NBR1-mediated selective autophagy of ARF7 modulates root branching.

Auxin dictates root architecture via the Auxin Response Factor (ARF) family of transcription factors, which control lateral root (LR) formation. In Arabidopsis, ARF7 regulates the specification of prebranch sites (PBS) generating LRs through gene expression oscillations and plays a pivotal role during LR initiation. Despite the importance of ARF7 in this process, there is a surprising lack of knowledge about how ARF7 turnover is regulated and how this impacts root architecture. Here, we show that ARF7 accumulates in autophagy mutants and is degraded through NBR1-dependent selective autophagy. We demonstrate that the previously reported rhythmic changes to ARF7 abundance in roots are modulated via autophagy and might occur in other tissues. In addition, we show that the level of co-localization between ARF7 and autophagy markers oscillates and can be modulated by auxin to trigger ARF7 turnover. Furthermore, we observe that autophagy impairment prevents ARF7 oscillation and reduces both PBS establishment and LR formation. In conclusion, we report a novel role for autophagy during development, namely by enacting auxin-induced selective degradation of ARF7 to optimize periodic root branching.

Brassinosteroids Inhibit Autotropic Root Straightening by Modifying Filamentous-Actin Organization and Dynamics.

When positioned horizontally, roots grow down toward the direction of gravity. This phenomenon, called gravitropism, is influenced by most of the major plant hormones including brassinosteroids. Epi-brassinolide (eBL) was previously shown to enhance root gravitropism, a phenomenon similar to the response of roots exposed to the actin inhibitor, latrunculin B (LatB). This led us to hypothesize that eBL might enhance root gravitropism through its effects on filamentous-actin (F-actin). This hypothesis was tested by comparing gravitropic responses of maize (Zea mays) roots treated with eBL or LatB. LatB- and eBL-treated roots displayed similar enhanced downward growth compared with controls when vertical roots were oriented horizontally. Moreover, the effects of the two compounds on root growth directionality were more striking on a slowly-rotating two-dimensional clinostat. Both compounds inhibited autotropism, a process in which the root straightened after the initial gravistimulus was withdrawn by clinorotation. Although eBL reduced F-actin density in chemically-fixed Z. mays roots, the impact was not as strong as that of LatB. Modification of F-actin organization after treatment with both compounds was also observed in living roots of barrel medic (Medicago truncatula) seedlings expressing genetically encoded F-actin reporters. Like in fixed Z. mays roots, eBL effects on F-actin in living M. truncatula roots were modest compared with those of LatB. Furthermore, live cell imaging revealed a decrease in global F-actin dynamics in hypocotyls of etiolated M. truncatula seedlings treated with eBL compared to controls. Collectively, our data indicate that eBL-and LatB-induced enhancement of root gravitropism can be explained by inhibited autotropic root straightening, and that eBL affects this process, in part, by modifying F-actin organization and dynamics.

GRANAR, a Computational Tool to Better Understand the Functional Importance of Monocotyledon Root Anatomy.

Root hydraulic conductivity is a limiting factor along the water pathways between the soil and the leaf, and root radial conductivity is itself defined by cell-scale hydraulic properties and anatomical features. However, quantifying the influence of anatomical features on the radial conductivity remains challenging due to complex time-consuming experimental procedures. We present an open-source computational tool, the Generator of Root Anatomy in R (GRANAR; http://granar.github.io), that can be used to rapidly generate digital versions of contrasted monocotyledon root anatomical networks. GRANAR uses a limited set of root anatomical parameters, easily acquired with existing image analysis tools. The generated anatomical network can then be used in combination with hydraulic models to estimate the corresponding hydraulic properties. We used GRANAR to reanalyze large maize (Zea mays) anatomical datasets from the literature. Our model was successful at creating virtual anatomies for each experimental observation. We also used GRANAR to generate anatomies not observed experimentally over wider ranges of anatomical parameters. The generated anatomies were then used to estimate the corresponding radial conductivities with the hydraulic model MECHA (model of explicit cross-section hydraulic architecture). Our simulations highlight the large importance of the width of the stele and the cortex. GRANAR is a computational tool that generates root anatomical networks from experimental data. It enables the quantification of the effect of individual anatomical features on the root radial conductivity.

Two Wheat Cultivars with Contrasting Post-Embryonic Root Biomass Differ in Shoot Re-Growth after Defoliation: Implications for Breeding Grazing Resilient Forages.

