A temporal analysis and response to nitrate availability of 3D root system architecture in diverse pennycress (Thlaspi arvense L.) accessions.

Introduction: Roots have a central role in plant resource capture and are the interface between the plant and the soil that affect multiple ecosystem processes. Field pennycress (Thlaspi arvense L.) is a diploid annual cover crop species that has potential utility for reducing soil erosion and nutrient losses; and has rich seeds (30-35% oil) amenable to biofuel production and as a protein animal feed. The objective of this research was to (1) precisely characterize root system architecture and development, (2) understand plastic responses of pennycress roots to nitrate nutrition, (3) and determine genotypic variance available in root development and nitrate plasticity. Methods: Using a root imaging and analysis pipeline, the 4D architecture of the pennycress root system was characterized under four nitrate regimes, ranging from zero to high nitrate concentrations. These measurements were taken at four time points (days 5, 9, 13, and 17 after sowing). Results: Significant nitrate condition response and genotype interactions were identified for many root traits, with the greatest impact observed on lateral root traits. In trace nitrate conditions, a greater lateral root count, length, density, and a steeper lateral root angle was observed compared to high nitrate conditions. Additionally, genotype-by-nitrate condition interaction was observed for root width, width:depth ratio, mean lateral root length, and lateral root density. Discussion: These findings illustrate root trait variance among pennycress accessions. These traits could serve as targets for breeding programs aimed at developing improved cover crops that are responsive to nitrate, leading to enhanced productivity, resilience, and ecosystem service.

Whole-plant phenotypic engineering: moving beyond ratios for multi-objective optimization of nutrient use efficiency.

Nutrient use efficiency (NUE) is typically measured as the ratio of yield to soil nutrient availability but ignores contributions of underlying plant traits. Relevant plant traits can be grouped as root acquisition efficiency, shoot radiation use efficiency, and plant metabolic efficiency. The intentional integration of these traits will lead to synergistic improvements of NUE. Recent progress in trait-focused research includes phenotyping root nutrient uptake rates and respiration, engineering reduced photorespiration, and identification of nutrient assimilation pathways. Traits need to be conceptualized in agricultural systems contexts to improve synchrony of plant demand and soil supply of nutrients, including consideration of crop mixtures. Use of simulation modeling and multi-objective optimization will allow accelerating NUE gains beyond selection for a single ratio.

Interactions among rooting traits for deep water and nitrogen uptake in upland and lowland ecotypes of switchgrass (Panicum virgatum L.).

The response of plant growth and development to nutrient and water availability is an important adaptation for abiotic stress tolerance. Roots need to intercept both passing nutrients and water while foraging into new soil layers for further resources. Substantial amounts of nitrate can be lost in the field when leaching into groundwater, yet very little is known about how deep rooting affects this process. Here, we phenotyped root system traits and deep 15N nitrate capture across 1.5 m vertical profiles of solid media using tall mesocosms in switchgrass (Panicum virgatum L.), a promising cellulosic bioenergy feedstock. Root and shoot biomass traits, photosynthesis and respiration measures, and nutrient uptake and accumulation traits were quantified in response to a water and nitrate stress factorial experiment for switchgrass upland (VS16) and lowland (AP13) ecotypes. The two switchgrass ecotypes shared common plastic abiotic responses to nitrogen (N) and water availability, and yet had substantial genotypic variation for root and shoot traits. A significant interaction between N and water stress combination treatments for axial and lateral root traits represents a complex and shared root development strategy for stress mitigation. Deep root growth and 15N capture were found to be closely linked to aboveground growth. Together, these results represent the wide genetic pool of switchgrass and show that deep rooting promotes nitrate capture, plant productivity, and sustainability.

Optimisation of root traits to provide enhanced ecosystem services in agricultural systems: A focus on cover crops.

Roots are the interface between the plant and the soil and play a central role in multiple ecosystem processes. With intensification of agricultural practices, rhizosphere processes are being disrupted and are causing degradation of the physical, chemical and biotic properties of soil. However, cover crops, a group of plants that provide ecosystem services, can be utilised during fallow periods or used as an intercrop to restore soil health. The effectiveness of ecosystem services provided by cover crops varies widely as very little breeding has occurred in these species. Improvement of ecosystem service performance is rarely considered as a breeding trait due to the complexities and challenges of belowground evaluation. Advancements in root phenotyping and genetic tools are critical in accelerating ecosystem service improvement in cover crops. In this study, we provide an overview of the range of belowground ecosystem services provided by cover crop roots: (1) soil structural remediation, (2) capture of soil resources and (3) maintenance of the rhizosphere and building of organic matter content. Based on the ecosystem services described, we outline current and promising phenotyping technologies and breeding strategies in cover crops that can enhance agricultural sustainability through improvement of root traits.

