Construction and characterisation of a structured, tuneable, and transparent 3D culture platform for soil bacteria.

We have developed a tuneable workflow for the study of soil microbes in an imitative 3D soil environment that is compatible with routine and advanced optical imaging, is chemically customisable, and is reliably refractive index matched based on the carbon catabolism of the study organism. We demonstrate our transparent soil pipeline with two representative soil organisms, Bacillus subtilis and Streptomyces coelicolor, and visualise their colonisation behaviours using fluorescence microscopy and mesoscopy. This spatially structured, 3D approach to microbial culture has the potential to further study the behaviour of bacteria in conditions matching their native environment and could be expanded to study microbial interactions, such as competition and warfare.

Mobility and growth in confined spaces are important mechanisms for the establishment of Bacillus subtilis in the rhizosphere.

The rhizosphere hosts complex and abundant microbiomes whose structure and composition are now well described by metagenomic studies. However, the dynamic mechanisms that enable micro-organisms to establish along a growing plant root are poorly characterized. Here, we studied how a motile bacterium utilizes the microhabitats created by soil pore space to establish in the proximity of plant roots. We have established a model system consisting of Bacillus subtilis and lettuce seedlings co-inoculated in transparent soil microcosms. We carried out live imaging experiments and developed image analysis pipelines to quantify the abundance of the bacterium as a function of time and position in the pore space. Results showed that the establishment of the bacterium in the rhizosphere follows a precise sequence of events where small islands of mobile bacteria were first seen forming near the root tip within the first 12-24 h of inoculation. Biofilm was then seen forming on the root epidermis at distances of about 700-1000 µm from the tip. Bacteria accumulated predominantly in confined pore spaces within 200 µm from the root or the surface of a particle. Using probabilistic models, we could map the complete sequence of events and propose a conceptual model of bacterial establishment in the pore space. This study therefore advances our understanding of the respective role of growth and mobility in the efficient colonization of bacteria in the rhizosphere.

Substrate heterogeneities cause root growth trajectories to switch from vertical to oblique.

Hard pans, soil compaction, soil aggregation and stones create physical barriers that can affect the development of a root system. Roots are known to exploit paths of least resistance to avoid such obstacles, but the mechanism through which this is achieved is not well understood. Here, we combined 3D-printed substrates with a high-throughput live imaging platform to study the responses of plant roots to a range of physical barriers. Using image analysis algorithms, we determined the properties of growth trajectories and identified how the presence of rigid circular obstacles affects the ability of a primary root to maintain its vertical trajectory. Results showed the types of growth responses were limited, both vertical and oblique trajectories were found to be stable and influenced by the size of the obstacles. When obstacles were of intermediate sizes, trajectories were unstable and changed in nature through time. We formalised the conditions for root trajectory to change from vertical to oblique, linking the angle at which the root detaches from the obstacle to the root curvature due to gravitropism. Exploitation of paths of least resistance by a root may therefore be constrained by the ability of the root to curve and respond to gravitropic signals.

Root architecture and rhizosphere-microbe interactions.

Plant roots fulfil crucial tasks during a plant's life. As roots encounter very diverse conditions while exploring the soil for resources, their growth and development must be responsive to changes in the rhizosphere, resulting in root architectures that are tailor-made for all prevailing circumstances. Using multi-disciplinary approaches, we are gaining more intricate insights into the regulatory mechanisms directing root system architecture. This Special Issue provides insights into our advancement of knowledge on different aspects of root development and identifies opportunities for future research.

Particle-based model shows complex rearrangement of tissue mechanical properties are needed for roots to grow in hard soil.

When exposed to increased mechanical resistance from the soil, plant roots display non-linear growth responses that cannot be solely explained by mechanical principles. Here, we aim to investigate how changes in tissue mechanical properties are biologically regulated in response to soil strength. A particle-based model was developed to solve root-soil mechanical interactions at the cellular scale, and a detailed numerical study explored factors that affect root responses to soil resistance. Results showed how softening of root tissues at the tip may contribute to root responses to soil impedance, a mechanism likely linked to soil cavity expansion. The model also predicted the shortening and decreased anisotropy of the zone where growth occurs, which may improve the mechanical stability of the root against axial forces. The study demonstrates the potential of advanced modeling tools to help identify traits that confer plant resistance to abiotic stress.

