Natural variation of TBR confers plant zinc toxicity tolerance through root cell wall pectin methylesterification.

Zinc (Zn) is an essential micronutrient but can be cytotoxic when present in excess. Plants have evolved mechanisms to tolerate Zn toxicity. To identify genetic loci responsible for natural variation of plant tolerance to Zn toxicity, we conduct genome-wide association studies for root growth responses to high Zn and identify 21 significant associated loci. Among these loci, we identify Trichome Birefringence (TBR) allelic variation determining root growth variation in high Zn conditions. Natural alleles of TBR determine TBR transcript and protein levels which affect pectin methylesterification in root cell walls. Together with previously published data showing that pectin methylesterification increase goes along with decreased Zn binding to cell walls in TBR mutants, our findings lead to a model in which TBR allelic variation enables Zn tolerance through modulating root cell wall pectin methylesterification. The role of TBR in Zn tolerance is conserved across dicot and monocot plant species.

Arabidopsis transcriptome responses to low water potential using high-throughput plate assays.

Soil-free assays that induce water stress are routinely used to investigate drought responses in the plant Arabidopsis thaliana. Due to their ease of use, the research community often relies on polyethylene glycol (PEG), mannitol, and salt (NaCl) treatments to reduce the water potential of agar media, and thus induce drought conditions in the laboratory. However, while these types of stress can create phenotypes that resemble those of water deficit experienced by soil-grown plants, it remains unclear how these treatments compare at the transcriptional level. Here, we demonstrate that these different methods of lowering water potential elicit both shared and distinct transcriptional responses in Arabidopsis shoot and root tissue. When we compared these transcriptional responses to those found in Arabidopsis roots subject to vermiculite drying, we discovered many genes induced by vermiculite drying were repressed by low water potential treatments on agar plates (and vice versa). Additionally, we also tested another method for lowering water potential of agar media. By increasing the nutrient content and tensile strength of agar, we show the 'hard agar' (HA) treatment can be leveraged as a high-throughput assay to investigate natural variation in Arabidopsis growth responses to low water potential.

Nutrient levels control root growth responses to high ambient temperature in plants.

Global warming will lead to significantly increased temperatures on earth. Plants respond to high ambient temperature with altered developmental and growth programs, termed thermomorphogenesis. Here we show that thermomorphogenesis is conserved in Arabidopsis, soybean, and rice and that it is linked to a decrease in the levels of the two macronutrients nitrogen and phosphorus. We also find that low external levels of these nutrients abolish root growth responses to high ambient temperature. We show that in Arabidopsis, this suppression is due to the function of the transcription factor ELONGATED HYPOCOTYL 5 (HY5) and its transcriptional regulation of the transceptor NITRATE TRANSPORTER 1.1 (NRT1.1). Soybean and Rice homologs of these genes are expressed consistently with a conserved role in regulating temperature responses in a nitrogen and phosphorus level dependent manner. Overall, our data show that root thermomorphogenesis is a conserved feature in species of the two major groups of angiosperms, monocots and dicots, that it leads to a reduction of nutrient levels in the plant, and that it is dependent on environmental nitrogen and phosphorus supply, a regulatory process mediated by the HY5-NRT1.1 module.

Assessing Temperature Responses in Roots.

Due to global warming, it is important to understand how plants respond to high ambient temperature. Plant growth responses to high ambient temperature are termed thermomophogenesis and have been explored for more than a decade. However, this was mostly focused on the above-ground part of plants, the shoot. In this chapter, we describe a simple method to assess root growth phenotype to high ambient temperatures. In principle, this protocol can be applied for any other treatments to test overall seedling growth.

PAT (Periderm Assessment Toolkit): A Quantitative and Large-Scale Screening Method for Periderm Measurements.