Dark Respiration Measurement from Arabidopsis Shoots.

Dark respiration refers to experimental measures of leaf respiration in the absence of light, done to distinguish it from the photorespiration that occurs during photosynthesis. Dark aerobic respiration reactions occur solely in the mitochondria and convert glucose molecules from cytoplasmatic glycolysis and oxygen into carbon dioxide and water, with the generation of ATP molecules. Previous methods typically use oxygen sensors to measure oxygen depletion or complicated and expensive photosynthesis instruments to measure CO2 accumulation. Here, we provide a detailed, step-by-step approach to measure dark respiration in plants by recording CO2 fluxes of Arabidopsis shoot and root tissues. Briefly, plants are dark acclimated for 1 hour, leaves and roots are excised and placed separately in airtight chambers, and CO2 accumulation is measured over time with standard infrared gas analyzers. The time-series data is processed with R scripts to produce dark respiration rates, which can be standardized by fresh or dry tissue mass. The current method requires inexpensive infrared gas analyzers, off-the-shelf parts for chambers, and publicly available data analysis scripts.

RhizoVision Explorer: open-source software for root image analysis and measurement standardization.

Roots are central to the function of natural and agricultural ecosystems by driving plant acquisition of soil resources and influencing the carbon cycle. Root characteristics like length, diameter and volume are critical to measure to understand plant and soil functions. RhizoVision Explorer is an open-source software designed to enable researchers interested in roots by providing an easy-to-use interface, fast image processing and reliable measurements. The default broken roots mode is intended for roots sampled from pots and soil cores, washed and typically scanned on a flatbed scanner, and provides measurements like length, diameter and volume. The optional whole root mode for complete root systems or root crowns provides additional measurements such as angles, root depth and convex hull. Both modes support providing measurements grouped by defined diameter ranges, the inclusion of multiple regions of interest and batch analysis. RhizoVision Explorer was successfully validated against ground truth data using a new copper wire image set. In comparison, the current reference software, the commercial WinRhizo™, drastically underestimated volume when wires of different diameters were in the same image. Additionally, measurements were compared with WinRhizo™ and IJ_Rhizo using a simulated root image set, showing general agreement in software measurements, except for root volume. Finally, scanned root image sets acquired in different labs for the crop, herbaceous and tree species were used to compare results from RhizoVision Explorer with WinRhizo™. The two software showed general agreement, except that WinRhizo™ substantially underestimated root volume relative to RhizoVision Explorer. In the current context of rapidly growing interest in root science, RhizoVision Explorer intends to become a reference software, improve the overall accuracy and replicability of root trait measurements and provide a foundation for collaborative improvement and reliable access to all.

A multiple ion-uptake phenotyping platform reveals shared mechanisms affecting nutrient uptake by roots.

Nutrient uptake is critical for crop growth and is determined by root foraging in soil. Growth and branching of roots lead to effective root placement to acquire nutrients, but relatively little is known about absorption of nutrients at the root surface from the soil solution. This knowledge gap could be alleviated by understanding sources of genetic variation for short-term nutrient uptake on a root length basis. A modular platform called RhizoFlux was developed for high-throughput phenotyping of multiple ion-uptake rates in maize (Zea mays L.). Using this system, uptake rates were characterized for the crop macronutrients nitrate, ammonium, potassium, phosphate, and sulfate among the Nested Association Mapping (NAM) population founder lines. The data revealed substantial genetic variation for multiple ion-uptake rates in maize. Interestingly, specific nutrient uptake rates (nutrient uptake rate per length of root) were found to be both heritable and distinct from total uptake and plant size. The specific uptake rates of each nutrient were positively correlated with one another and with specific root respiration (root respiration rate per length of root), indicating that uptake is governed by shared mechanisms. We selected maize lines with high and low specific uptake rates and performed an RNA-seq analysis, which identified key regulatory components involved in nutrient uptake. The high-throughput multiple ion-uptake kinetics pipeline will help further our understanding of nutrient uptake, parameterize holistic plant models, and identify breeding targets for crops with more efficient nutrient acquisition.