Plant-environment microscopy tracks interactions of Bacillus subtilis with plant roots across the entire rhizosphere.

Our understanding of plant-microbe interactions in soil is limited by the difficulty of observing processes at the microscopic scale throughout plants' large volume of influence. Here, we present the development of three-dimensional live microscopy for resolving plant-microbe interactions across the environment of an entire seedling growing in a transparent soil in tailor-made mesocosms, maintaining physical conditions for the culture of both plants and microorganisms. A tailor-made, dual-illumination light sheet system acquired photons scattered from the plant while fluorescence emissions were simultaneously captured from transparent soil particles and labeled microorganisms, allowing the generation of quantitative data on samples ∼3,600 mm3 in size, with as good as 5 µm resolution at a rate of up to one scan every 30 min. The system tracked the movement of Bacillus subtilis populations in the rhizosphere of lettuce plants in real time, revealing previously unseen patterns of activity. Motile bacteria favored small pore spaces over the surface of soil particles, colonizing the root in a pulsatile manner. Migrations appeared to be directed toward the root cap, the point of "first contact," before the subsequent colonization of mature epidermis cells. Our findings show that microscopes dedicated to live environmental studies present an invaluable tool to understand plant-microbe interactions.

In situ control of root-bacteria interactions using optical trapping in transparent soil.

Bacterial attachment on root surfaces is an important step preceding the colonization or internalization and subsequent infection of plants by pathogens. Unfortunately, bacterial attachment is not well understood because the phenomenon is difficult to observe. Here we assessed whether this limitation could be overcome using optical trapping approaches. We have developed a system based on counter-propagating beams and studied its ability to guide Pectobacterium atrosepticum (Pba) cells to different root cell types within the interstices of transparent soils. Bacterial cells were successfully trapped and guided to root hair cells, epidermal cells, border cells, and tissues damaged by laser ablation. Finally, we used the system to quantify the bacterial cell detachment rate of Pba cells on root surfaces following reversible attachment. Optical trapping techniques could greatly enhance our ability to deterministically characterize mechanisms linked to attachment and formation of biofilms in the rhizosphere.

The helical motions of roots are linked to avoidance of particle forces in soil.

Limitation to root growth results from forces required to overcome soil resistance to deformation. The variations in individual particle forces affects root development and often deflects the growth trajectory. We have developed transparent soil and optical projection tomography microscopy systems where measurements of growth trajectory and particle forces can be acquired in a granular medium at a range of confining pressures. We developed image-processing pipelines to analyse patterns in root trajectories and a stochastic-mechanical theory to establish how root deflections relate to particle forces and thickening of the root. Root thickening compensates for the increase in mean particle forces but does not prevent deflections from 5% of most extreme individual particle forces causing root deflection. The magnitude of deflections increases with pressure but they assemble into helices of conserved wavelength in response linked to gravitropism. The study reveals mechanisms for the understanding of root growth in mechanically impeding soil conditions and provides insights relevant to breeding of drought-resistant crops.

Genetical and comparative genomics of Brassica under altered Ca supply identifies Arabidopsis Ca-transporter orthologs.

Although Ca transport in plants is highly complex, the overexpression of vacuolar Ca(2+) transporters in crops is a promising new technology to improve dietary Ca supplies through biofortification. Here, we sought to identify novel targets for increasing plant Ca accumulation using genetical and comparative genomics. Expression quantitative trait locus (eQTL) mapping to 1895 cis- and 8015 trans-loci were identified in shoots of an inbred mapping population of Brassica rapa (IMB211 × R500); 23 cis- and 948 trans-eQTLs responded specifically to altered Ca supply. eQTLs were screened for functional significance using a large database of shoot Ca concentration phenotypes of Arabidopsis thaliana. From 31 Arabidopsis gene identifiers tagged to robust shoot Ca concentration phenotypes, 21 mapped to 27 B. rapa eQTLs, including orthologs of the Ca(2+) transporters At-CAX1 and At-ACA8. Two of three independent missense mutants of BraA.cax1a, isolated previously by targeting induced local lesions in genomes, have allele-specific shoot Ca concentration phenotypes compared with their segregating wild types. BraA.CAX1a is a promising target for altering the Ca composition of Brassica, consistent with prior knowledge from Arabidopsis. We conclude that multiple-environment eQTL analysis of complex crop genomes combined with comparative genomics is a powerful technique for novel gene identification/prioritization.