The periderm is a vital protective tissue found in the roots, stems, and woody elements of diverse plant species. It plays an important function in these plants by assuming the role of the epidermis as the outermost layer. Despite its critical role for protecting plants from environmental stresses and pathogens, research on root periderm development has been limited due to its late formation during root development, its presence only in mature root regions, and its impermeability. One of the most straightforward measurements for comparing periderm formation between different genotypes and treatments is periderm (phellem) length. We have developed PAT (Periderm Assessment Toolkit), a high-throughput user-friendly pipeline that integrates an efficient staining protocol, automated imaging, and a deep-learning-based image analysis approach to accurately detect and measure periderm length in the roots of Arabidopsis thaliana. The reliability and reproducibility of our method was evaluated using a diverse set of 20 Arabidopsis natural accessions. Our automated measurements exhibited a strong correlation with human-expert-generated measurements, achieving a 94% efficiency in periderm length quantification. This robust PAT pipeline streamlines large-scale periderm measurements, thereby being able to facilitate comprehensive genetic studies and screens. Although PAT proves highly effective with automated digital microscopes in Arabidopsis roots, its application may pose challenges with nonautomated microscopy. Although the workflow and principles could be adapted for other plant species, additional optimization would be necessary. While we show that periderm length can be used to distinguish a mutant impaired in periderm development from wild type, we also find it is a plastic trait. Therefore, care must be taken to include sufficient repeats and controls, to minimize variation, and to ensure comparability of periderm length measurements between different genotypes and growth conditions.

Fast and Efficient Root Phenotyping via Pose Estimation.

Image segmentation is commonly used to estimate the location and shape of plants and their external structures. Segmentation masks are then used to localize landmarks of interest and compute other geometric features that correspond to the plant's phenotype. Despite its prevalence, segmentation-based approaches are laborious (requiring extensive annotation to train) and error-prone (derived geometric features are sensitive to instance mask integrity). Here, we present a segmentation-free approach that leverages deep learning-based landmark detection and grouping, also known as pose estimation. We use a tool originally developed for animal motion capture called SLEAP (Social LEAP Estimates Animal Poses) to automate the detection of distinct morphological landmarks on plant roots. Using a gel cylinder imaging system across multiple species, we show that our approach can reliably and efficiently recover root system topology at high accuracy, few annotated samples, and faster speed than segmentation-based approaches. In order to make use of this landmark-based representation for root phenotyping, we developed a Python library (sleap-roots) for trait extraction directly comparable to existing segmentation-based analysis software. We show that pose-derived root traits are highly accurate and can be used for common downstream tasks including genotype classification and unsupervised trait mapping. Altogether, this work establishes the validity and advantages of pose estimation-based plant phenotyping. To facilitate adoption of this easy-to-use tool and to encourage further development, we make sleap-roots, all training data, models, and trait extraction code available at: https://github.com/talmolab/sleap-roots and https://osf.io/k7j9g/.

Identification of mebendazole as an ethylene signaling activator reveals a role of ethylene signaling in the regulation of lateral root angles.

The lateral root angle or gravitropic set-point angle (GSA) is an important trait for root system architecture (RSA) that determines the radial expansion of the root system. The GSA therefore plays a crucial role for the ability of plants to access nutrients and water in the soil. Only a few regulatory pathways and mechanisms that determine GSA are known. These mostly relate to auxin and cytokinin pathways. Here, we report the identification of a small molecule, mebendazole (MBZ), that modulates GSA in Arabidopsis thaliana roots and acts via the activation of ethylene signaling. MBZ directly acts on the serine/threonine protein kinase CTR1, which is a negative regulator of ethylene signaling. Our study not only shows that the ethylene signaling pathway is essential for GSA regulation but also identifies a small molecular modulator of RSA that acts downstream of ethylene receptors and that directly activates ethylene signaling.

Spatial IMA1 regulation restricts root iron acquisition on MAMP perception.

