Ethylene inhibits rice root elongation in compacted soil via ABA- and auxin-mediated mechanisms.

Soil compaction represents a major agronomic challenge, inhibiting root elongation and impacting crop yields. Roots use ethylene to sense soil compaction as the restricted air space causes this gaseous signal to accumulate around root tips. Ethylene inhibits root elongation and promotes radial expansion in compacted soil, but its mechanistic basis remains unclear. Here, we report that ethylene promotes abscisic acid (ABA) biosynthesis and cortical cell radial expansion. Rice mutants of ABA biosynthetic genes had attenuated cortical cell radial expansion in compacted soil, leading to better penetration. Soil compaction-induced ethylene also up-regulates the auxin biosynthesis gene OsYUC8. Mutants lacking OsYUC8 are better able to penetrate compacted soil. The auxin influx transporter OsAUX1 is also required to mobilize auxin from the root tip to the elongation zone during a root compaction response. Moreover, osaux1 mutants penetrate compacted soil better than the wild-type roots and do not exhibit cortical cell radial expansion. We conclude that ethylene uses auxin and ABA as downstream signals to modify rice root cell elongation and radial expansion, causing root tips to swell and reducing their ability to penetrate compacted soil.

Root angle is controlled by EGT1 in cereal crops employing an antigravitropic mechanism.

Root angle in crops represents a key trait for efficient capture of soil resources. Root angle is determined by competing gravitropic versus antigravitropic offset (AGO) mechanisms. Here we report a root angle regulatory gene termed ENHANCED GRAVITROPISM1 (EGT1) that encodes a putative AGO component, whose loss-of-function enhances root gravitropism. Mutations in barley and wheat EGT1 genes confer a striking root phenotype, where every root class adopts a steeper growth angle. EGT1 encodes an F-box and Tubby domain-containing protein that is highly conserved across plant species. Haplotype analysis found that natural allelic variation at the barley EGT1 locus impacts root angle. Gravitropic assays indicated that Hvegt1 roots bend more rapidly than wild-type. Transcript profiling revealed Hvegt1 roots deregulate reactive oxygen species (ROS) homeostasis and cell wall-loosening enzymes and cofactors. ROS imaging shows that Hvegt1 root basal meristem and elongation zone tissues have reduced levels. Atomic force microscopy measurements detected elongating Hvegt1 root cortical cell walls are significantly less stiff than wild-type. In situ analysis identified HvEGT1 is expressed in elongating cortical and stele tissues, which are distinct from known root gravitropic perception and response tissues in the columella and epidermis, respectively. We propose that EGT1 controls root angle by regulating cell wall stiffness in elongating root cortical tissue, counteracting the gravitropic machinery's known ability to bend the root via its outermost tissues. We conclude that root angle is controlled by EGT1 in cereal crops employing an antigravitropic mechanism.

The auxin efflux carrier PIN1a regulates vascular patterning in cereal roots.

Barley (Hordeum vulgare) is an important global cereal crop and a model in genetic studies. Despite advances in characterising barley genomic resources, few mutant studies have identified genes controlling root architecture and anatomy, which plays a critical role in capturing soil resources. Our phenotypic screening of a TILLING mutant collection identified line TM5992 exhibiting a short-root phenotype compared with wild-type (WT) Morex background. Outcrossing TM5992 with barley variety Proctor and subsequent SNP array-based bulk segregant analysis, fine mapped the mutation to a cM scale. Exome sequencing pinpointed a mutation in the candidate gene HvPIN1a, further confirming this by analysing independent mutant alleles. Detailed analysis of root growth and anatomy in Hvpin1a mutant alleles exhibited a slower growth rate, shorter apical meristem and striking vascular patterning defects compared to WT. Expression and mutant analyses of PIN1 members in the closely related cereal brachypodium (Brachypodium distachyon) revealed that BdPIN1a and BdPIN1b were redundantly expressed in root vascular tissues but only Bdpin1a mutant allele displayed root vascular defects similar to Hvpin1a. We conclude that barley PIN1 genes have sub-functionalised in cereals, compared to Arabidopsis (Arabidopsis thaliana), where PIN1a sequences control root vascular patterning.

