Craig J. Sturrock

RooTrak: Automated Recovery of Three-Dimensional Plant Root Architecture in Soil from X-Ray Microcomputed Tomography Images Using Visual Tracking

X-ray microcomputed tomography (μCT) is an invaluable tool for visualizing plant root systems within their natural soil environment noninvasively. However, variations in the x-ray attenuation values of root material and the overlap in attenuation values between roots and soil caused by water and organic materials represent major challenges to data recovery. We report the development of automatic root segmentation methods and software that view μCT data as a sequence of images through which root objects appear to move as the x-y cross sections are traversed along the z axis of the image stack. Previous approaches have employed significant levels of user interaction and/or fixed criteria to distinguish root and nonroot material. RooTrak exploits multiple, local models of root appearance, each built while tracking a specific segment, to identify new root material. It requires minimal user interaction and is able to adapt to changing root density estimates. The model-guided search for root material arising from the adoption of a visual-tracking framework makes RooTrak less sensitive to the natural ambiguity of x-ray attenuation data. We demonstrate the utility of RooTrak using μCT scans of maize (Zea mays), wheat (Triticum aestivum), and tomato (Solanum lycopersicum) grown in a range of contrasting soil textures. Our results demonstrate that RooTrak can successfully extract a range of root architectures from the surrounding soil and promises to facilitate future root phenotyping efforts.

Plant roots use a patterning mechanism to position lateral root branches toward available water

Significance Few studies have asked at what spatial scale environmental stimuli regulate plant development and when during the patterning process these signals act. We have discovered that plant roots can sense microscale heterogeneity in water availability across their circumference, which causes dramatic differences in the patterning of tissues along this axis. Root branching is a target of such hydropatterning; lateral roots only form on the side of the main root contacting water in soil or agar. We show that hydropatterning is a conserved process in Arabidopsis, maize, and rice and reveal the importance of auxin biosynthesis and transport in regulating this process. The architecture of the branched root system of plants is a major determinant of vigor. Water availability is known to impact root physiology and growth; however, the spatial scale at which this stimulus influences root architecture is poorly understood. Here we reveal that differences in the availability of water across the circumferential axis of the root create spatial cues that determine the position of lateral root branches. We show that roots of several plant species can distinguish between a wet surface and air environments and that this also impacts the patterning of root hairs, anthocyanins, and aerenchyma in a phenomenon we describe as hydropatterning. This environmental response is distinct from a touch response and requires available water to induce lateral roots along a contacted surface. X-ray microscale computed tomography and 3D reconstruction of soil-grown root systems demonstrate that such responses also occur under physiologically relevant conditions. Using early-stage lateral root markers, we show that hydropatterning acts before the initiation stage and likely determines the circumferential position at which lateral root founder cells are specified. Hydropatterning is independent of endogenous abscisic acid signaling, distinguishing it from a classic water-stress response. Higher water availability induces the biosynthesis and transport of the lateral root-inductive signal auxin through local regulation of TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 and PIN-FORMED 3, both of which are necessary for normal hydropatterning. Our work suggests that water availability is sensed and interpreted at the suborgan level and locally patterns a wide variety of developmental processes in the root.

Recovering complete plant root system architectures from soil via X-ray μ-Computed Tomography.

BackgroundX-ray micro-Computed Tomography (μCT) offers the ability to visualise the three-dimensional structure of plant roots growing in their natural environment – soil. Recovery of root architecture descriptions from X-ray CT data is, however, challenging. The X-ray attenuation values of roots and soil overlap, and the attenuation values of root material vary. Any successful root identification method must both explicitly target root material and be able to adapt to local changes in root properties.RooTrak meets these requirements by combining the level set method with a visual tracking framework and has been shown to be capable of segmenting a variety of plant roots from soil in X-ray μCT images. The approach provides high quality root descriptions, but tracks root systems top to bottom and so omits upward-growing (plagiotropic) branches.ResultsWe present an extension to RooTrak which allows it to extract plagiotropic roots. An additional backward-looking step revisits the previous image, marking possible upward-growing roots. These are then tracked, leading to efficient and more complete recovery of the root system. Results show clear improvement in root extraction, without which key architectural traits would be underestimated.ConclusionsThe visual tracking framework adopted in RooTrak provides the focus and flexibility needed to separate roots from soil in X-ray CT imagery and can be extended to detect plagiotropic roots. The extended software tool produces more complete descriptions of plant root structure and supports more accurate computation of architectural traits.