Functional phenomics and genetics of the root economics space in winter wheat using high-throughput phenotyping of respiration and architecture.

The root economics space is a useful framework for plant ecology but is rarely considered for crop ecophysiology. In order to understand root trait integration in winter wheat, we combined functional phenomics with trait economic theory, utilizing genetic variation, high-throughput phenotyping, and multivariate analyses. We phenotyped a diversity panel of 276 genotypes for root respiration and architectural traits using a novel high-throughput method for CO2 flux and the open-source software RhizoVision Explorer to analyze scanned images. We uncovered substantial variation in specific root respiration (SRR) and specific root length (SRL), which were primary indicators of root metabolic and structural costs. Multiple linear regression analysis indicated that lateral root tips had the greatest SRR, and the residuals from this model were used as a new trait. Specific root respiration was negatively correlated with plant mass. Network analysis, using a Gaussian graphical model, identified root weight, SRL, diameter, and SRR as hub traits. Univariate and multivariate genetic analyses identified genetic regions associated with SRR, SRL, and root branching frequency, and proposed gene candidates. Combining functional phenomics and root economics is a promising approach to improving our understanding of crop ecophysiology. We identified root traits and genomic regions that could be harnessed to breed more efficient crops for sustainable agroecosystems.

Application of Synthetic Peptide CEP1 Increases Nutrient Uptake Rates Along Plant Roots.

The root system of a plant provides vital functions including resource uptake, storage, and anchorage in soil. The uptake of macro-nutrients like nitrogen (N), phosphorus (P), potassium (K), and sulphur (S) from the soil is critical for plant growth and development. Small signaling peptide (SSP) hormones are best known as potent regulators of plant growth and development with a few also known to have specialized roles in macronutrient utilization. Here we describe a high throughput phenotyping platform for testing SSP effects on root uptake of multiple nutrients. The SSP, CEP1 (C-TERMINALLY ENCODED PEPTIDE) enhanced nitrate uptake rate per unit root length in Medicago truncatula plants deprived of N in the high-affinity transport range. Single structural variants of M. truncatula and Arabidopsis thaliana specific CEP1 peptides, MtCEP1D1:hyp4,11 and AtCEP1:hyp4,11, enhanced uptake not only of nitrate, but also phosphate and sulfate in both model plant species. Transcriptome analysis of Medicago roots treated with different MtCEP1 encoded peptide domains revealed that hundreds of genes respond to these peptides, including several nitrate transporters and a sulfate transporter that may mediate the uptake of these macronutrients downstream of CEP1 signaling. Likewise, several putative signaling pathway genes including LEUCINE-RICH REPEAT RECPTOR-LIKE KINASES and Myb domain containing transcription factors, were induced in roots by CEP1 treatment. Thus, a scalable method has been developed for screening synthetic peptides of potential use in agriculture, with CEP1 shown to be one such peptide.

RhizoVision Crown: An Integrated Hardware and Software Platform for Root Crown Phenotyping.

Root crown phenotyping measures the top portion of crop root systems and can be used for marker-assisted breeding, genetic mapping, and understanding how roots influence soil resource acquisition. Several imaging protocols and image analysis programs exist, but they are not optimized for high-throughput, repeatable, and robust root crown phenotyping. The RhizoVision Crown platform integrates an imaging unit, image capture software, and image analysis software that are optimized for reliable extraction of measurements from large numbers of root crowns. The hardware platform utilizes a backlight and a monochrome machine vision camera to capture root crown silhouettes. The RhizoVision Imager and RhizoVision Analyzer are free, open-source software that streamline image capture and image analysis with intuitive graphical user interfaces. The RhizoVision Analyzer was physically validated using copper wire, and features were extensively validated using 10,464 ground-truth simulated images of dicot and monocot root systems. This platform was then used to phenotype soybean and wheat root crowns. A total of 2,799 soybean (Glycine max) root crowns of 187 lines and 1,753 wheat (Triticum aestivum) root crowns of 186 lines were phenotyped. Principal component analysis indicated similar correlations among features in both species. The maximum heritability was 0.74 in soybean and 0.22 in wheat, indicating that differences in species and populations need to be considered. The integrated RhizoVision Crown platform facilitates high-throughput phenotyping of crop root crowns and sets a standard by which open plant phenotyping platforms can be benchmarked.

Iron-Sulfur Cluster Protein NITROGEN FIXATION S-LIKE1 and Its Interactor FRATAXIN Function in Plant Immunity.