Novel Insights Into the Architecture of Macro and Microstructures in Cattle Ossa Cordis.

Ossa cordis, bones located within the heart trigones, are often classified as heterotopic or ectopic bones. Despite their high prevalence in cattle and some other bovids, little is known about their structure or development. Scanning electron microscopy, X-ray microtomography, gross dissections, and measurements showed the anatomical locations, prevalence, shapes, and measurements of the cardiac bones in both Egyptian Baladi cattle and Holstein-Friesians. All cattle (n = 12) had an Ossa cordis dextrum (average = 50.70 × 20.91 × 5.40 mm). Additionally, 80% Egyptian Baladi and 57% Holstein-Friesian had a smaller Ossa cordis sinistrum (average = 24.94 × 12.75 × 4.12 mm). Egyptian Baladi Ossa cordis were smaller than observed in Holstein-Friesians. Energy-dispersive X-ray analysis showed the elemental constitution (carbon, oxygen, calcium, nitrogen, phosphorus, sodium, and magnesium) of Ossa cordis and Cartilago cordis. These imaging techniques, plus four histological stains (hematoxylin and eosin, Crossman's trichrome, Alcian blue with Van Gieson, and Sirius Red) and microscopy, demonstrated osteoblasts, osteocytes, osteoclasts, astrocytes, blood vessels, bone marrow, lamellar and woven bone, cortical bone, trabeculations with pores and canaliculi, and fibrous components including collagen in the Ossa cordis dextrum and sinistrum. Hyaline cartilage and fibrocartilage (chondrocytes and cartilage matrix) were found within and surrounding the Ossa cordis. These findings were additionally compared against other cattle breeds and species.

The interaction between wheat roots and soil pores in structured field soil.

Wheat (Triticum aestivum L.) root growth in the subsoil is usually constrained by soil strength, although roots can use macropores to elongate to deeper layers. The quantitative relationship between the elongation of wheat roots and the soil pore system, however, is still to be determined. We studied the depth distribution of roots of six wheat varieties and explored their relationship with soil macroporosity from samples with the field structure preserved. Undisturbed soil cores (to a depth of 100 cm) were collected from the field and then non-destructively imaged using X-ray computed tomography (at a spatial resolution of 90 µm) to quantify soil macropore structure and root number density (the number of roots cm-2 within a horizontal cross-section of a soil core). Soil macroporosity changed significantly with depth but not between the different wheat lines. There was no significant difference in root number density between wheat varieties. In the subsoil, wheat roots used macropores, especially biopores (i.e. former root or earthworm channels) to grow into deeper layers. Soil macroporosity explained 59% of the variance in root number density. Our data suggested that the development of the wheat root system in the field was more affected by the soil macropore system than by genotype. On this basis, management practices which enhance the porosity of the subsoil may therefore be an effective strategy to improve deep rooting of wheat.

CEP receptor signalling controls root system architecture in Arabidopsis and Medicago.

Root system architecture (RSA) influences the effectiveness of resources acquisition from soils but the genetic networks that control RSA remain largely unclear. We used rhizoboxes, X-ray computed tomography, grafting, auxin transport measurements and hormone quantification to demonstrate that Arabidopsis and Medicago CEP (C-TERMINALLY ENCODED PEPTIDE)-CEP RECEPTOR signalling controls RSA, the gravitropic set-point angle (GSA) of lateral roots (LRs), auxin levels and auxin transport. We showed that soil-grown Arabidopsis and Medicago CEP receptor mutants have a narrower RSA, which results from a steeper LR GSA. Grafting showed that CEPR1 in the shoot controls GSA. CEP receptor mutants exhibited an increase in rootward auxin transport and elevated shoot auxin levels. Consistently, the application of auxin to wild-type shoots induced a steeper GSA and auxin transport inhibitors counteracted the CEP receptor mutant's steep GSA phenotype. Concordantly, CEP peptides increased GSA and inhibited rootward auxin transport in wild-type but not in CEP receptor mutants. The results indicated that CEP-CEP receptor-dependent signalling outputs in Arabidopsis and Medicago control overall RSA, LR GSA, shoot auxin levels and rootward auxin transport. We propose that manipulating CEP signalling strength or CEP receptor downstream targets may provide means to alter RSA.

