Droplet Microfluidic-Based In Situ Analyzer for Monitoring Free Nitrate in Soil.

Monitoring nutrients in the soil can provide valuable information for understanding their spatiotemporal variability and informing precise soil management. Here, we describe an autonomous in situ analyzer for the real-time monitoring of nitrate in soil. The analyzer can sample soil nitrate using either microdialysis or ultrafiltration probes placed within the soil and quantify soil nitrate using droplet microfluidics and colorimetric measurement. Compared with traditional manual sampling and lab analysis, the analyzer features low reagent consumption (96 µL per measurement), low maintenance requirement (monthly), and high measurement frequency (2 or 4 measurements per day), providing nondrifting lab-quality data with errors of less than 10% using a microdialysis probe and 2-3% for ultrafiltration. The analyzer was deployed at both the campus garden and forest for different periods of time, being able to capture changes in free nitrate levels in response to manual perturbation by the addition of nitrate standard solutions and natural perturbation by rainfall events.

Multimodal correlative imaging and modelling of phosphorus uptake from soil by hyphae of mycorrhizal fungi.

Phosphorus (P) is essential for plant growth. Arbuscular mycorrhizal fungi (AMF) aid its uptake by acquiring P from sources distant from roots in return for carbon. Little is known about how AMF colonise soil pore-space, and models of AMF-enhanced P-uptake are poorly validated. We used synchrotron X-ray computed tomography to visualize mycorrhizas in soil and synchrotron X-ray fluorescence/X-ray absorption near edge structure (XRF/XANES) elemental mapping for P, sulphur (S) and aluminium (Al) in combination with modelling. We found that AMF inoculation had a suppressive effect on colonisation by other soil fungi and identified differences in structure and growth rate between hyphae of AMF and nonmycorrhizal fungi. Our results showed that AMF co-locate with areas of high P and low Al, and preferentially associate with organic-type P species over Al-rich inorganic P. We discovered that AMF avoid Al-rich areas as a source of P. Sulphur-rich regions were found to be correlated with higher hyphal density and an increased organic-associated P-pool, whilst oxidized S-species were found close to AMF hyphae. Increased S oxidation close to AMF suggested the observed changes were microbiome-related. Our experimentally-validated model led to an estimate of P-uptake by AMF hyphae that is an order of magnitude lower than rates previously estimated - a result with significant implications for the modelling of plant-soil-AMF interactions.

Space and time-resolved monitoring of phosphorus release from a fertilizer pellet and its mobility in soil using microdialysis and X-ray computed tomography.

Phosphorus is an essential nutrient for crops. Precise spatiotemporal application of P fertilizer can improve plant P acquisition and reduce run-off losses of P. Optimizing application would benefit from understanding the dynamics of P release from a fertilizer pellet into bulk soil, which requires space- and time-resolved measurements of P concentration in soil solutions. In this study, we combined microdialysis and X-ray computed tomography to investigate P transport in soil. Microdialysis probes enabled repeated solute sampling from one location with minimal physical disturbance, and their small dimensions permitted spatially resolved monitoring. We observed a rapid initial release of P from the source, producing high dissolved P concentrations within the first 24 h, followed by a decrease in dissolved P over time compatible with adsorption onto soil particles. Soils with greater bulk density (i.e., reduced soil porosity) impeded the P pulse movement, which resulted in a less homogeneous distribution of total P in the soil column at the end of the experiment. The model fit to the data showed that the observed phenomena can be explained by diffusion and adsorption. The results showed that compared with conventional measurement techniques (e.g., suction cups), microdialysis measurements present a less invasive alternative. The time-resolved measurements ultimately highlighted rapid P dynamics that require more attention for improving P use efficiency.

Linking root structure to functionality: the impact of root system architecture on citrate-enhanced phosphate uptake.