Branching Out in Roots: uncovering form, function and regulation

The diversity of postembryonic root forms and their functions add to our understanding of the genes, signals and mechanisms regulating lateral and adventitious root branching in the plant models Arabidopsis and rice. Root branching is critical for plants to secure anchorage and ensure the supply of water, minerals, and nutrients. To date, research on root branching has focused on lateral root development in young seedlings. However, many other programs of postembryonic root organogenesis exist in angiosperms. In cereal crops, the majority of the mature root system is composed of several classes of adventitious roots that include crown roots and brace roots. In this Update, we initially describe the diversity of postembryonic root forms. Next, we review recent advances in our understanding of the genes, signals, and mechanisms regulating lateral root and adventitious root branching in the plant models Arabidopsis (Arabidopsis thaliana), maize (Zea mays), and rice (Oryza sativa). While many common signals, regulatory components, and mechanisms have been identified that control the initiation, morphogenesis, and emergence of new lateral and adventitious root organs, much more remains to be done. We conclude by discussing the challenges and opportunities facing root branching research.

Investigation of growth responses in saprophytic fungi to charred biomass.

We present the results of a study testing the response of two saprophytic white-rot fungi species, Pleurotus pulmonarius and Coriolus versicolor, to charred biomass (charcoal) as a growth substrate. We used a combination of optical microscopy, scanning electron microscopy, elemental abundance measurements, and isotope ratio mass spectrometry (13C and 15N) to investigate fungal colonisation of control and incubated samples of Scots Pine (Pinus sylvestris) wood, and charcoal from the same species produced at 300 °C and 400 °C. Both species of fungi colonise the surface and interior of wood and charcoals over time periods of less than 70 days; however, distinctly different growth forms are evident between the exterior and interior of the charcoal substrate, with hyphal penetration concentrated along lines of structural weakness. Although the fungi were able to degrade and metabolise the pine wood, charcoal does not form a readily available source of fungal nutrients at least for these species under the conditions used in this study.

Three‐dimensional quantification of soil hydraulic properties using X‐ray Computed Tomography and image‐based modeling

We demonstrate the application of a high-resolution X-ray Computed Tomography (CT) method to quantify water distribution in soil pores under successive reductive drying. We focus on the wet end of the water release characteristic (WRC) (0 to −75 kPa) to investigate changes in soil water distribution in contrasting soil textures (sand and clay) and structures (sieved and field structured) and to determine the impact of soil structure on hydraulic behavior. The 3-D structure of each soil was obtained from the CT images (at a 10 μm resolution). Stokes equations for flow were solved computationally for each measured structure to estimate hydraulic conductivity. The simulated values obtained compared extremely well with the measured saturated hydraulic conductivity values. By considering different sample sizes we were able to identify the smallest possible representative sample size which is required to determine a globally valid hydraulic conductivity.

To what extent can zero tillage lead to a reduction in greenhouse gas emissions from temperate soils

Soil tillage practices have a profound influence on the physical properties of soil and the greenhouse gas (GHG) balance. However there have been very few integrated studies on the emission of carbon dioxide (CO2), methane (CH4) and nitrous oxide (N2O) and soil biophysical and chemical characteristics under different soil management systems. We recorded a significantly higher net global warming potential under conventional tillage systems (26–31% higher than zero tillage systems). Crucially the 3-D soil pore network, imaged using X-ray Computed Tomography, modified by tillage played a significant role in the flux of CO2 and CH4. In contrast, N2O flux was determined mainly by microbial biomass carbon and soil moisture content. Our work indicates that zero tillage could play a significant role in minimising emissions of GHGs from soils and contribute to efforts to mitigate against climate change.

