Pitfalls and potential of high-throughput plant phenotyping platforms.

Automated high-throughput plant phenotyping (HTPP) enables non-invasive, fast and standardized evaluations of a large number of plants for size, development, and certain physiological variables. Many research groups recognize the potential of HTPP and have made significant investments in HTPP infrastructure, or are considering doing so. To make optimal use of limited resources, it is important to plan and use these facilities prudently and to interpret the results carefully. Here we present a number of points that users should consider before purchasing, building or utilizing such equipment. They relate to (1) the financial and time investment for acquisition, operation, and maintenance, (2) the constraints associated with such machines in terms of flexibility and growth conditions, (3) the pros and cons of frequent non-destructive measurements, (4) the level of information provided by proxy traits, and (5) the utilization of calibration curves. Using data from an Arabidopsis experiment, we demonstrate how diurnal changes in leaf angle can impact plant size estimates from top-view cameras, causing deviations of more than 20% over the day. Growth analysis data from another rosette species showed that there was a curvilinear relationship between total and projected leaf area. Neglecting this curvilinearity resulted in linear calibration curves that, although having a high r2 (> 0.92), also exhibited large relative errors. Another important consideration we discussed is the frequency at which calibration curves need to be generated and whether different treatments, seasons, or genotypes require distinct calibration curves. In conclusion, HTPP systems have become a valuable addition to the toolbox of plant biologists, provided that these systems are tailored to the research questions of interest, and users are aware of both the possible pitfalls and potential involved.

Deep-water uptake under drought improved due to locally increased root conductivity in maize, but not in faba bean.

Moderate soil drying can cause a strong decrease in the soil-root system conductance. The resulting impact on root water uptake depends on the spatial distribution of the altered conductance relatively to remaining soil water resources, which is largely unknown. Here, we analyzed the vertical distribution of conductance across root systems using a novel, noninvasive sensor technology on pot-grown faba bean and maize plants. Withholding water for 4 days strongly enhanced the vertical gradient in soil water potential. Therefore, roots in upper and deeper soil layers were affected differently: In drier, upper layers, root conductance decreased by 66%-72%, causing an amplification of the drop in leaf water potential. In wetter, deeper layers, root conductance increased in maize but not in faba bean. The consequently facilitated deep-water uptake in maize contributed up to 21% of total water uptake at the end of the measurement. Analysis of root length distributions with MRI indicated that the locally increased conductance was mainly caused by an increased intrinsic conductivity and not by additional root growth. Our findings show that plants can partly compensate for a reduced root conductance in upper, drier soil layers by locally increasing root conductivity in wetter layers, thereby improving deep-water uptake.

Stomatal conductance tracks soil-to-leaf hydraulic conductance in faba bean and maize during soil drying.

Although regulation of stomatal conductance is widely assumed to be the most important plant response to soil drying, the picture is incomplete when hydraulic conductance from soil to the leaf, upstream of the stomata, is not considered. Here, we investigated to what extent soil drying reduces the conductance between soil and leaf, whether this reduction differs between species, how it affects stomatal regulation, and where in the hydraulic pathway it occurs. To this end, we noninvasively and continuously measured the total root water uptake rate, soil water potential, leaf water potential, and stomatal conductance of 4-week-old, pot-grown maize (Zea mays) and faba bean (Vicia faba) plants during 4 days of water restriction. In both species, the soil-plant conductance, excluding stomatal conductance, declined exponentially with soil drying and was reduced to 50% above a soil water potential of -0.1 MPa, which is far from the permanent wilting point. This loss of conductance has immediate consequences for leaf water potential and the associated stomatal regulation. Both stomatal conductance and soil-plant conductance declined at a higher rate in faba bean than in maize. Estimations of the water potential at the root surface and an incomplete recovery 22 h after rewatering indicate that the loss of conductance, at least partly, occurred inside the plants, for example, through root suberization or altered aquaporin gene expression. Our findings suggest that differences in the stomatal sensitivity among plant species are partly explained by the sensitivity of root hydraulic conductance to soil drying.