Root citrate exudation is thought to be important for phosphate solubilization. Previous research has concluded that cluster-like roots benefit most from this exudation in terms of increased phosphate uptake, suggesting that root structure plays an important role in citrate-enhanced uptake (additional phosphate uptake due to citrate exudation). Time-resolved computed tomography images of wheat root systems were used as the geometry for 3D citrate-phosphate solubilization models. Citrate-enhanced uptake was correlated with morphological measures of the root systems to determine which had the most benefit. A large variation of citrate-enhanced uptake over 11 root structures was observed. Root surface area dominated absolute phosphate uptake, but did not explain citrate-enhanced uptake. Number of exuding root tips correlated well with citrate-enhanced uptake. Root tips in close proximity could collectively exude high amounts of citrate, resulting in a delayed spike in citrate-enhanced uptake. Root system architecture plays an important role in citrate-enhanced uptake. Singular morphological measurements of the root systems cannot entirely explain variations in citrate-enhanced uptake. Root systems with many tips would benefit greatly from citrate exudation. Quantifying citrate-enhanced uptake experimentally is difficult as variations in root surface area would overwhelm citrate benefits.

Root-induced soil deformation influences Fe, S and P: rhizosphere chemistry investigated using synchrotron XRF and XANES.

Rhizosphere soil has distinct physical and chemical properties from bulk soil. However, besides root-induced physical changes, chemical changes have not been extensively measured in situ on the pore scale. In this study, we couple structural information, previously obtained using synchrotron X-ray computed tomography (XCT), with synchrotron X-ray fluorescence microscopy (XRF) and X-ray absorption near-edge structure (XANES) to unravel chemical changes induced by plant roots. Our results suggest that iron (Fe) and sulfur (S) increase notably in the direct vicinity of the root via solubilization and microbial activity. XANES further shows that Fe is slightly reduced, S is increasingly transformed into sulfate (SO42- ) and phosphorus (P) is increasingly adsorbed to humic substances in this enrichment zone. In addition, the ferrihydrite fraction decreases drastically, suggesting the preferential dissolution and the formation of more stable Fe oxides. Additionally, the increased transformation of organic S to sulfate indicates that the microbial activity in this zone is increased. These changes in soil chemistry correspond to the soil compaction zone as previously measured via XCT. The fact that these changes are colocated near the root and the compaction zone suggests that decreased permeability as a result of soil structural changes acts as a barrier creating a zone with increased rhizosphere chemical interactions via surface-mediated processes, microbial activity and acidification.

Imaging microstructure of the barley rhizosphere: particle packing and root hair influences.

Soil adjacent to roots has distinct structural and physical properties from bulk soil, affecting water and solute acquisition by plants. Detailed knowledge on how root activity and traits such as root hairs affect the three-dimensional pore structure at a fine scale is scarce and often contradictory. Roots of hairless barley (Hordeum vulgare L. cv Optic) mutant (NRH) and its wildtype (WT) parent were grown in tubes of sieved (<250 µm) sandy loam soil under two different water regimes. The tubes were scanned by synchrotron-based X-ray computed tomography to visualise pore structure at the soil-root interface. Pore volume fraction and pore size distribution were analysed vs distance within 1 mm of the root surface. Less dense packing of particles at the root surface was hypothesised to cause the observed increased pore volume fraction immediately next to the epidermis. The pore size distribution was narrower due to a decreased fraction of larger pores. There were no statistically significant differences in pore structure between genotypes or moisture conditions. A model is proposed that describes the variation in porosity near roots taking into account soil compaction and the surface effect at the root surface.

Soil carbon dioxide venting through rice roots.

The growth of rice in submerged soils depends on its ability to form continuous gas channels-aerenchyma-through which oxygen (O2 ) diffuses from the shoots to aerate the roots. Less well understood is the extent to which aerenchyma permits venting of respiratory carbon dioxide (CO2 ) in the opposite direction. Large, potentially toxic concentrations of dissolved CO2 develop in submerged rice soils. We show using X-ray computed tomography and image-based mathematical modelling that CO2 venting through rice roots is far greater than thought hitherto. We found rates of venting equivalent to a third of the daily CO2 fixation in photosynthesis. Without this venting through the roots, the concentrations of CO2 and associated bicarbonate (HCO3- ) in root cells would have been well above levels known to be toxic to roots. Removal of CO2 and hence carbonic acid (H2 CO3 ) from the soil was sufficient to increase the pH in the rhizosphere close to the roots by 0.7 units, which is sufficient to solubilize or immobilize various nutrients and toxicants. A sensitivity analysis of the model showed that such changes are expected for a wide range of plant and soil conditions.