Iron is critical during host-microorganism interactions1-4. Restriction of available iron by the host during infection is an important defence strategy, described as nutritional immunity5. However, this poses a conundrum for externally facing, absorptive tissues such as the gut epithelium or the plant root epidermis that generate environments that favour iron bioavailability. For example, plant roots acquire iron mostly from the soil and, when iron deficient, increase iron availability through mechanisms that include rhizosphere acidification and secretion of iron chelators6-9. Yet, the elevated iron bioavailability would also be beneficial for the growth of bacteria that threaten plant health. Here we report that microorganism-associated molecular patterns such as flagellin lead to suppression of root iron acquisition through a localized degradation of the systemic iron-deficiency signalling peptide Iron Man 1 (IMA1) in Arabidopsis thaliana. This response is also elicited when bacteria enter root tissues, but not when they dwell on the outer root surface. IMA1 itself has a role in modulating immunity in root and shoot, affecting the levels of root colonization and the resistance to a bacterial foliar pathogen. Our findings reveal an adaptive molecular mechanism of nutritional immunity that affects iron bioavailability and uptake, as well as immune responses.

GLK transcription factors accompany ELONGATED HYPOCOTYL5 to orchestrate light-induced seedling development in Arabidopsis.

Light-induced de-etiolation is an important aspect of seedling photomorphogenesis. GOLDEN2 LIKE (GLK) transcriptional regulators are involved in chloroplast development, but to what extent they participate in photomorphogenesis is not clear. Here, we show that ELONGATED HYPOCOTYL5 (HY5) binds to GLK promoters to activate their expression, and also interacts with GLK proteins in Arabidopsis (Arabidopsis thaliana). The chlorophyll content in the de-etiolating Arabidopsis seedlings of the hy5 glk2 double mutants was lower than that in the hy5 single mutant. GLKs inhibited hypocotyl elongation, and the phenotype could superimpose on the hy5 phenotype. Correspondingly, GLK2 regulated the expression of photosynthesis and cell elongation genes partially independent of HY5. Before exposure to light, DE-ETIOLATED 1 (DET1) affected accumulation of GLK proteins. The enhanced etioplast development and photosystem gene expression observed in the det1 mutant were attenuated in the det1 glk2 double mutant. Our study reveals that GLKs act downstream of HY5, or additive to HY5, and are likely quantitatively adjusted by DET1, to orchestrate multiple developmental traits during the light-induced skotomorphogenesis-to-photomorphogenesis transition in Arabidopsis.

Root Walker: an automated pipeline for large scale quantification of early root growth responses at high spatial and temporal resolution.

Plants are sessile organisms that constantly adapt to their changing environment. The root is exposed to numerous environmental signals ranging from nutrients and water to microbial molecular patterns. These signals can trigger distinct responses including the rapid increase or decrease of root growth. Consequently, using root growth as a readout for signal perception can help decipher which external cues are perceived by roots, and how these signals are integrated. To date, studies measuring root growth responses using large numbers of roots have been limited by a lack of high-throughput image acquisition, poor scalability of analytical methods, or low spatiotemporal resolution. Here, we developed the Root Walker pipeline, which uses automated microscopes to acquire time-series images of many roots exposed to controlled treatments with high spatiotemporal resolution, in conjunction with fast and automated image analysis software. We demonstrate the power of Root Walker by quantifying root growth rate responses at different time and throughput scales upon treatment with natural auxin and two mitogen-associated protein kinase cascade inhibitors. We find a concentration-dependent root growth response to auxin and reveal the specificity of one MAPK inhibitor. We further demonstrate the ability of Root Walker to conduct genetic screens by performing a genome-wide association study on 260 accessions in under 2 weeks, revealing known and unknown root growth regulators. Root Walker promises to be a useful toolkit for the plant science community, allowing large-scale screening of root growth dynamics for a variety of purposes, including genetic screens for root sensing and root growth response mechanisms.

Fast and efficient root phenotyping via pose estimation.