Rice increases phosphorus uptake in strongly sorbing soils by intra-root facilitation.

Upland rice (Oryza sativa) is adapted to strongly phosphorus (P) sorbing soils. The mechanisms underlying P acquisition, however, are not well understood, and models typically underestimate uptake. This complicates root ideotype development and trait-based selection for further improvement. We present a novel model, which correctly simulates the P uptake by a P-efficient rice genotype measured over 48 days of growth. The model represents root morphology at the local rhizosphere scale, including root hairs and fine S-type laterals. It simulates fast- and slowly reacting soil P and the P-solubilizing effect of root-induced pH changes in the soil. Simulations predict that the zone of pH changes and P solubilization around a root spreads further into the soil than the zone of P depletion. A root needs to place laterals outside its depletion- but inside its solubilization zone to maximize P uptake. S-type laterals, which are short but hairy, appear to be the key root structures to achieve that. Thus, thicker roots facilitate the P uptake by fine lateral roots. Uptake can be enhanced through longer root hairs and greater root length density but was less sensitive to total root length and root class proportions.

Simulating Crop Root Systems Using OpenSimRoot.

Functional-structural plant models are valuable modeling tools in analyzing plant development. A functional-structural plant model combines a three-dimensional representation of plant structure with models for physiological functions in order to better understand plant development. We present a guide to simulating crop root systems with OpenSimRoot, a feature-rich, highly cited, and open-source functional-structural root architecture model. We describe in detail how to create your own input files in conjunction with some examples. The aim of this guide is to highlight the potential of computational modeling in biology and to make modeling more accessible to the plant science community.

Assessing the Storage Root Development of Cassava with a New Analysis Tool.

Storage roots of cassava plants crops are one of the main providers of starch in many South American, African, and Asian countries. Finding varieties with high yields is crucial for growing and breeding. This requires a better understanding of the dynamics of storage root formation, which is usually done by repeated manual evaluation of root types, diameters, and their distribution in excavated roots. We introduce a newly developed method that is capable to analyze the distribution of root diameters automatically, even if root systems display strong variations in root widths and clustering in high numbers. An application study was conducted with cassava roots imaged in a video acquisition box. The root diameter distribution was quantified automatically using an iterative ridge detection approach, which can cope with a wide span of root diameters and clustering. The approach was validated with virtual root models of known geometries and then tested with a time-series of excavated root systems. Based on the retrieved diameter classes, we show plausibly that the dynamics of root type formation can be monitored qualitatively and quantitatively. We conclude that this new method reliably determines important phenotypic traits from storage root crop images. The method is fast and robustly analyses complex root systems and thereby applicable in high-throughput phenotyping and future breeding.

A starting guide to root ecology: strengthening ecological concepts and standardising root classification, sampling, processing and trait measurements.

In the context of a recent massive increase in research on plant root functions and their impact on the environment, root ecologists currently face many important challenges to keep on generating cutting-edge, meaningful and integrated knowledge. Consideration of the below-ground components in plant and ecosystem studies has been consistently called for in recent decades, but methodology is disparate and sometimes inappropriate. This handbook, based on the collective effort of a large team of experts, will improve trait comparisons across studies and integration of information across databases by providing standardised methods and controlled vocabularies. It is meant to be used not only as starting point by students and scientists who desire working on below-ground ecosystems, but also by experts for consolidating and broadening their views on multiple aspects of root ecology. Beyond the classical compilation of measurement protocols, we have synthesised recommendations from the literature to provide key background knowledge useful for: (1) defining below-ground plant entities and giving keys for their meaningful dissection, classification and naming beyond the classical fine-root vs coarse-root approach; (2) considering the specificity of root research to produce sound laboratory and field data; (3) describing typical, but overlooked steps for studying roots (e.g. root handling, cleaning and storage); and (4) gathering metadata necessary for the interpretation of results and their reuse. Most importantly, all root traits have been introduced with some degree of ecological context that will be a foundation for understanding their ecological meaning, their typical use and uncertainties, and some methodological and conceptual perspectives for future research. Considering all of this, we urge readers not to solely extract protocol recommendations for trait measurements from this work, but to take a moment to read and reflect on the extensive information contained in this broader guide to root ecology, including sections I-VII and the many introductions to each section and root trait description. Finally, it is critical to understand that a major aim of this guide is to help break down barriers between the many subdisciplines of root ecology and ecophysiology, broaden researchers' views on the multiple aspects of root study and create favourable conditions for the inception of comprehensive experiments on the role of roots in plant and ecosystem functioning.