Can VEGFC Form Turing Patterns in the Zebrafish Embryo?

This paper is concerned with a late stage of lymphangiogenesis in the trunk of the zebrafish embryo. At 48 hours post-fertilisation (HPF), a pool of parachordal lymphangioblasts (PLs) lies in the horizontal myoseptum. Between 48 and 168 HPF, the PLs spread from the horizontal myoseptum to form the thoracic duct, dorsal longitudinal lymphatic vessel, and parachordal lymphatic vessel. This paper deals with the potential of vascular endothelial growth factor C (VEGFC) to guide the differentiation of PLs into the mature lymphatic endothelial cells that form the vessels. We built a mathematical model to describe the biochemical interactions between VEGFC, collagen I, and matrix metalloproteinase 2 (MMP2). We also carried out a linear stability analysis of the model and computer simulations of VEGFC patterning. The results suggest that VEGFC can form Turing patterns due to its relations with MMP2 and collagen I, but the zebrafish embryo needs a separate control mechanism to create the right physiological conditions. Furthermore, this control mechanism must ensure that the VEGFC patterns are useful for lymphangiogenesis: stationary, steep gradients, and reasonably fast forming. Generally, the combination of a patterning species, a matrix protein, and a remodelling species is a new patterning mechanism.

Multiple Scale Homogenisation of Nutrient Movement and Crop Growth in Partially Saturated Soil.

In this paper, we use multiple scale homogenisation to derive a set of averaged macroscale equations that describe the movement of nutrients in partially saturated soil that contains growing potato tubers. The soil is modelled as a poroelastic material, which is deformed by the growth of the tubers, where the growth of each tuber is dependent on the uptake of nutrients via a sink term within the soil representing root nutrient uptake. Special attention is paid to the reduction in void space, resulting change in local water content and the impact on nutrient diffusion within the soil as the tubers increase in size. To validate the multiple scale homogenisation procedure, we compare the system of homogenised equations to the original set of equations and find that the solutions between the two models differ by [Formula: see text]. However, we find that the computation time between the two sets of equations differs by several orders of magnitude. This is due to the combined effects of the complex three-dimensional geometry and the implementation of a moving boundary condition to capture tuber growth.

Quantification of root water uptake in soil using X-ray computed tomography and image-based modelling.

Spatially averaged models of root-soil interactions are often used to calculate plant water uptake. Using a combination of X-ray computed tomography (CT) and image-based modelling, we tested the accuracy of this spatial averaging by directly calculating plant water uptake for young wheat plants in two soil types. The root system was imaged using X-ray CT at 2, 4, 6, 8 and 12 d after transplanting. The roots were segmented using semi-automated root tracking for speed and reproducibility. The segmented geometries were converted to a mesh suitable for the numerical solution of Richards' equation. Richards' equation was parameterized using existing pore scale studies of soil hydraulic properties in the rhizosphere of wheat plants. Image-based modelling allows the spatial distribution of water around the root to be visualized and the fluxes into the root to be calculated. By comparing the results obtained through image-based modelling to spatially averaged models, the impact of root architecture and geometry in water uptake was quantified. We observed that the spatially averaged models performed well in comparison to the image-based models with <2% difference in uptake. However, the spatial averaging loses important information regarding the spatial distribution of water near the root system.

A Model of Uranium Uptake by Plant Roots Allowing for Root-Induced Changes in the soil.

We develop a model with which to study the poorly understood mechanisms of uranium (U) uptake by plants. The model is based on equations for transport and reaction of U and acids and bases in the rhizosphere around cylindrical plant roots. It allows for the speciation of U with hydroxyl, carbonate, and organic ligands in the soil solution; the nature and kinetics of sorption reactions with the soil solid; and the effects of root-induced changes in rhizosphere pH. A sensitivity analysis showed the importance of soil sorption and speciation parameters as influenced by pH and CO2 pressure; and of root geometry and root-induced acid-base changes linked to the form of nitrogen taken up by the root. The root absorbing coefficient for U, relating influx to the concentration of U species in solution at the root surface, was also important. Simplified empirical models of U uptake by different plant species and soil types need to account for these effects.