The role of plant species and soil condition in the structural development of the rhizosphere.

Roots naturally exert axial and radial pressures during growth, which alter the structural arrangement of soil at the root-soil interface. However, empirical models suggest soil densification, which can have negative impacts on water and nutrient uptake, occurs at the immediate root surface with decreasing distance from the root. Here, we spatially map structural gradients in the soil surrounding roots using non-invasive imaging, to ascertain the role of root growth in early stage formation of soil structure. X-ray computed tomography provided a means not only to visualize a root system in situ and in 3-D but also to assess the precise root-induced alterations to soil structure close to, and at selected distances away from the root-soil interface. We spatially quantified the changes in soil structure generated by three common but contrasting plant species (pea, tomato, and wheat) under different soil texture and compaction treatments. Across the three plant types, significant increases in porosity at the immediate root surface were found in both clay loam and loamy sand soils and not soil densification, the currently assumed norm. Densification of the soil was recorded, at some distance away from the root, dependent on soil texture and plant type. There was a significant soil texture × bulk density × plant species interaction for the root convex hull, a measure of the extent to which root systems explore the soil, which suggested pea and wheat grew better in the clay soil when at a high bulk density, compared with tomato, which preferred lower bulk density soils. These results, only revealed by high resolution non-destructive imagery, show that although the root penetration mechanisms can lead to soil densification (which could have a negative impact on growth), the immediate root-soil interface is actually a zone of high porosity, which is very important for several key rhizosphere processes occurring at this scale including water and nutrient uptake and gaseous diffusion.

LARGE ROOT ANGLE1, encoding OsPIN2, is involved in root system architecture in rice.

Root system architecture is very important for plant growth and crop yield. It is essential for nutrient and water uptake, anchoring, and mechanical support. Root growth angle (RGA) is a vital constituent of root system architecture and is used as a parameter for variety evaluation in plant breeding. However, little is known about the underlying molecular mechanisms that determine root growth angle in rice (Oryza sativa). In this study, a rice mutant large root angle1 (lra1) was isolated and shown to exhibit a large RGA and reduced sensitivity to gravity. Genome resequencing and complementation assays identified OsPIN2 as the gene responsible for the mutant phenotypes. OsPIN2 was mainly expressed in roots and the base of shoots, and showed polar localization in the plasma membrane of root epidermal and cortex cells. OsPIN2 was shown to play an important role in mediating root gravitropic responses in rice and was essential for plants to produce normal RGAs. Taken together, our findings suggest that OsPIN2 plays an important role in root gravitropic responses and determining the root system architecture in rice by affecting polar auxin transport in the root tip.

Soil seal development under simulated rainfall: Structural, physical and hydrological dynamics.