The ability of forages to quickly resume aboveground growth after grazing is a trait that enables farmers to better manage their livestock for maximum profitability. Leaf removal impairs root growth. As a consequence of a deficient root system, shoot re-growth is inhibited leading to poor pasture performance. Despite the importance of roots for forage productivity, they have not been considered as breeding targets for improving grazing resilience due in large part to the lack of knowledge on the relationship between roots and aboveground biomass re-growth. Winter wheat (Triticum aestivum) is extensively used as forage source in temperate climates worldwide. Here, we investigated the impact of leaf clipping on specific root traits, and how these influence shoot re-growth in two winter wheat cultivars (i.e., Duster and Cheyenne) with contrasting root and shoot biomass. We found that root growth angle and post-embryonic root growth in both cultivars are strongly influenced by defoliation. We discovered that Duster, which had less post-embryonic roots before defoliation, reestablished its root system faster after leaf cutting compared with Cheyenne, which had a more extensive pre-defoliation post-embryonic root system. Rapid resumption of root growth in Duster after leaf clipping was associated with faster aboveground biomass re-growth even after shoot overcutting. Taken together, our results suggest that lower investments in the production of post-embryonic roots presents an important ideotype to consider when breeding for shoot re-growth vigor in dual purpose wheat.

Demystifying roots: A need for clarification and extended concepts in root phenotyping.

Plant roots have major roles in plant anchorage, resource acquisition and offer environmental benefits including carbon sequestration and soil erosion mitigation. As such, the study of root system architecture, anatomy and functional properties is of crucial interest to plant breeding, with the aim of sustainable yield production and environmental stewardship. Due to the importance of the root system studies, there is a need for clarification of terms and concepts in the root phenotyping community. In particular in this contribution, we advocate for the use of a reference naming system (ontologies) for roots and root phenes. Such uniformity would not only allow better understanding of research results, but would also enable a better sharing of data. In addition, we highlight the need to incorporate the concept of plasticity in breeding programs, as it is an essential component of root system development in heterogeneous environments.

Fluorescence Imaging of the Cytoskeleton in Plant Roots.

During the past two decades the use of live cytoskeletal probes has increased dramatically due to the introduction of the green fluorescent protein. However, to make full use of these live cell reporters it is necessary to implement simple methods to maintain plant specimens in optimal growing conditions during imaging. To image the cytoskeleton in living Arabidopsis roots, we rely on a system involving coverslips coated with nutrient supplemented agar where the seeds are directly germinated. This coverslip system can be conveniently transferred to the stage of a confocal microscope with minimal disturbance to the growth of the seedling. For roots with a larger diameter such as Medicago truncatula, seeds are first germinated in moist paper, grown vertically in between plastic trays, and roots mounted on glass slides for confocal imaging. Parallel with our live cell imaging approaches, we routinely process fixed plant material via indirect immunofluorescence. For these methods we typically use non-embedded vibratome-sectioned and whole mount permeabilized root tissue. The clearly defined developmental regions of the root provide us with an elegant system to further understand the cytoskeletal basis of plant development.

Root Traits and Phenotyping Strategies for Plant Improvement.

Roots are crucial for nutrient and water acquisition and can be targeted to enhance plant productivity under a broad range of growing conditions. A current challenge for plant breeding is the limited ability to phenotype and select for desirable root characteristics due to their underground location. Plant breeding efforts aimed at modifying root traits can result in novel, more stress-tolerant crops and increased yield by enhancing the capacity of the plant for soil exploration and, thus, water and nutrient acquisition. Available approaches for root phenotyping in laboratory, greenhouse and field encompass simple agar plates to labor-intensive root digging (i.e., shovelomics) and soil boring methods, the construction of underground root observation stations and sophisticated computer-assisted root imaging. Here, we summarize root architectural traits relevant to crop productivity, survey root phenotyping strategies and describe their advantages, limitations and practical value for crop and forage breeding programs.

Role of actin cytoskeleton in brassinosteroid signaling and in its integration with the auxin response in plants.

In plants, developmental programs and tropisms are modulated by the phytohormone auxin. Auxin reconfigures the actin cytoskeleton, which controls polar localization of auxin transporters such as PIN2 and thus determines cell-type-specific responses. In conjunction with a second growth-promoting phytohormone, brassinosteroid (BR), auxin synergistically enhances growth and gene transcription. We show that BR alters actin configuration and PIN2 localization in a manner similar to that of auxin. We describe a BR constitutive-response mutant that bears an allele of the ACTIN2 gene and shows altered actin configuration, PIN2 delocalization, and a broad array of phenotypes that recapitulate BR-treated plants. Moreover, we show that actin filament reconfiguration is sufficient to activate BR signaling, which leads to an enhanced auxin response. Our results demonstrate that the actin cytoskeleton functions as an integration node for the BR signaling pathway and auxin responsiveness.