High-resolution synchrotron imaging shows that root hairs influence rhizosphere soil structure formation.

In this paper, we provide direct evidence of the importance of root hairs on pore structure development at the root-soil interface during the early stage of crop establishment. This was achieved by use of high-resolution (c. 5 µm) synchrotron radiation computed tomography (SRCT) to visualise both the structure of root hairs and the soil pore structure in plant-soil microcosms. Two contrasting genotypes of barley (Hordeum vulgare), with and without root hairs, were grown for 8 d in microcosms packed with sandy loam soil at 1.2 g cm-3 dry bulk density. Root hairs were visualised within air-filled pore spaces, but not in the fine-textured soil regions. We found that the genotype with root hairs significantly altered the porosity and connectivity of the detectable pore space (> 5 µm) in the rhizosphere, as compared with the no-hair mutants. Both genotypes showed decreasing pore space between 0.8 and 0.1 mm from the root surface. Interestingly the root-hair-bearing genotype had a significantly greater soil pore volume-fraction at the root-soil interface. Effects of pore structure on diffusion and permeability were estimated to be functionally insignificant under saturated conditions when simulated using image-based modelling.

A Mathematical Model of Lymphangiogenesis in a Zebrafish Embryo.

The lymphatic system of a vertebrate is important in health and diseases. We propose a novel mathematical model to elucidate the lymphangiogenic processes in zebrafish embryos. Specifically, we are interested in the period when lymphatic endothelial cells (LECs) exit the posterior cardinal vein and migrate to the horizontal myoseptum of a zebrafish embryo. We wonder whether vascular endothelial growth factor C (VEGFC) is a morphogen and a chemotactic factor for these LECs. The model considers the interstitial flow driving convection, the reactive transport of VEGFC, and the changing dynamics of the extracellular matrix in the embryo. Simulations of the model illustrate that VEGFC behaves very differently in diffusion and convection-dominant scenarios. In the former case, it must bind to the matrix to establish a functional morphogen gradient. In the latter case, the opposite is true and the pressure field is the key determinant of what VEGFC may do to the LECs. Degradation of collagen I, a matrix component, by matrix metallopeptidase 2 controls the spatiotemporal dynamics of VEGFC. It controls whether diffusion or convection is dominant in the embryo; it can create channels of abundant VEGFC and scarce collagen I to facilitate lymphangiogenesis; when collagen I is insufficient, VEGFC cannot influence the LECs at all. We predict that VEGFC is a morphogen for the migrating LECs, but it is not a chemotactic factor for them.

An Explicit Structural Model of Root Hair and Soil Interactions Parameterised by Synchrotron X-ray Computed Tomography.

The rhizosphere is a zone of fundamental importance for understanding the dynamics of nutrient acquisition by plant roots. The canonical difficulty of experimentally investigating the rhizosphere led long ago to the adoption of mathematical models, the most sophisticated of which now incorporate explicit representations of root hairs and rhizosphere soil. Mathematical upscaling regimes, such as homogenisation, offer the possibility of incorporating into larger-scale models the important mechanistic processes occurring at the rhizosphere scale. However, we lack concrete descriptions of all the features required to fully parameterise models at the rhizosphere scale. By combining synchrotron X-ray computed tomography (SRXCT) and a novel root growth assay, we derive a three-dimensional description of rhizosphere soil structure suitable for use in multi-scale modelling frameworks. We describe an approach to mitigate sub-optimal root hair detection via structural root hair growth modelling. The growth model is explicitly parameterised with SRXCT data and simulates three-dimensional root hair ideotypes in silico, which are suitable for both ideotypic analysis and parameterisation of 3D geometry in mathematical models. The study considers different hypothetical conditions governing root hair interactions with soil matrices, with their respective effects on hair morphology being compared between idealised and image-derived soil/root geometries. The studies in idealised geometries suggest that packing arrangement of soil affects hair tortuosity more than the particle diameter. Results in field-derived soil suggest that hair access to poorly mobile nutrients is particularly sensitive to the physical interaction between the growing hairs and the phase of the soil in which soil water is present (i.e. the hydrated textural phase). The general trends in fluid-coincident hair length with distance from the root, and their dependence on hair/soil interaction mechanisms, are conserved across Cartesian and cylindrical geometries.