This study delivers new insights into rainfall-induced seal formation through a novel approach in the use of X-ray Computed Tomography (CT). Up to now seal and crust thickness have been directly quantified mainly through visual examination of sealed/crusted surfaces, and there has been no quantitative method to estimate this important property. X-ray CT images were quantitatively analysed to derive formal measures of seal and crust thickness. A factorial experiment was established in the laboratory using open-topped microcosms packed with soil. The factors investigated were soil type (three soils: silty clay loam - ZCL, sandy silt loam - SZL, sandy loam - SL) and rainfall duration (2-14 min). Surface seal formation was induced by applying artificial rainfall events, characterised by variable duration, but constant kinetic energy, intensity, and raindrop size distribution. Soil porosities derived from CT scans were used to quantify the thickness of the rainfall-induced surface seals and reveal temporal seal micro-morphological variations with increasing rainfall duration. In addition, the water repellency and infiltration dynamics of the developing seals were investigated by measuring water drop penetration time (WDPT) and unsaturated hydraulic conductivity (Kun). The range of seal thicknesses detected varied from 0.6 to 5.4 mm. Soil textural characteristics and OM content played a central role in the development of rainfall-induced seals, with coarser soil particles and lower OM content resulting in thicker seals. Two different trends in soil porosity vs. depth were identified: i) for SL soil porosity was lowest at the immediate soil surface, it then increased constantly with depth till the median porosity of undisturbed soil was equalled; ii) for ZCL and SL the highest reduction in porosity, as compared to the median porosity of undisturbed soil, was observed in a well-defined zone of maximum porosity reduction c. 0.24-0.48 mm below the soil surface. This contrasting behaviour was related to different dynamics and processes of seal formation which depended on the soil properties. The impact of rainfall-induced surface sealing on the hydrological behaviour of soil (as represented by WDTP and Kun) was rapid and substantial: an average 60% reduction in Kun occurred for all soils between 2 and 9 min rainfall, and water repellent surfaces were identified for SZL and ZCL. This highlights that the condition of the immediate surface of agricultural soils involving rainfall-induced structural seals has a strong impact in the overall ability of soil to function as water reservoir.

Roots-eye view: Using microdialysis and microCT to non-destructively map root nutrient depletion and accumulation zones.

Improvement in fertilizer use efficiency is a key aspect for achieving sustainable agriculture in order to minimize costs, greenhouse gas emissions, and pollution from nutrient run-off. To optimize root architecture for nutrient uptake and efficiency, we need to understand what the roots encounter in their environment. Traditional methods of nutrient sampling, such as salt extractions can only be done at the end of an experiment, are impractical for sampling locations precisely and give total nutrient values that can overestimate the nutrients available to the roots. In contrast, microdialysis provides a non-invasive, continuous method for sampling available nutrients in the soil. Here, for the first time, we have used microCT imaging to position microdialysis probes at known distances from the roots and then measured the available nitrate and ammonium. We found that nitrate accumulated close to roots whereas ammonium was depleted demonstrating that this combination of complementary techniques provides a unique ability to measure root-available nutrients non-destructively and in almost real time.

A Herbivore Tag-and-Trace System Reveals Contact- and Density-Dependent Repellence of a Root Toxin.

Foraging behavior of root feeding organisms strongly affects plant-environment-interactions and ecosystem processes. However, the impact of plant chemistry on root herbivore movement in the soil is poorly understood. Here, we apply a simple technique to trace the movement of soil-dwelling insects in their habitats without disturbing or restricting their interactions with host plants. We tagged the root feeding larvae of Melolontha melolontha with a copper ring and repeatedly located their position in relation to their preferred host plant, Taraxacum officinale, using a commercial metal detector. This method was validated and used to study the influence of the sesquiterpene lactone taraxinic acid ß-D-glucopyranosyl ester (TA-G) on the foraging of M. melolontha. TA-G is stored in the latex of T. officinale and protects the roots from herbivory. Using behavioral arenas with TA-G deficient and control plants, we tested the impact of physical root access and plant distance on the effect of TA-G on M. melolontha. The larvae preferred TA-G deficient plants to control plants, but only when physical root contact was possible and the plants were separated by 5 cm. Melolontha melolontha showed no preference for TA-G deficient plants when the plants were grown 15 cm apart, which may indicate a trade-off between the cost of movement and the benefit of consuming less toxic food. We demonstrate that M. melolontha integrates host plant quality and distance into its foraging patterns and suggest that plant chemistry affects root herbivore behavior in a plant-density dependent manner.

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

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.

Extracting multiple interacting root systems using X-ray microcomputed tomography.