Iron-sulfur (Fe-S) clusters are inorganic cofactors that are present in all kingdoms of life as part of a large number of proteins involved in several cellular processes, including DNA replication and metabolism. In this work, we demonstrate an additional role for two Fe-S cluster genes in biotic stress responses in plants. Eleven Fe-S cluster genes, including the NITROGEN FIXATION S-LIKE1 (NFS1) and its interactor FRATAXIN (FH), when silenced in Nicotiana benthamiana, compromised nonhost resistance to Pseudomonas syringae pv. tomato T1. NbNFS1 expression was induced by pathogens and salicylic acid. Arabidopsis (Arabidopsis thaliana) atnfs and atfh mutants, with reduced AtNFS1 or AtFH gene expression, respectively, showed increased susceptibility to both host and nonhost pathogen infection. Arabidopsis AtNFS1 and AtFH overexpressor lines displayed decreased susceptibility to infection by host pathogen P syringae pv. tomato DC3000. The AtNFS1 overexpression line exhibited constitutive upregulation of several defense-related genes and enrichment of gene ontology terms related to immunity and salicylic acid responses. Our results demonstrate that NFS1 and its interactor FH are involved not only in nonhost resistance but also in basal resistance, suggesting a new role of the Fe-S cluster pathway in plant immunity.

Three Dimensional Root CT Segmentation using Multi-Resolution Encoder-Decoder Networks.

We address the complex problem of reliably segmenting root structure from soil in X-ray Computed Tomography (CT) images. We utilise a deep learning approach, and propose a state-of-the-art multi-resolution architecture based on encoderdecoders. While previous work in encoder-decoders implies the use of multiple resolutions simply by downsampling and upsampling images, we make this process explicit, with branches of the network tasked separately with obtaining local high-resolution segmentation, and wider low-resolution contextual information. The complete network is a memory efficient implementation that is still able to resolve small root detail in large volumetric images. We compare against a number of different encoder-decoder based architectures from the literature, as well as a popular existing image analysis tool designed for root CT segmentation. We show qualitatively and quantitatively that a multi-resolution approach offers substantial accuracy improvements over a both a small receptive field size in a deep network, or a larger receptive field in a shallower network. We then further improve performance using an incremental learning approach, in which failures in the original network are used to generate harder negative training examples. Our proposed method requires no user interaction, is fully automatic, and identifies large and fine root material throughout the whole volume.

Targeting Root Ion Uptake Kinetics to Increase Plant Productivity and Nutrient Use Efficiency.

Root system architecture has received increased attention in recent years; however, significant knowledge gaps remain for physiological phenes, or units of phenotype, that have been relatively less studied. Ion uptake kinetics studies have been invaluable in uncovering distinct nutrient uptake systems in plants with the use of Michaelis-Menten kinetic modeling. This review outlines the theoretical framework behind ion uptake kinetics, provides a meta-analysis for macronutrient uptake parameters, and proposes new strategies for using uptake kinetics parameters as selection criteria for breeding crops with improved resource acquisition capability. Presumably, variation in uptake kinetics is caused by variation in type and number of transporters, assimilation machinery, and anatomical features that can vary greatly within and among species. Critically, little is known about what determines transporter properties at the molecular level or how transporter properties scale to the entire root system. A meta-analysis of literature containing measures of crop nutrient uptake kinetics provides insights about the need for standardization of reporting, the differences among crop species, and the relationships among various uptake parameters and experimental conditions. Therefore, uptake kinetics parameters are proposed as promising target phenes that integrate several processes for functional phenomics and genetic analysis, which will lead to a greater understanding of this fundamental plant process. Exploiting this genetic and phenotypic variation has the potential to greatly advance breeding efforts for improved nutrient use efficiency in crops.

Combining semi-automated image analysis techniques with machine learning algorithms to accelerate large-scale genetic studies.

Genetic analyses of plant root systems require large datasets of extracted architectural traits. To quantify such traits from images of root systems, researchers often have to choose between automated tools (that are prone to error and extract only a limited number of architectural traits) or semi-automated ones (that are highly time consuming). We trained a Random Forest algorithm to infer architectural traits from automatically extracted image descriptors. The training was performed on a subset of the dataset, then applied to its entirety. This strategy allowed us to (i) decrease the image analysis time by 73% and (ii) extract meaningful architectural traits based on image descriptors. We also show that these traits are sufficient to identify the quantitative trait loci that had previously been discovered using a semi-automated method. We have shown that combining semi-automated image analysis with machine learning algorithms has the power to increase the throughput of large-scale root studies. We expect that such an approach will enable the quantification of more complex root systems for genetic studies. We also believe that our approach could be extended to other areas of plant phenotyping.