The Application of Contrast Media for In Vivo Feature Enhancement in X-Ray Computed Tomography of Soil-Grown Plant Roots.

The use of in vivo X-ray microcomputed tomography (µCT) to study plant root systems has become routine, but is often hampered by poor contrast between roots, soil, soil water, and soil organic matter. In clinical radiology, imaging of poorly contrasting regions is frequently aided by the use of radio-opaque contrast media. In this study, we present evidence for the utility of iodinated contrast media (ICM) in the study of plant root systems using µCT. Different dilutions of an ionic and nonionic ICM (Gastrografin 370 and Niopam 300) were perfused into the aerial vasculature of juvenile pea plants via a leaf flap (Pisum sativum). The root systems were imaged via µCT, and a variety of image-processing approaches used to quantify and compare the magnitude of the contrast enhancement between different regions. Though the treatment did not appear to significantly aid extraction of full root system architectures from the surrounding soil, it did allow the xylem and phloem units of seminal roots and the vascular morphology within rhizobial nodules to be clearly visualized. The nonionic, low-osmolality contrast agent Niopam appeared to be well tolerated by the plant, whereas Gastrografin showed evidence of toxicity. In summary, the use of iodine-based contrast media allows usually poorly contrasting root structures to be visualized nondestructively using X-ray µCT. In particular, the vascular structures of roots and rhizobial nodules can be clearly visualized in situ.

Measurement of strains experienced by viscerofugal nerve cell bodies during mechanosensitive firing using digital image correlation.

Mechanosensory neurons detect physical events in the local environments of the tissues that they innervate. Studies of mechanosensitivity of neurons or nerve endings in the gut have related their firing to strain, wall tension, or pressure. Digital image correlation (DIC) is a technique from materials engineering that can be adapted to measure the local physical environments of afferent neurons at high resolution. Flat-sheet preparations of guinea pig distal colon were set up with arrays of tissue markers in vitro. Firing of single viscerofugal neurons was identified in extracellular colonic nerve recordings. The locations of viscerofugal nerve cell bodies were inferred by mapping firing responses to focal application of the nicotinic receptor agonist 1,1-dimethyl-4-phenylpiperazinium iodide. Mechanosensory firing was recorded during load-evoked uniaxial or biaxial distensions. Distension caused movement of surface markers which was captured by video imaging. DIC tracked the markers, interpolating the mechanical state of the gut at the location of the viscerofugal nerve cell body. This technique revealed heterogeneous load-evoked strain within preparations. Local strains at viscerofugal nerve cell bodies were usually smaller than global strain measurements and correlated more closely with mechanosensitive firing. Both circumferential and longitudinal strain activated viscerofugal neurons. Simultaneous loading in circumferential and longitudinal axes caused the highest levels of viscerofugal neuron firing. Multiaxial strains, reflecting tissue shearing and changing area, linearly correlated with mechanosensory firing of viscerofugal neurons. Viscerofugal neurons were mechanically sensitive to both local circumferential and local longitudinal gut strain, and appear to lack directionality in their stretch sensitivity.

Image-based modelling of nutrient movement in and around the rhizosphere.

In this study, we developed a spatially explicit model for nutrient uptake by root hairs based on X-ray computed tomography images of the rhizosphere soil structure. This work extends our previous work to larger domains and hence is valid for longer times. Unlike the model used previously, which considered only a small region of soil about the root, we considered an effectively infinite volume of bulk soil about the rhizosphere. We asked the question: At what distance away from root surfaces do the specific structural features of root-hair and soil aggregate morphology not matter because average properties start dominating the nutrient transport? The resulting model was used to capture bulk and rhizosphere soil properties by considering representative volumes of soil far from the root and adjacent to the root, respectively. By increasing the size of the volumes that we considered, the diffusive impedance of the bulk soil and root uptake were seen to converge. We did this for two different values of water content. We found that the size of region for which the nutrient uptake properties converged to a fixed value was dependent on the water saturation. In the fully saturated case, the region of soil we needed to consider was only of radius 1.1mm for poorly soil-mobile species such as phosphate. However, in the case of a partially saturated medium (relative saturation 0.3), we found that a radius of 1.4mm was necessary. This suggests that, in addition to the geometrical properties of the rhizosphere, there is an additional effect of soil moisture properties, which extends further from the root and may relate to other chemical changes in the rhizosphere. The latter were not explicitly included in our model.