Motherly care: How Leymus chinensis ramets support their offspring exposed to saline-alkali and clipping stresses.

BACKGROUND: While clonal integration can improve the performance of rhizomatous plants, it remains unclear whether their clonal integration strategy changes under contrasting clipping and saline-alkali homogeneous and heterogeneous environments. Leymus chinensis is a clonal grass native to the Songnen grassland where heavy grazing and patchy saline-alkali stress are serious environmental and ecological problems. We hypothesized that L. chinensis overcomes these stresses through clonal integration, in particular the transfer of nitrogen and carbohydrates. METHODS: A pot experiment was carried out with 15N isotope soil labeling method to study clonal integration strategy in the connected mother and daughter ramets of L. chinensis. The connected ramet pairs were grown in homogeneous (both connected ramets were treated) and heterogeneous (only daughter ramets were treated) environments with four treatments: control, clipping (60% aboveground biomass removal), saline-alkali (3.45 g of NaCl, NaHCO3, and Na2CO3 per pot), and clipping × saline-alkali. RESULTS: A significant amount (22.5%) of 15N was transferred from mother to daughter ramets under non-stressed conditions. When homogeneously stressing both mother and daughter ramets, N transfer was significantly reduced to 8.5--14.6%, independent of the nature of the stress. When only daughters were stressed (heterogeneous stress), saline-alkali stress led to a division of labor where daughters had enhanced photosynthesis, and mother ramets had increased 15N uptake and growth. Clipping only daughters reduced biomass and 15N uptake of both daughter and mother ramets. CONCLUSIONS: Our results demonstrated that clonal integration also occurs in homogeneous favorable environments but is reduced under homogeneous stress. In heterogeneous environments, clonal integration is used to translocate resource after clipping and a division of labor is established to overcome saline-alkali stress. Clonal integration continued even when daughters were severely stressed by the combined treatments. Our findings suggest that these mechanisms are key to the success of L. chinensis in the Songnen grassland.

Root traits as drivers of plant and ecosystem functioning: current understanding, pitfalls and future research needs.

The effects of plants on the biosphere, atmosphere and geosphere are key determinants of terrestrial ecosystem functioning. However, despite substantial progress made regarding plant belowground components, we are still only beginning to explore the complex relationships between root traits and functions. Drawing on the literature in plant physiology, ecophysiology, ecology, agronomy and soil science, we reviewed 24 aspects of plant and ecosystem functioning and their relationships with a number of root system traits, including aspects of architecture, physiology, morphology, anatomy, chemistry, biomechanics and biotic interactions. Based on this assessment, we critically evaluated the current strengths and gaps in our knowledge, and identify future research challenges in the field of root ecology. Most importantly, we found that belowground traits with the broadest importance in plant and ecosystem functioning are not those most commonly measured. Also, the estimation of trait relative importance for functioning requires us to consider a more comprehensive range of functionally relevant traits from a diverse range of species, across environments and over time series. We also advocate that establishing causal hierarchical links among root traits will provide a hypothesis-based framework to identify the most parsimonious sets of traits with the strongest links on functions, and to link genotypes to plant and ecosystem functioning.

Dividing the pie: A quantitative review on plant density responses.