Root system interactions and competition for resources are active areas of research that contribute to our understanding of how roots perceive and react to environmental conditions. Recent research has shown this complex suite of processes can now be observed in a natural environment (i.e. soil) through the use of X-ray microcomputed tomography (µCT), which allows non-destructive analysis of plant root systems. Due to their similar X-ray attenuation coefficients and densities, the roots of different plants appear as similar greyscale intensity values in µCT image data. Unless they are manually and carefully traced, it has not previously been possible to automatically label and separate different root systems grown in the same soil environment. We present a technique, based on a visual tracking approach, which exploits knowledge of the shape of root cross-sections to automatically recover from X-ray µCT data three-dimensional descriptions of multiple, interacting root architectures growing in soil. The method was evaluated on both simulated root data and real images of two interacting winter wheat Cordiale (Triticumaestivum L.) plants grown in a single soil column, demonstrating that it is possible to automatically segment different root systems from within the same soil sample. This work supports the automatic exploration of supportive and competitive foraging behaviour of plant root systems in natural soil environments.

X-ray computed tomography: A novel non-invasive approach for the detection of microplastics in sediments?

As a non-invasive imaging technique, this study explores the application of Computed Tomography (CT) in microplastics research, assessing its potential to distinguish different types and sizes of microplastics (polypropylene, polyethylene terephthalate, polyethylene, and polyvinyl chloride) from homogenised river-estuarine sediment. When examined in layers within artificial cores, all microplastic types could be observed by CT imagery, with good contrast in X-ray attenuation (based on image gray level intensity) against background sediments. Large microplastics (4 mm diameter) were also detectable when distributed randomly amongst the sediment. These spiked cores had sufficient difference in attenuation to allow segmentation between type, and therefore isolate individual microplastics. Due to limitations on scan resolution, smaller microplastics (≤125 µm diameter) could not be detected in spiked cores. Scans of two sediment cores from a Thames River tributary (UK) revealed two distinctive sediment structures which could influence microplastic accumulation. This information would be lost using conventional recovery procedures.

ERFVII action and modulation through oxygen-sensing in Arabidopsis thaliana.

Oxygen is a key signalling component of plant biology, and whilst an oxygen-sensing mechanism was previously described in Arabidopsis thaliana, key features of the associated PLANT CYSTEINE OXIDASE (PCO) N-degron pathway and Group VII ETHYLENE RESPONSE FACTOR (ERFVII) transcription factor substrates remain untested or unknown. We demonstrate that ERFVIIs show non-autonomous activation of root hypoxia tolerance and are essential for root development and survival under oxygen limiting conditions in soil. We determine the combined effects of ERFVIIs in controlling gene expression and define genetic and environmental components required for proteasome-dependent oxygen-regulated stability of ERFVIIs through the PCO N-degron pathway. Using a plant extract, unexpected amino-terminal cysteine sulphonic acid oxidation level of ERFVIIs was observed, suggesting a requirement for additional enzymatic activity within the pathway. Our results provide a holistic understanding of the properties, functions and readouts of this oxygen-sensing mechanism defined through its role in modulating ERFVII stability.

Anatomy, histology, development and functions of Ossa cordis: A review.

This systematic review highlights the similarities and variations in Ossa cordis prevalence, histology and anatomical location between differing veterinary species and in humans. In addition, it also identifies associated factors such as aging and cardiovascular disease for each species in relation to functional roles and developmental mechanisms that these bone structures may play. The potential functions of Ossa cordis are presented, ranging from aiding cardiac contraction and conduction, providing cardiac structure, and protecting components of the heart, through to counteracting high mechanical stress. Furthermore, this review discusses the evidence and rationale behind the theories regarding the formation and development of Ossa cordis in different veterinary species and in people.

Hydraulic flux-responsive hormone redistribution determines root branching.

Plant roots exhibit plasticity in their branching patterns to forage efficiently for heterogeneously distributed resources, such as soil water. The xerobranching response represses lateral root formation when roots lose contact with water. Here, we show that xerobranching is regulated by radial movement of the phloem-derived hormone abscisic acid, which disrupts intercellular communication between inner and outer cell layers through plasmodesmata. Closure of these intercellular pores disrupts the inward movement of the hormone signal auxin, blocking lateral root branching. Once root tips regain contact with moisture, the abscisic acid response rapidly attenuates. Our study reveals how roots adapt their branching pattern to heterogeneous soil water conditions by linking changes in hydraulic flux with dynamic hormone redistribution.