3D deformation field in growing plant roots reveals both mechanical and biological responses to axial mechanical forces.

Strong regions and physical barriers in soils may slow root elongation, leading to reduced water and nutrient uptake and decreased yield. In this study, the biomechanical responses of roots to axial mechanical forces were assessed by combining 3D live imaging, kinematics and a novel mechanical sensor. This system quantified Young's elastic modulus of intact poplar roots (32MPa), a rapid <0.2 mN touch-elongation sensitivity, and the critical elongation force applied by growing roots that resulted in bending. Kinematic analysis revealed a multiphase bio-mechanical response of elongation rate and curvature in 3D. Measured critical elongation force was accurately predicted from an Euler buckling model, indicating that no biologically mediated accommodation to mechanical forces influenced bending during this short period of time. Force applied by growing roots increased more than 15-fold when buckling was prevented by lateral bracing of the root. The junction between the growing and the mature zones was identified as a zone of mechanical weakness that seemed critical to the bending process. This work identified key limiting factors for root growth and buckling under mechanical constraints. The findings are relevant to crop and soil sciences, and advance our understanding of root growth in heterogeneous structured soils.

Analysis of root growth from a phenotyping data set using a density-based model.

Major research efforts are targeting the improved performance of root systems for more efficient use of water and nutrients by crops. However, characterizing root system architecture (RSA) is challenging, because roots are difficult objects to observe and analyse. A model-based analysis of RSA traits from phenotyping image data is presented. The model can successfully back-calculate growth parameters without the need to measure individual roots. The mathematical model uses partial differential equations to describe root system development. Methods based on kernel estimators were used to quantify root density distributions from experimental image data, and different optimization approaches to parameterize the model were tested. The model was tested on root images of a set of 89 Brassica rapa L. individuals of the same genotype grown for 14 d after sowing on blue filter paper. Optimized root growth parameters enabled the final (modelled) length of the main root axes to be matched within 1% of their mean values observed in experiments. Parameterized values for elongation rates were within ±4% of the values measured directly on images. Future work should investigate the time dependency of growth parameters using time-lapse image data. The approach is a potentially powerful quantitative technique for identifying crop genotypes with more efficient root systems, using (even incomplete) data from high-throughput phenotyping systems.

A scanner system for high-resolution quantification of variation in root growth dynamics of Brassica rapa genotypes.

The potential exists to breed for root system architectures that optimize resource acquisition. However, this requires the ability to screen root system development quantitatively, with high resolution, in as natural an environment as possible, with high throughput. This paper describes the construction of a low-cost, high-resolution root phenotyping platform, requiring no sophisticated equipment and adaptable to most laboratory and glasshouse environments, and its application to quantify environmental and temporal variation in root traits between genotypes of Brassica rapa L. Plants were supplied with a complete nutrient solution through the wick of a germination paper. Images of root systems were acquired without manual intervention, over extended periods, using multiple scanners controlled by customized software. Mixed-effects models were used to describe the sources of variation in root traits contributing to root system architecture estimated from digital images. It was calculated that between one and 43 replicates would be required to detect a significant difference (95% CI 50% difference between traits). Broad-sense heritability was highest for shoot biomass traits (>0.60), intermediate (0.25-0.60) for the length and diameter of primary roots and lateral root branching density on the primary root, and lower (<0.25) for other root traits. Models demonstrate that root traits show temporal variations of various types. The phenotyping platform described here can be used to quantify environmental and temporal variation in traits contributing to root system architecture in B. rapa and can be extended to screen the large populations required for breeding for efficient resource acquisition.

Light Sheet Tomography (LST) for in situ imaging of plant roots.