Plant population density is an important variable in agronomy and forestry and offers an experimental way to better understand plant-plant competition. We made a meta-analysis of responses of even-aged mono-specific stands to population density by quantifying for 3 stand and 33 individual plant variables in 334 experiments how much both plant biomass and phenotypic traits change with a doubling in density. Increasing density increases standing crop per area, but decreases the mean size of its individuals, mostly through reduced tillering and branching. Among the phenotypic traits, stem diameter is negatively affected, but plant height remains remarkably similar, partly due to an increased stem length-to-mass ratio and partly by increased allocation to stems. The reduction in biomass is caused by a lower photosynthetic rate, mainly due to shading of part of the foliage. Total seed mass per plant is also strongly reduced, marginally by lower mass per seed, but mainly because of lower seed numbers. Plants generally have fewer shoot-born roots, but their overall rooting depth seems hardly affected. The phenotypic plasticity responses to high densities correlate strongly with those to low light, and less with those to low nutrients, suggesting that at high density, shading affects plants more than nutrient depletion.

An Analysis of Soil Coring Strategies to Estimate Root Depth in Maize (Zea mays) and Common Bean (Phaseolus vulgaris).

A soil coring protocol was developed to cooptimize the estimation of root length distribution (RLD) by depth and detection of functionally important variation in root system architecture (RSA) of maize and bean. The functional-structural model OpenSimRoot was used to perform in silico soil coring at six locations on three different maize and bean RSA phenotypes. Results were compared to two seasons of field soil coring and one trench. Two one-sided T-test (TOST) analysis of in silico data suggests a between-row location 5 cm from plant base (location 3), best estimates whole-plot RLD/D of deep, intermediate, and shallow RSA phenotypes, for both maize and bean. Quadratic discriminant analysis indicates location 3 has ~70% categorization accuracy for bean, while an in-row location next to the plant base (location 6) has ~85% categorization accuracy in maize. Analysis of field data suggests the more representative sampling locations vary by year and species. In silico and field studies suggest location 3 is most robust, although variation is significant among seasons, among replications within a field season, and among field soil coring, trench, and simulations. We propose that the characterization of the RLD profile as a dynamic rhizo canopy effectively describes how the RLD profile arises from interactions among an individual plant, its neighbors, and the pedosphere.

Spatio-Temporal Variation in Water Uptake in Seminal and Nodal Root Systems of Barley Plants Grown in Soil.

The spatial and temporal dynamics of root water uptake in nodal and seminal roots are poorly understood, especially in relation to root system development and aging. Here we non-destructively quantify 1) root water uptake and 2) root length of nodal and seminal roots of barley in three dimensions during 43 days of growth. We developed a concentric split root system to hydraulically and physically isolate the seminal and nodal root systems. Using magnetic resonance imaging (MRI), roots were visualized, root length was determined, and soil water depletion in both compartments was measured. From 19 days after germination and onwards, the nodal root system had greater water uptake compared to the seminal root system due to both greater root length and greater root conductivity. At 29 days after germination onwards, the average age of the seminal and nodal root systems was similar and no differences were observed in water uptake per root length between seminal and nodal root systems, indicating the importance of embryonic root systems for seedling establishment and nodal root systems in more mature plants. Since nodal roots perform the majority of water uptake at 29 days after germination and onwards, nodal root phenes merit consideration as a selection target to improve water capture in barley and possibly other crops.

Spatially Resolved Root Water Uptake Determination Using a Precise Soil Water Sensor.

To answer long-standing questions about how plants use and regulate water, an affordable, noninvasive way to determine local root water uptake (RWU) is required. Here, we present a sensor, the soil water profiler (SWaP), which can determine local soil water content (θ) with a precision of 6.10-5 cm3 ⋅ cm-3, an accuracy of 0.002 cm3 ⋅ cm-3, a temporal resolution of 24 min, and a one-dimensional spatial resolution of 1 cm. The sensor comprises two copper sheets, integrated into a sleeve and connected to a coil, which form a resonant circuit. A vector network analyzer, inductively coupled to the resonant circuit, measures the resonance frequency, against which θ was calibrated. The sensors were integrated into a positioning system, which measures θ along the depth of cylindrical tubes. When combined with modulating light (4-h period) and resultant modulating plant transpiration, the SWaP enables quantification of the component of RWU distribution that varies proportionally with total plant water uptake, and distinguishes it from soil water redistribution via soil pores and roots. Additionally, as a young, growing maize (Zea mays) plant progressively tapped its soil environment dry, we observed clear changes in plant-driven RWU and soil water redistribution profiles. Our SWaP setup can measure the RWU and redistribution of sandy-soil water content with unprecedented precision. The SWaP is therefore a promising device offering new insights into soil-plant hydrology, with applications for functional root phenotyping in nonsaline, temperature-controlled conditions, at low cost.

