Root systems play an essential role in ensuring plant productivity. Experiments conducted in controlled environments and simulation models suggest that root geometry and responses of root architecture to environmental factors should be studied as a priority. However, compared with aboveground plant organs, roots are not easily accessible by non-invasive analyses and field research is still based almost completely on manual, destructive methods. Contributing to reducing the gap between laboratory and field experiments, we present a novel phenotyping system (GROWSCREEN-Rhizo), which is capable of automatically imaging roots and shoots of plants grown in soil-filled rhizotrons (up to a volume of ~18 L) with a throughput of 60 rhizotrons per hour. Analysis of plants grown in this setup is restricted to a certain plant size (up to a shoot height of 80 cm and root-system depth of 90 cm). We performed validation experiments using six different species and for barley and maize, we studied the effect of moderate soil compaction, which is a relevant factor in the field. First, we found that the portion of root systems that is visible through the rhizotrons’ transparent plate is representative of the total root system. The percentage of visible roots decreases with increasing average root diameter of the plant species studied and depends, to some extent, on environmental conditions. Second, we could measure relatively minor changes in root-system architecture induced by a moderate increase in soil compaction. Taken together, these findings demonstrate the good potential of this methodology to characterise root geometry and temporal growth responses with relatively high spatial accuracy and resolution for both monocotyledonous and dicotyledonous species. Our prototype will allow the design of high-throughput screening methodologies simulating environmental scenarios that are relevant in the field and will support breeding efforts towards improved resource use efficiency and stability of crop yields.

Root phenotyping is a challenging task, mainly because of the hidden nature of this organ. Only recently, imaging technologies have become available that allow us to elucidate the dynamic establishment of root structure and function in the soil. In root tips, optical analysis of the relative elemental growth rates in root expansion zones of hydroponically-grown plants revealed that it is the maximum intensity of cellular growth processes rather than the length of the root growth zone that control the acclimation to dynamic changes in temperature. Acclimation of entire root systems was studied at high throughput in agar-filled Petri dishes. In the present study, optical analysis of root system architecture showed that low temperature induced smaller branching angles between primary and lateral roots, which caused a reduction in the volume that roots access at lower temperature. Simulation of temperature gradients similar to natural soil conditions led to differential responses in basal and apical parts of the root system, and significantly affected the entire root system. These results were supported by first data on the response of root structure and carbon transport to different root zone temperatures. These data were acquired by combined magnetic resonance imaging (MRI) and positron emission tomography (PET). They indicate acclimation of root structure and geometry to temperature and preferential accumulation of carbon near the root tip at low root zone temperatures. Overall, this study demonstrated the value of combining different phenotyping technologies that analyse processes at different spatial and temporal scales. Only such an integrated approach allows us to connect differences between genotypes obtained in artificial high throughput conditions with specific characteristics relevant for field performance. Thus, novel routes may be opened up for improved plant breeding as well as for mechanistic understanding of root structure and function.

Significance: To date, the potential of utilizing root traits in plant breeding remains largely untapped. In this study, we cloned and characterized theENHANCED GRAVITROPISM2(EGT2) gene of barley that encodes a STERILE ALPHA MOTIF domain–containing protein. We demonstrated thatEGT2is a key gene of root growth angle regulation in response to gravity, which is conserved in barley and wheat and could be a promising target for crop improvement in cereals.

New techniques and approaches have been developed for root phenotyping recently; however, rapid and repeatable non-invasive root phenotyping remains challenging. Here, we present GrowScreen-PaGe, a non-invasive, high-throughput phenotyping system (4 plants min–1) based on flat germination paper. GrowScreen-PaGe allows the acquisition of time series of the developing root systems of 500 plants, thereby enabling to quantify short-term variations in root system. The choice of germination paper was found to be crucial and paper ? root interaction should be considered when comparing data from different studies on germination paper. The system is suitable for phenotyping dicot and monocot plant species. The potential of the system for high-throughput phenotyping was shown by investigating phenotypic diversity of root traits in a collection of 180 rapeseed accessions and of 52 barley genotypes grown under control and nutrient-starved conditions. Most traits showed a large variation linked to both genotype and treatment. In general, root length traits contributed more than shape and branching related traits in separating the genotypes. Overall, results showed that GrowScreen-PaGe will be a powerful resource to investigate root systems and root plasticity of large sets of plants and to explore the molecular and genetic root traits of various species including for crop improvement programs.

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 termedENHANCED GRAVITROPISM1(EGT1) that encodes a putative AGO component, whose loss-of-function enhances root gravitropism. Mutations in barley and wheatEGT1genes confer a striking root phenotype, where every root class adopts a steeper growth angle.EGT1encodes an F-box and Tubby domain-containing protein that is highly conserved across plant species. Haplotype analysis found that natural allelic variation at the barleyEGT1locus impacts root angle. Gravitropic assays indicated thatHvegt1roots bend more rapidly than wild-type. Transcript profiling revealedHvegt1roots deregulate reactive oxygen species (ROS) homeostasis and cell wall-loosening enzymes and cofactors. ROS imaging shows thatHvegt1root basal meristem and elongation zone tissues have reduced levels. Atomic force microscopy measurements detected elongatingHvegt1root cortical cell walls are significantly less stiff than wild-type. In situ analysis identifiedHvEGT1is 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 byEGT1in cereal crops employing an antigravitropic mechanism.

Seedling establishment is the first stage of crop productivity, and root phenotypes at seed emergence are critical to a successful start of shoot growth as well as for water and nutrient uptake. In this study, we investigate seedling establishment in winter wheat utilizing a newly developed workflow based on magnetic resonance imaging (MRI). Using the eight parents of the MAGIC (multi-parent advanced generation inter-cross) population we analysed the 4D root architecture of 288 individual seedlings grown in natural soils with plant neighbors over 3 d of development. Time of root and shoot emergence, total length, angle, and depth of the axile roots varied significantly among these genotypes. The temporal data resolved rates of elongation of primary roots and first and second seminal root pairs. Genotypes with slowly elongating primary roots had rapidly elongating first and second seminal root pairs and vice versa, resulting in variation in root system architecture mediated not only by root angle but also by initiation and relative elongation of axile roots. We demonstrated that our novel MRI workflow with a unique planting design and automated measurements allowed medium throughput phenotyping of wheat roots in 4D and could give new insights into regulation of root system architecture.