The production of crops capable of efficient nutrient use is essential for addressing the problem of global food security. The ability of a plant's root system to interact with the soil micro-environment determines how effectively it can extract water and nutrients. In order to assess this ability and develop the fast and cost effective phenotyping techniques which are needed to establish efficient root systems, in situ imaging in soil is required. To date this has not been possible due to the high density of scatterers and absorbers in soil or because other growth substrates do not sufficiently model the heterogeneity of a soil's microenvironment. We present here a new form of light sheet imaging with novel transparent soil containing refractive index matched particles. This imaging method does not rely on fluorescence, but relies solely on scattering from root material. We term this form of imaging Light Sheet Tomography (LST). We have tested LST on a range of materials and plant roots in transparent soil and gel. Due to the low density of root structures, i.e. relatively large spaces between adjacent roots, long-term monitoring of lettuce root development in situ with subsequent quantitative analysis was achieved.

In situ laser manipulation of root tissues in transparent soil.

AIMS: Laser micromanipulation such as dissection or optical trapping enables remote physical modification of the activity of tissues, cells and organelles. To date, applications of laser manipulation to plant roots grown in soil have been limited. Here, we show laser manipulation can be applied in situ when plant roots are grown in transparent soil. METHODS: We have developed a Q-switched laser manipulation and imaging instrument to perform controlled dissection of roots and to study light-induced root growth responses. We performed a detailed characterisation of the properties of the cutting beams through the soil, studying dissection and optical ablation. Furthermore, we also studied the use of low light doses to control the root elongation rate of lettuce seedlings (Lactuca sativa) in air, agar, gel and transparent soil. RESULTS: We show that whilst soil inhomogeneities affect the thickness and circularity of the beam, those distortions are not inherently limiting. The ability to induce changes in root elongation or complete dissection of microscopic regions of the root is robust to substrate heterogeneity and microscopy set up and is maintained following the limited distortions induced by the transparent soil environment. CONCLUSIONS: Our findings show that controlled in situ laser dissection of root tissues is possible with a simple and low-cost optical set-up. We also show that, in the absence of dissection, a reduced laser light power density can provide reversible control of root growth, achieving a precise "point and shoot" method for root manipulation.

Framework for Quantification of the Dynamics of Root Colonization by Pseudomonas fluorescens Isolate SBW25.

Colonization of the root surface, or rhizoplane, is one of the first steps for soil-borne bacteria to become established in the plant microbiome. However, the relative contributions of processes, such as bacterial attachment and proliferation is not well characterized, and this limits our ability to comprehend the complex dynamics of microbial communities in the rhizosphere. The work presented here addresses this knowledge gap. A model system was developed to acquire quantitative data on the colonization process of lettuce (Lactuca sativa L. cultivar. All Year Round) roots by Pseudomonas fluorescens isolate SBW25. A theoretical framework is proposed to calculate attachment rate and quantify the relative contribution of bacterial attachment to colonization. This allows the assessment of attachment rates on the root surface beyond the short time period during which it can be quantified experimentally. All techniques proposed are generic and similar analyses could be applied to study various combinations of plants and bacteria, or to assess competition between species. In the future this could allow for selection of microbial traits that improve early colonization and maintenance of targeted isolates in cropping systems, with potential applications for the development of biological fertilizers.

Dynamic biospeckle analysis, a new tool for the fast screening of plant nematicide selectivity.

BACKGROUND: Plant feeding, free-living nematodes cause extensive damage to plant roots by direct feeding and, in the case of some trichodorid and longidorid species, through the transmission of viruses. Developing more environmentally friendly, target-specific nematicides is currently impeded by slow and laborious methods of toxicity testing. Here, we developed a bioactivity assay based on the dynamics of light 'speckle' generated by living cells and we demonstrate its application by assessing chemicals' toxicity to different nematode trophic groups. RESULTS: Free-living nematode populations extracted from soil were exposed to methanol and phenyl isothiocyanate (PEITC). Biospeckle analysis revealed differing behavioural responses as a function of nematode feeding groups. Trichodorus nematodes were less sensitive than were bacterial feeding nematodes or non-trichodorid plant feeding nematodes. Following 24 h of exposure to PEITC, bioactivity significantly decreased for plant and bacterial feeders but not for Trichodorus nematodes. Decreases in movement for plant and bacterial feeders in the presence of PEITC also led to measurable changes to the morphology of biospeckle patterns. CONCLUSIONS: Biospeckle analysis can be used to accelerate the screening of nematode bioactivity, thereby providing a fast way of testing the specificity of potential nematicidal compounds. With nematodes' distinctive movement and activity levels being visible in the biospeckle pattern, the technique has potential to screen the behavioural responses of diverse trophic nematode communities. The method discriminates both behavioural responses, morphological traits and activity levels and hence could be used to assess the specificity of nematicidal compounds.

