Samuel D. Keyes

High resolution synchrotron imaging of wheat root hairs growing in soil and image based modelling of phosphate uptake

· Root hairs are known to be highly important for uptake of sparingly soluble nutrients, particularly in nutrient deficient soils. Development of increasingly sophisticated mathematical models has allowed uptake characteristics to be quantified. However, modelling has been constrained by a lack of methods for imaging live root hairs growing in real soils. · We developed a plant growth protocol and used Synchrotron Radiation X-ray Tomographic Microscopy (SRXTM) to uncover the three-dimensional (3D) interactions of root hairs in real soil. We developed a model of phosphate uptake by root hairs based directly on the geometry of hairs and associated soil pores as revealed by imaging. · Previous modelling studies found that root hairs dominate phosphate uptake. By contrast, our study suggests that hairs and roots contribute equally. We show that uptake by hairs is more localized than by roots and strongly dependent on root hair and aggregate orientation. · The ability to image hair-soil interactions enables a step change in modelling approaches, allowing a more realistic treatment of processes at the scale of individual root hairs in soil pores.

Challenges in imaging and predictive modeling of rhizosphere processes

BackgroundPlant-soil interaction is central to human food production and ecosystem function. Thus, it is essential to not only understand, but also to develop predictive mathematical models which can be used to assess how climate and soil management practices will affect these interactions.ScopeIn this paper we review the current developments in structural and chemical imaging of rhizosphere processes within the context of multiscale mathematical image based modeling. We outline areas that need more research and areas which would benefit from more detailed understanding.ConclusionsWe conclude that the combination of structural and chemical imaging with modeling is an incredibly powerful tool which is fundamental for understanding how plant roots interact with soil. We emphasize the need for more researchers to be attracted to this area that is so fertile for future discoveries. Finally, model building must go hand in hand with experiments. In particular, there is a real need to integrate rhizosphere structural and chemical imaging with modeling for better understanding of the rhizosphere processes leading to models which explicitly account for pore scale processes.

Root hair formation in rice (Oryza sativa L.) differs between root types and is altered in artificial growth conditions

Root hairs are important sites for nutrient uptake, especially in P limiting conditions. Here we provide first insights into root hair development for the diverse root types of rice grown under different conditions, and show the first in situ images of rice root hairs in intact soil. Roots of plants grown in upland fields produced short root hairs that showed little responsiveness to P deficiency, and had a higher root hair density in the high P condition. These results were reproducible in rhizoboxes under greenhouse conditions. Synchrotron-based in situ analysis of root hairs in intact soil further confirmed this pattern of root hair formation. In contrast, plants grown in nutrient solution produced more and longer root hairs in low P conditions, but these were unequally distributed among the different root types. While nutrient solution-grown main roots had longer hairs compared to upland field-grown main roots, second order lateral roots did not form any root hairs in nutrient solution-grown plants. Furthermore, root hair formation for plants grown in flooded lowland fields revealed few similarities with those grown in nutrient solution, thus defining nutrient solution as a possible measure of maximal, but not natural root hair development. By combining root hair length and density as a measure for root hair impact on the whole soil-grown root system we show that lateral roots provided the majority of root hair surface.

Three Dimensional Imaging of Paraffin Embedded Human Lung Tissue Samples by Micro-Computed Tomography

Background Understanding the three-dimensional (3-D) micro-architecture of lung tissue can provide insights into the pathology of lung disease. Micro computed tomography (µCT) has previously been used to elucidate lung 3D histology and morphometry in fixed samples that have been stained with contrast agents or air inflated and dried. However, non-destructive microstructural 3D imaging of formalin-fixed paraffin embedded (FFPE) tissues would facilitate retrospective analysis of extensive tissue archives of lung FFPE lung samples with linked clinical data. Methods FFPE human lung tissue samples (n = 4) were scanned using a Nikon metrology µCT scanner. Semi-automatic techniques were used to segment the 3D structure of airways and blood vessels. Airspace size (mean linear intercept, Lm) was measured on µCT images and on matched histological sections from the same FFPE samples imaged by light microscopy to validate µCT imaging. Results The µCT imaging protocol provided contrast between tissue and paraffin in FFPE samples (15mm x 7mm). Resolution (voxel size 6.7 µm) in the reconstructed images was sufficient for semi-automatic image segmentation of airways and blood vessels as well as quantitative airspace analysis. The scans were also used to scout for regions of interest, enabling time-efficient preparation of conventional histological sections. The Lm measurements from µCT images were not significantly different to those from matched histological sections. Conclusion We demonstrated how non-destructive imaging of routinely prepared FFPE samples by laboratory µCT can be used to visualize and assess the 3D morphology of the lung including by morphometric analysis.

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

Summary 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.

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

Highlight Rigorous mathematical techniques and image-based modelling were used to quantify the effect of root hairs on nutrient uptake and to investigate how uptake is influenced by growing root hairs.

Mapping soil deformation around plant roots using in vivo 4D X-ray Computed Tomography and Digital Volume Correlation.

The mechanical impedance of soils inhibits the growth of plant roots, often being the most significant physical limitation to root system development. Non-invasive imaging techniques have recently been used to investigate the development of root system architecture over time, but the relationship with soil deformation is usually neglected. Correlative mapping approaches parameterised using 2D and 3D image data have recently gained prominence for quantifying physical deformation in composite materials including fibre-reinforced polymers and trabecular bone. Digital Image Correlation (DIC) and Digital Volume Correlation (DVC) are computational techniques which use the inherent material texture of surfaces and volumes, captured using imaging techniques, to map full-field deformation components in samples during physical loading. Here we develop an experimental assay and methodology for four-dimensional, in vivo X-ray Computed Tomography (XCT) and apply a Digital Volume Correlation (DVC) approach to the data to quantify deformation. The method is validated for a field-derived soil under conditions of uniaxial compression, and a calibration study is used to quantify thresholds of displacement and strain measurement. The validated and calibrated approach is then demonstrated for an in vivo test case in which an extending maize root in field-derived soil was imaged hourly using XCT over a growth period of 19h. This allowed full-field soil deformation data and 3D root tip dynamics to be quantified in parallel for the first time. This fusion of methods paves the way for comparative studies of contrasting soils and plant genotypes, improving our understanding of the fundamental mechanical processes which influence root system development.

Measurement of micro-scale soil deformation around roots using four-dimensional synchrotron tomography and image correlation

This study applied time lapse (four-dimensional) synchrotron X-ray computed tomography to observe micro-scale interactions between plant roots and soil. Functionally contrasting maize root tips were repeatedly imaged during ingress into soil columns of varying water content and compaction. This yielded sequences of three-dimensional densiometric data, representing time-resolved geometric soil and root configurations at the micronmetre scale. These data were used as inputs for two full-field kinematic quantification methods, which enabled the analysis of three-dimensional soil deformation around elongating roots. Discrete object tracking was used to track rigid mineral grains, while continuum digital volume correlation was used to track grey-level patterns within local sub-volumes. These techniques both allowed full-field soil displacements to be quantified at an intra-rhizosphere spatial sampling scale of less than 300 µm. Significant differences in deformation mechanisms were identified around different phenotypes under different soil conditions. A uniquely strong contrast was observed between intact and de-capped roots grown in dry, compacted soil. This provides evidence that functional traits of the root cap significantly reduce the amount of soil disturbance per unit of root elongation, with this effect being particularly significant in drier soil.

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.