods. They are secreted by the roots into the rhizosphere, a complex and dynamic environment where intense nutrient exchange occurs between plants and microbes. Here we modeled, expressed, purified, and characterized Zea mays and Oryza sativa root chitinases, and the chitinase of a symbiotic bacterium, Chitinophaga oryzae 1303 for their activities with chitin, di-, tri-, and tetra-saccharides and Aspergillus niger, with the goal of determining their role(s) in the rhizosphere and better understanding the molecular mechanisms underlying plant-microbe interactions. We show that Zea mays basic endochitinase (ZmChi19A) and Oryza sativa chitinase (OsChi19A) are from the GH19 chitinase family. The Chitinophaga oryzae 1303 chitinase (CspCh18A) belongs to the GH18 family. The three enzymes have similar apparent K M values of (20-40 µM) for the substrate 4-MU-GlcNAc 3 . They vary in their pH and temperature optima with OsChi19A activity optimal between pH 5–7 and 30–40°C while ZmChi19A and CspCh18A activities were optimal at pH 7-9 and 50–60°C. Modeling and site-directed mutation of ZmChi19A identified the catalytic cleft and the active residues E147 and E169 strategically positioned at ~8.6Å from each other in the folded protein. Cleavage of 4-MU-GlcNAc 3 was unaffected by the absence of the CBD but diminished in the absence of the flexible C-terminal domain. However, unlike for the soluble substrate, the CBD and the newly identified flexible C-terminal domain were vital for inhibiting Aspergillus niger growth. The results are consistent with the involvement of the plant chitinases in defense against pathogens like fungi that have chitin exoskeletons. In summary, we have characterized the functional features and structural domains necessary for the activity of two plant root chitinases that are believed to be involved in plant defense and a bacterial chitinase that, along with the plant chitinases, may participate in nutrient recycling in the rhizosphere.
Extensive cytocompatibility testing of 2D nanocarbon materials including graphene oxide (GO) has been performed, but results remain contradictory. Literature has yet to account for settling—although sedimentation is visible to the eye and physics suggests that even individual graphenic flakes will settle. To investigate settling, a series of functional graphenic materials (FGMs) with differing oxidation levels, functionalities, and physical dimensions were synthesized. Though zeta potential indicated colloidal stability, significant gravitational settling of the FGMs was theoretically and experimentally demonstrated. By creating a setup to culture cells in traditional and inverted orientations in the same well, a “blanket effect” was unequivocally demonstrated in which FGMs settle out of solution and cover cells at the bottom of the well, limiting nutrient exchange and viability. Inverted cells protected from the blanket effect are unaffected. Therefore, these results demonstrate that settling is a crucial factor that must be considered for FGM cytocompatibility experiments.
Coastal environments are dynamic interfaces that mediate carbon and nutrient exchanges between terrestrial landscapes and open waters, and understanding the biogeochemical factors controlling these exchanges, particularly iron (Fe) redox transformations, is crucial for predicting coastal ecosystem functions. Here, we investigated the mechanisms controlling Fe speciation changes across upland-to-shoreline gradients in freshwater and estuarine soils using Fe K-edge X-ray absorption spectroscopy, solid and porewater composition analysis, and 16S rRNA sequencing analysis. We show that Fe transformations depend primarily on inundation patterns. In unsaturated uplands, Fe occurs as Fe(III) oxyhydroxides, mainly goethite (9–35 %), Fe(II,III)-phyllosilicates (39–89 %), and Fe(III)-organic species (0–61 %). Soils influenced by estuarine waters exhibit porewater sulfide concentrations reaching up to 221 μM, Fe- and S-cycling bacteria, and up to 81 % pyrite (FeS 2 ), indicating that sulfur-driven redox dynamics control Fe transformations. In lacustrine wetlands, Fe(III) reduction is indicated by porewater Fe(II) concentrations increasing to 1.0–2.1 mM, and ~10–15 % of Fe as Fe(II,III)-(hydr)oxides (green rust), vivianite (Fe 3 (PO 4 ) 2 ·8H 2 O), and/or adsorbed Fe(II) species. EXAFS data also indicate reduction of structural Fe(III) to Fe(II) in phyllosilicates. The presence of Fe- and S-cycling bacteria, as well as sulfide (0–10 μM), suggests that Fe-cycling is microbially driven and potentially coupled with cryptic S-cycling. Fe(II) oxidation was indicated above/near the water table by the presence of Fe(III) oxyhydroxides (ferrihydrite, lepidocrocite). Furthermore, negligible Fe(III) or sulfate reduction was observed at some water-saturated sites located at the upland-wetland transition, likely due to oxic (sub-)surface water inputs. Overall, our results highlight the importance of considering both Fe-speciation and hydro-biogeochemical dynamics when predicting Fe-cycling at coastal interfaces.