Call for Participation: Collaborative Benchmarking of Functional-Structural Root Architecture Models. The Case of Root Water Uptake.

Three-dimensional models of root growth, architecture and function are becoming important tools that aid the design of agricultural management schemes and the selection of beneficial root traits. However, while benchmarking is common in many disciplines that use numerical models, such as natural and engineering sciences, functional-structural root architecture models have never been systematically compared. The following reasons might induce disagreement between the simulation results of different models: different representation of root growth, sink term of root water and solute uptake and representation of the rhizosphere. Presently, the extent of discrepancies is unknown, and a framework for quantitatively comparing functional-structural root architecture models is required. We propose, in a first step, to define benchmarking scenarios that test individual components of complex models: root architecture, water flow in soil and water flow in roots. While the latter two will focus mainly on comparing numerical aspects, the root architectural models have to be compared at a conceptual level as they generally differ in process representation. Therefore, defining common inputs that allow recreating reference root systems in all models will be a key challenge. In a second step, benchmarking scenarios for the coupled problems are defined. We expect that the results of step 1 will enable us to better interpret differences found in step 2. This benchmarking will result in a better understanding of the different models and contribute toward improving them. Improved models will allow us to simulate various scenarios with greater confidence and avoid bugs, numerical errors or conceptual misunderstandings. This work will set a standard for future model development.

Crop Improvement from Phenotyping Roots: Highlights Reveal Expanding Opportunities.

Root systems determine the water and nutrients for photosynthesis and harvested products, underpinning agricultural productivity. We highlight 11 programs that integrated root traits into germplasm for breeding, relying on phenotyping. Progress was successful but slow. Today's phenotyping technologies will speed up root trait improvement. They combine multiple new alleles in germplasm for target environments, in parallel. Roots and shoots are detected simultaneously and nondestructively, seed to seed measures are automated, and field and laboratory technologies are increasingly linked. Available simulation models can aid all phenotyping decisions. This century will see a shift from single root traits to rhizosphere selections that can be managed dynamically on farms and a shift to phenotype-based improvement to accommodate the dynamic complexity of whole crop systems.

Soil compaction and the architectural plasticity of root systems.

Soil compaction is a serious global problem, and is a major cause of inadequate rooting and poor yield in crops around the world. Root system architecture (RSA) describes the spatial arrangement of root components within the soil and determines the plant's exploration of the soil. Soil strength restricts root growth and may slow down root system development. RSA plasticity may have an adaptive value, providing environmental tolerance to soil compaction. However, it is challenging to distinguish developmental retardation (apparent plasticity) or responses to severe stress from those root architectural changes that may provide an actual environmental tolerance (adaptive plasticity). In this review, we outline the consequences of soil compaction on the rooting environment and extensively review the various root responses reported in the literature. Finally, we discuss which responses enhance root exploration capabilities in tolerant genotypes, and to what extent these responses might be useful for breeding. We conclude that RSA plasticity in response to soil compaction is complex and can be targeted in breeding to increase the performance of crops under specific agronomical conditions.

Co-optimization of axial root phenotypes for nitrogen and phosphorus acquisition in common bean.