The effects of biochar on soil physical properties and winter wheat growth

Biochar has been reported to improve soil quality and crop yield; however, less is known about its effects on the physical and, in particular, structural properties of soil. This study examines the potential ability of biochar to improve water retention and crop growth through a pot trial using biochar concentrations of 0%, 1·5%, 2·5% and 5% w/w. X-ray computed tomography was used to measure soil structure via pore size characteristics; this showed that pore size is significantly affected by biochar concentration. Increasing biochar is associated with decreasing average pore size, which we hypothesise would impact heavily on hydraulic performance. At the end of the experiment, average pore size had decreased from 0·07 mm 2 in the 0% biochar soil to 0·046 mm 2 in the 5% biochar soil. Increased biochar concentration also significantly decreases saturated hydraulic conductivity and soil bulk density. It was also observed that increased biochar significantly decreases soil water repellency. Increased water retention was also observed at low matric potentials, where it was shown that increased biochar is able to retain more water as the soil dried out. The application of biochar had little effect on short-term (

Effects of X-Ray Dose On Rhizosphere Studies Using X-Ray Computed Tomography

X-ray Computed Tomography (CT) is a non-destructive imaging technique originally designed for diagnostic medicine, which was adopted for rhizosphere and soil science applications in the early 1980s. X-ray CT enables researchers to simultaneously visualise and quantify the heterogeneous soil matrix of mineral grains, organic matter, air-filled pores and water-filled pores. Additionally, X-ray CT allows visualisation of plant roots in situ without the need for traditional invasive methods such as root washing. However, one routinely unreported aspect of X-ray CT is the potential effect of X-ray dose on the soil-borne microorganisms and plants in rhizosphere investigations. Here we aimed to i) highlight the need for more consistent reporting of X-ray CT parameters for dose to sample, ii) to provide an overview of previously reported impacts of X-rays on soil microorganisms and plant roots and iii) present new data investigating the response of plant roots and microbial communities to X-ray exposure. Fewer than 5% of the 126 publications included in the literature review contained sufficient information to calculate dose and only 2.4% of the publications explicitly state an estimate of dose received by each sample. We conducted a study involving rice roots growing in soil, observing no significant difference between the numbers of root tips, root volume and total root length in scanned versus unscanned samples. In parallel, a soil microbe experiment scanning samples over a total of 24 weeks observed no significant difference between the scanned and unscanned microbial biomass values. We conclude from the literature review and our own experiments that X-ray CT does not impact plant growth or soil microbial populations when employing a low level of dose (<30 Gy). However, the call for higher throughput X-ray CT means that doses that biological samples receive are likely to increase and thus should be closely monitored.

Seeing space: visualization and quantification of plant leaf structure using X-ray micro-computed tomography View Point

A mechanistic understanding of plant physiological processes occurring in the above-ground organs, such as photosynthesis in leaves, requires knowledge of the structural properties of the tissues, including leaf thickness (Hanba et al., 1999; Zhu et al., 2010; Niinemets et al., 2011), cell size, and stomatal distribution influencing gas exchange (Sage and Sage, 2009; Gong et al., 2011; Terashima et al., 2011). However, previous work has been unable to determine precisely how the three dimensional (3D) complexity of leaf structure impacts upon photosynthetic activity, partly due to a lack of appropriate methods for undisturbed visualization and quantification of inner structures at an appropriate high resolution (i.e. 5–10 μm). Although there is a wide range of traditional, optical-based techniques for visualization and quantification of plant morphology and anatomy, ranging from imaging of thin sections to confocal microscopy of intact specimens, their use is usually limited by the type and size of sample material. Furthermore, analysis is often time consuming, destructive, and constrained to two dimensions (2D) (classical histology) or limited in optical depth (confocal microscopy).