An Image-Based Model of Fluid Flow Through Lymph Nodes.

The lymphatic system returns fluid to the bloodstream from the tissues to maintain tissue fluid homeostasis. Lymph nodes distributed throughout the system filter the lymphatic fluid. The afferent and efferent lymph flow conditions of lymph nodes can be measured in experiments; however, it is difficult to measure the flow within the nodes. In this paper, we present an image-based modelling approach to investigating how the internal structure of the node affects the fluid flow pathways within the node. Selective plane illumination microscopy images of murine lymph nodes are used to identify the geometry and structure of the tissue within the node and to determine the permeability of the lymph node interstitium to lymphatic fluid. Experimental data are used to determine boundary conditions and optimise the parameters for the model. The numerical simulations conducted within the model are implemented in COMSOL Multiphysics, a commercial finite element analysis software. The parameter fitting resulted in the estimate that the average permeability for lymph node tissue is of the order of magnitude of [Formula: see text]. Our modelling shows that the flow predominantly takes a direct path between the afferent and efferent lymphatics and that fluid is both filtered and absorbed across the blood vessel boundaries. The amount that is absorbed or extravasated in the model is dependent on the efferent lymphatic lumen fluid pressure.

Assessing the influence of the rhizosphere on soil hydraulic properties using X-ray computed tomography and numerical modelling.

Understanding the dynamics of water distribution in soil is crucial for enhancing our knowledge of managing soil and water resources. The application of X-ray computed tomography (CT) to the plant and soil sciences is now well established. However, few studies have utilized the technique for visualizing water in soil pore spaces. Here this method is utilized to visualize the water in soil in situ and in three-dimensions at successive reductive matric potentials in bulk and rhizosphere soil. The measurements are combined with numerical modelling to determine the unsaturated hydraulic conductivity, providing a complete picture of the hydraulic properties of the soil. The technique was performed on soil cores that were sampled adjacent to established roots (rhizosphere soil) and from soil that had not been influenced by roots (bulk soil). A water release curve was obtained for the different soil types using measurements of their pore geometries derived from CT imaging and verified using conventional methods, such as pressure plates. The water, soil, and air phases from the images were segmented and quantified using image analysis. The water release characteristics obtained for the contrasting soils showed clear differences in hydraulic properties between rhizosphere and bulk soil, especially in clay soil. The data suggest that soils influenced by roots (rhizosphere soil) are less porous due to increased aggregation when compared with bulk soil. The information and insights obtained on the hydraulic properties of rhizosphere and bulk soil will enhance our understanding of rhizosphere biophysics and improve current water uptake models.

A Model for Interstitial Drainage Through a Sliding Lymphatic Valve.

This study investigates fluid flow and elastic deformation in tissues that are drained by the primary lymphatic system. A model is formulated based on the Rossi hypothesis that states that the primary lymphatic valves, which are formed by overlapping endothelial cells around the circumferential lining of lymphatic capillaries, open in response to swelling of the surrounding tissue. Tissue deformation and interstitial fluid flow through the tissue are treated using the Biot equations of poroelasticity and, the fluid flux (into the interstitium) across the walls of the blood capillaries, is assumed to be linearly related to the pressure difference across the walls via a constant of proportionality (the vascular permeability). The resulting model is solved in a periodic domain containing one blood capillary and one lymphatic capillary starting from a configuration in which the tissue is undeformed. On imposition of a constant pressure difference between blood and lymphatic capillaries, the solutions are found to settle to a steady state. Given that the magnitude of pressure fluctuations in the lymphatic system is much smaller than this pressure difference between blood and lymph, it is postulated that the resulting steady-state solution gives a good representation of the state of the tissue under physiological conditions. The effects of changes to the Young's modulus of the tissue, the blood-lymphatic pressure difference, vascular permeability and valve dimensions on the steady state are investigated and discussed in terms of their effects on oedema in the context of age- and pregnancy-related changes to the body.