Background and Aims: Root architecture is a primary determinant of soil resource acquisition. We hypothesized that root architectural phenes will display both positive and negative interactions with each other for soil resource capture because of competition for internal resources and functional trade-offs in soil exploration. Methods: We employed the functional-structural plant model SimRoot to explore how interactions among architectural phenes in common bean determine the acquisition of phosphate and nitrate, two key soil resources contrasting in mobility. We evaluated the utility of basal root whorl number (BRWN) when basal root growth angle, hypocotyl-borne roots and lateral root branching density (LRBD) were varied, under varying availability of phosphate and nitrate. Key Results: Three basal root whorls were optimal in most phenotypes. This optimum shifted towards greater values when LRBD decreased and to smaller numbers when LRBD increased. The maximum biomass accumulated for a given BRWN phenotype in a given limiting nutrient scenario depended upon root growth angle. Under phosphorus stress shallow phenotypes grew best, whereas under nitrate stress fanned phenotypes grew best. The effect of increased hypocotyl-borne roots depended upon BRWN as well as the limiting nutrient. Greater production of axial roots due to BRWN or hypocotyl-borne roots reduced rooting depth, leading to reduced biomass under nitrate-limiting conditions. Increased BRWN as well as greater LRBD increased root carbon consumption, resulting in reduced shoot biomass. Conclusions: We conclude that the utility of a root architectural phenotype is determined by whether the constituent phenes are synergistic or antagonistic. Competition for internal resources and trade-offs for external resources result in multiple phenotypes being optimal under a given nutrient regime. We also find that no single phenotype is optimal across contrasting environments. These results have implications for understanding plant evolution and also for the breeding of more stress-tolerant crop phenotypes.

Ethylene modulates root cortical senescence in barley.

Background and Aims: Root cortical senescence (RCS) is a poorly understood phenomenon with implications for adaptation to edaphic stress. It was hypothesized that RCS in barley (Hordeum vulgare L.) is (1) accelerated by exogenous ethylene exposure; (2) accompanied by differential expression of ethylene synthesis and signalling genes; and (3) associated with differential expression of programmed cell death (PCD) genes. Methods: Gene expression of root segments from four barley genotypes with and without RCS was evaluated using quantitative real-time PCR (qRT-PCR). The progression of RCS was manipulated with root zone ethylene and ethylene inhibitor applications. Key Results: The results demonstrate that ethylene modulates RCS. Four genes related to ethylene synthesis and signalling were upregulated during RCS in optimal, low nitrogen and low phosphorus nutrient regimes. RCS was accelerated by root zone ethylene treatment, and this effect was reversed by an ethylene action inhibitor. Roots treated with exogenous ethylene had 35 and 46 % more cortical senescence compared with the control aeration treatment in seminal and nodal roots, respectively. RCS was correlated with expression of two genes related to programmed cell death (PCD). Conclusions: The development of RCS is similar to root cortical aerenchyma formation with respect to ethylene modulation of the PCD process.

Root Cortical Senescence Improves Growth under Suboptimal Availability of N, P, and K.

Root cortical senescence (RCS) in Triticeae reduces nutrient uptake, nutrient content, respiration, and radial hydraulic conductance of root tissue. We used the functional-structural model SimRoot to evaluate the functional implications of RCS in barley (Hordeum vulgare) under suboptimal nitrate, phosphorus, and potassium availability. The utility of RCS was evaluated using sensitivity analyses in contrasting nutrient regimes. At flowering (80 d), RCS increased simulated plant growth by up to 52%, 73%, and 41% in nitrate-, phosphorus-, and potassium-limiting conditions, respectively. Plants with RCS had reduced nutrient requirement of root tissue for optimal plant growth, reduced total cumulative cortical respiration, and increased total carbon reserves. Nutrient reallocation during RCS had a greater effect on simulated plant growth than reduced respiration or nutrient uptake. Under low nutrient availability, RCS had greater benefit in plants with fewer tillers. RCS had greater benefit in phenotypes with fewer lateral roots at low nitrate availability, but the opposite was true in low phosphorus or potassium availability. Additionally, RCS was quantified in field-grown barley in different nitrogen regimes. Field and virtual soil coring simulation results demonstrated that living cortical volume per root length (an indicator of RCS) decreased with depth in younger plants, while roots of older plants had very little living cortical volume per root length. RCS may be an adaptive trait for nutrient acquisition by reallocating nutrients from senescing tissue and secondarily by reducing root respiration. These simulated results suggest that RCS merits investigation as a breeding target for enhanced soil resource acquisition and edaphic stress tolerance.