Non-invasive Imaging of Rice Roots in Non-compacted and Compacted Soil.

Roots are the prime organ for nutrient and water uptake and are therefore fundamental to the growth and development of plants. However, physical challenges of a heterogeneous environment and diverse edaphic stresses affect root growth in soil. Compacted soil is a serious global problem, causing inhibition of root elongation, which reduces surface area and impacts resource foraging. Visualisation and quantification of roots in soil is difficult due to this growth substrate's opaque nature; however, non-destructive imaging technologies are now becoming more widely available to plant and soil scientists working to address this challenge. We have recently developed an integrated approach, combining X-ray Computed Tomography (X-ray CT) and confocal microscopy to image roots grown in compacted soil conditions from a plant to a cellular scale. The method is suited to visualize cellular responses of root tips grown in both non-compacted and compacted soils. This protocol presents a fully integrated workflow, including soil column preparation, creation of compaction conditions, plant growth, imaging, and quantification of root adaptive responses at a cellular scale.

Ploidy influences wheat mesophyll cell geometry, packing and leaf function.

Leaf function is influenced by leaf structure, which is itself related not only to the spatial arrangement of constituent mesophyll cells, but also their size and shape. In this study, we used confocal microscopy to image leaves of Triticum genotypes varying in ploidy level to extract 3D information on individual mesophyll cell size and geometry. Combined with X-ray Computed Tomography and gas exchange analysis, the effect of changes in wheat mesophyll cell geometry upon leaf structure and function were investigated. Mesophyll cell size and shape were found to have changed during the course of wheat evolution. An unexpected linear relationship between mesophyll cell surface area and volume was discovered, suggesting anisotropic scaling of mesophyll cell geometry with increasing ploidy. Altered mesophyll cell size and shape were demonstrated to be associated with changes in mesophyll tissue architecture. Under experimental growth conditions, CO2 assimilation did not vary with ploidy, but stomatal conductance was lower in hexaploid plants, conferring a greater instantaneous water-use efficiency. We propose that as wheat mesophyll cells have become larger with increased ploidy, this has been accompanied by changes in cell geometry and packing which limit water loss while maintaining carbon assimilation.

Determination of wheat spike and spikelet architecture and grain traits using X-ray Computed Tomography imaging.

BACKGROUND: Wheat spike architecture is a key determinant of multiple grain yield components and detailed examination of spike morphometric traits is beneficial to explain wheat grain yield and the effects of differing agronomy and genetics. However, quantification of spike morphometric traits has been very limited because it relies on time-consuming manual measurements. RESULTS: In this study, using X-ray Computed Tomography imaging, we proposed a method to efficiently detect the 3D architecture of wheat spikes and component spikelets by clustering grains based on their Euclidean distance and relative positions. Morphometric characteristics of wheat spikelets and grains, e.g., number, size and spatial distribution along the spike can be determined. Two commercial wheat cultivars, one old, Maris Widgeon, and one modern, Siskin, were studied as examples. The average grain volume of Maris Widgeon and Siskin did not differ, but Siskin had more grains per spike and therefore greater total grain volume per spike. The spike length and spikelet number were not statistically different between the two cultivars. However, Siskin had a higher spikelet density (number of spikelets per unit spike length), with more grains and greater grain volume per spikelet than Maris Widgeon. Spatial distribution analysis revealed the number of grains, the average grain volume and the total grain volume of individual spikelets varied along the spike. Siskin had more grains and greater grain volumes per spikelet from spikelet 6, but not spikelet 1-5, compared with Maris Widgeon. The distribution of average grain volume along the spike was similar for the two wheat cultivars. CONCLUSION: The proposed method can efficiently extract spike, spikelet and grain morphometric traits of different wheat cultivars, which can contribute to a more detailed understanding of the sink of wheat grain yield.