New live screening of plant-nematode interactions in the rhizosphere.

Free living nematodes (FLN) are microscopic worms found in all soils. While many FLN species are beneficial to crops, some species cause significant damage by feeding on roots and vectoring viruses. With the planned legislative removal of traditionally used chemical treatments, identification of new ways to manage FLN populations has become a high priority. For this, more powerful screening systems are required to rapidly assess threats to crops and identify treatments efficiently. Here, we have developed new live assays for testing nematode responses to treatment by combining transparent soil microcosms, a new light sheet imaging technique termed Biospeckle Selective Plane Illumination Microscopy (BSPIM) for fast nematode detection, and Confocal Laser Scanning Microscopy for high resolution imaging. We show that BSPIM increased signal to noise ratios by up to 60 fold and allowed the automatic detection of FLN in transparent soil samples of 1.5 mL. Growing plant root systems were rapidly scanned for nematode abundance and activity, and FLN feeding behaviour and responses to chemical compounds observed in soil-like conditions. This approach could be used for direct monitoring of FLN activity either to develop new compounds that target economically damaging herbivorous nematodes or ensuring that beneficial species are not negatively impacted.

Non-invasive Protocol for Kinematic Monitoring of Root Growth under Infrared Light.

Phenotyping the dynamics of root responses to environmental cues is necessary to understand plant acclimation to their environment. Continuous monitoring of root growth is challenging because roots normally grow belowground and are very sensitive to their growth environment. This protocol combines infrared imaging with hydroponic cultivation for kinematic analyses. It allows continuous imaging at fine spatiotemporal resolution and disturbs roots minimally. Examples are provided of how the procedure and materials can be adapted for 3D monitoring and of how environmental stress may be manipulated for experimental purposes.

Accelerating root system phenotyping of seedlings through a computer-assisted processing pipeline.

BACKGROUND: There are numerous systems and techniques to measure the growth of plant roots. However, phenotyping large numbers of plant roots for breeding and genetic analyses remains challenging. One major difficulty is to achieve high throughput and resolution at a reasonable cost per plant sample. Here we describe a cost-effective root phenotyping pipeline, on which we perform time and accuracy benchmarking to identify bottlenecks in such pipelines and strategies for their acceleration. RESULTS: Our root phenotyping pipeline was assembled with custom software and low cost material and equipment. Results show that sample preparation and handling of samples during screening are the most time consuming task in root phenotyping. Algorithms can be used to speed up the extraction of root traits from image data, but when applied to large numbers of images, there is a trade-off between time of processing the data and errors contained in the database. CONCLUSIONS: Scaling-up root phenotyping to large numbers of genotypes will require not only automation of sample preparation and sample handling, but also efficient algorithms for error detection for more reliable replacement of manual interventions.

Transparent soil microcosms allow 3D spatial quantification of soil microbiological processes in vivo.

The recently developed transparent soil consists of particles of Nafion, a polymer with a low refractive index (RI), which is prepared by milling and chemical treatment for use as a soil analog. After the addition of a RI-matched solution, confocal imaging can be carried out in vivo and without destructive sampling. In a previous study, we showed that the new substrate provides a good approximation of plant growth conditions found in natural soils. In this paper, we present further development of the techniques for detailed quantitative analysis of images of root-microbe interactions in situ. Using this system it was possible for the first time to analyze bacterial distribution along the roots and in the bulk substrate in vivo. These findings indicate that the coupling of transparent soil with light microscopy is an important advance toward the discovery of the mechanisms of microbial colonisation of the rhizosphere.