Spatial and temporal heterogeneity of nutrient exchange within the rhizosphere is a topic of increasing interest, although challenging to study due to limits in existing analytical capabilities. Here, we developed and demonstrated a new approach applying laser ablation sample introduction to capillary absorption spectroscopy (LA-CAS) to characterize carbon isotopic distribution within plant tissues, rhizosphere, and soil. We exposed switchgrass plants to 13 CO 2 to allow tracing of 13 C-labelled photosynthates within plant biomass and into the associated soil. The LA-CAS methods we describe leverage continuous measurements of a sample stream derived from laser ablation line scans (10-25 µm in width) over a sample surface which enables the user to produce an isotope map with a) higher data density or b) over larger spatial areas versus previously existing techniques. This versatility of LA-CAS is assessed through testing of a range of laser parameters (spot size, scan rates) on various materials (soil, plant biomass/tissues, and rhizosphere). Here, we demonstrate the ability of LA-CAS to provide near instantaneous $δ$ 13 C measurements over isotopically distinct surfaces to enable high spatially resolved mapping of 13 C-labelled material within the rhizosphere. Applying LA-CAS analysis to plant biomass, we observed higher $δ$ 13 C values concentrated within phloem structures, consistent with localized photosynthate transport. When mapping across the rhizosphere, 13 C-enriched soil was typically present within 5-10µm of root boundaries with a steep spatial increase in $δ$ 13 C when the scan approached the middle of the root. As with all LA approaches, care is required to ensure accurate results as phenomena linked to ablation, combustion, and isotopic measurement can impart artifacts if not carefully controlled. Still, taken as a whole, our demonstrations highlight the increased sample throughput, improved data density, and enhanced $δ$ 13 C capability of LA-CAS versus other LA techniques and emphasize the role this method can play in future plant and rhizosphere related studies.
Abstract Floodplain inundation links river and land systems through significant water, sediment, and nutrient exchanges. However, these two‐way interactions between land and river are currently missing in most Earth System Models. In this study, we introduced the two‐way hydrological coupling between the land component, E3SM Land Model, and the river component, Model for Scale Adaptive River Transport, in Energy Exascale Earth System Model (E3SM) to study the impacts of floodplain inundation on land and river processes. We calibrated the river channel geometry and developed a new data‐driven inundation scheme to improve the simulation of inundation dynamics in E3SM. The new inundation scheme captures 96% of the spatial variation of inundation area in a satellite inundation product at global scale, in contrast with 7% when the default inundation scheme of E3SM was used. Global simulations including the new inundation scheme performed at resolution with and without land‐river two‐way coupling was used to quantify the impact of coupling. Comparisons show that two‐way coupling modifies the water and energy cycle in 20% of the global land cells. Specifically, riverine inundation is reduced by two‐way coupling, but inland inundation is intensified. Wetter periods are more impacted by the two‐way coupling at the global scale, while regions with different climates exhibit different sensitivities. The two‐way exchange of water between the land and river components of E3SM provides the foundation for enabling two‐way coupling of land‐river sediment and biogeochemical fluxes. These capabilities will be used to improve understanding of the interactions between water and biogeochemical cycles and their response to human perturbations.