Deep machine learning provides state-of-the-art performance in image-based plant phenotyping.

In plant phenotyping, it has become important to be able to measure many features on large image sets in order to aid genetic discovery. The size of the datasets, now often captured robotically, often precludes manual inspection, hence the motivation for finding a fully automated approach. Deep learning is an emerging field that promises unparalleled results on many data analysis problems. Building on artificial neural networks, deep approaches have many more hidden layers in the network, and hence have greater discriminative and predictive power. We demonstrate the use of such approaches as part of a plant phenotyping pipeline. We show the success offered by such techniques when applied to the challenging problem of image-based plant phenotyping and demonstrate state-of-the-art results (>97% accuracy) for root and shoot feature identification and localization. We use fully automated trait identification using deep learning to identify quantitative trait loci in root architecture datasets. The majority (12 out of 14) of manually identified quantitative trait loci were also discovered using our automated approach based on deep learning detection to locate plant features. We have shown deep learning-based phenotyping to have very good detection and localization accuracy in validation and testing image sets. We have shown that such features can be used to derive meaningful biological traits, which in turn can be used in quantitative trait loci discovery pipelines. This process can be completely automated. We predict a paradigm shift in image-based phenotyping bought about by such deep learning approaches, given sufficient training sets.

Shaping 3D Root System Architecture.

Plants are sessile organisms rooted in one place. The soil resources that plants require are often distributed in a highly heterogeneous pattern. To aid foraging, plants have evolved roots whose growth and development are highly responsive to soil signals. As a result, 3D root architecture is shaped by myriad environmental signals to ensure resource capture is optimised and unfavourable environments are avoided. The first signals sensed by newly germinating seeds - gravity and light - direct root growth into the soil to aid seedling establishment. Heterogeneous soil resources, such as water, nitrogen and phosphate, also act as signals that shape 3D root growth to optimise uptake. Root architecture is also modified through biotic interactions that include soil fungi and neighbouring plants. This developmental plasticity results in a 'custom-made' 3D root system that is best adapted to forage for resources in each soil environment that a plant colonises.

OpenSimRoot: widening the scope and application of root architectural models.

OpenSimRoot is an open-source, functional-structural plant model and mathematical description of root growth and function. We describe OpenSimRoot and its functionality to broaden the benefits of root modeling to the plant science community. OpenSimRoot is an extended version of SimRoot, established to simulate root system architecture, nutrient acquisition and plant growth. OpenSimRoot has a plugin, modular infrastructure, coupling single plant and crop stands to soil nutrient and water transport models. It estimates the value of root traits for water and nutrient acquisition in environments and plant species. The flexible OpenSimRoot design allows upscaling from root anatomy to plant community to estimate the following: resource costs of developmental and anatomical traits; trait synergisms; and (interspecies) root competition. OpenSimRoot can model three-dimensional images from magnetic resonance imaging (MRI) and X-ray computed tomography (CT) of roots in soil. New modules include: soil water-dependent water uptake and xylem flow; tiller formation; evapotranspiration; simultaneous simulation of mobile solutes; mesh refinement; and root growth plasticity. OpenSimRoot integrates plant phenotypic data with environmental metadata to support experimental designs and to gain a mechanistic understanding at system scales.

Phenotyping pipeline reveals major seedling root growth QTL in hexaploid wheat.

Seedling root traits of wheat (Triticum aestivum L.) have been shown to be important for efficient establishment and linked to mature plant traits such as height and yield. A root phenotyping pipeline, consisting of a germination paper-based screen combined with image segmentation and analysis software, was developed and used to characterize seedling traits in 94 doubled haploid progeny derived from a cross between the winter wheat cultivars Rialto and Savannah. Field experiments were conducted to measure mature plant height, grain yield, and nitrogen (N) uptake in three sites over 2 years. In total, 29 quantitative trait loci (QTLs) for seedling root traits were identified. Two QTLs for grain yield and N uptake co-localize with root QTLs on chromosomes 2B and 7D, respectively. Of the 29 root QTLs identified, 11 were found to co-localize on 6D, with four of these achieving highly significant logarithm of odds scores (>20). These results suggest the presence of a major-effect gene regulating seedling root vigour/growth on chromosome 6D.