Image segmentation is commonly used to estimate the location and shape of plants and their external structures. Segmentation masks are then used to localize landmarks of interest and compute other geometric features that correspond to the plant's phenotype. Despite its prevalence, segmentation-based approaches are laborious (requiring extensive annotation to train), and error-prone (derived geometric features are sensitive to instance mask integrity). Here we present a segmentation-free approach which leverages deep learning-based landmark detection and grouping, also known as pose estimation. We use a tool originally developed for animal motion capture called SLEAP (Social LEAP Estimates Animal Poses) to automate the detection of distinct morphological landmarks on plant roots. Using a gel cylinder imaging system across multiple species, we show that our approach can reliably and efficiently recover root system topology at high accuracy, few annotated samples, and faster speed than segmentation-based approaches. In order to make use of this landmark-based representation for root phenotyping, we developed a Python library (sleap-roots) for trait extraction directly comparable to existing segmentation-based analysis software. We show that landmark-derived root traits are highly accurate and can be used for common downstream tasks including genotype classification and unsupervised trait mapping. Altogether, this work establishes the validity and advantages of pose estimation-based plant phenotyping. To facilitate adoption of this easy-to-use tool and to encourage further development, we make sleap-roots, all training data, models, and trait extraction code available at: https://github.com/talmolab/sleap-roots and https://osf.io/k7j9g/.

A lignin-derived material improves plant nutrient bioavailability and growth through its metal chelating capacity.

The lignocellulosic biorefinery industry can be an important contributor to achieving global carbon net zero goals. However, low valorization of the waste lignin severely limits the sustainability of biorefineries. Using a hydrothermal reaction, we have converted sulfuric acid lignin (SAL) into a water-soluble hydrothermal SAL (HSAL). Here, we show the improvement of HSAL on plant nutrient bioavailability and growth through its metal chelating capacity. We characterize HSAL's high ratio of phenolic hydroxyl groups to methoxy groups and its capacity to chelate metal ions. Application of HSAL significantly promotes root length and plant growth of both monocot and dicot plant species due to improving nutrient bioavailability. The HSAL-mediated increase in iron bioavailability is comparable to the well-known metal chelator ethylenediaminetetraacetic acid. Therefore, HSAL promises to be a sustainable nutrient chelator to provide an attractive avenue for sustainable utilization of the waste lignin from the biorefinery industry.

The AFB1 auxin receptor controls the cytoplasmic auxin response pathway in Arabidopsis thaliana.

The phytohormone auxin triggers root growth inhibition within seconds via a non-transcriptional pathway. Among members of the TIR1/AFB auxin receptor family, AFB1 has a primary role in this rapid response. However, the unique features that confer this specific function have not been identified. Here we show that the N-terminal region of AFB1, including the F-box domain and residues that contribute to auxin binding, is essential and sufficient for its specific role in the rapid response. Substitution of the N-terminal region of AFB1 with that of TIR1 disrupts its distinct cytoplasm-enriched localization and activity in rapid root growth inhibition by auxin. Importantly, the N-terminal region of AFB1 is indispensable for auxin-triggered calcium influx, which is a prerequisite for rapid root growth inhibition. Furthermore, AFB1 negatively regulates lateral root formation and transcription of auxin-induced genes, suggesting that it plays an inhibitory role in canonical auxin signaling. These results suggest that AFB1 may buffer the transcriptional auxin response, whereas it regulates rapid changes in cell growth that contribute to root gravitropism.

Plasmodesmata mediate cell-to-cell transport of brassinosteroid hormones.

Brassinosteroids (BRs) are steroidal phytohormones that are essential for plant growth, development and adaptation to environmental stresses. BRs act in a dose-dependent manner and do not travel over long distances; hence, BR homeostasis maintenance is critical for their function. Biosynthesis of bioactive BRs relies on the cell-to-cell movement of hormone precursors. However, the mechanism of the short-distance BR transport is unknown, and its contribution to the control of endogenous BR levels remains unexplored. Here we demonstrate that plasmodesmata (PD) mediate the passage of BRs between neighboring cells. Intracellular BR content, in turn, is capable of modulating PD permeability to optimize its own mobility, thereby manipulating BR biosynthesis and signaling. Our work uncovers a thus far unknown mode of steroid transport in eukaryotes and exposes an additional layer of BR homeostasis regulation in plants.

The Perpendicular Extraction - Visionary Concept with the Prospect of Generating the Perfect Extraction of Teeth, or a Misguided Vision that had to Fail Due to Anatomical Circumstances?

From the second half of the 18th century to the last third of the 19th century, a period of about 100 years, perpendicular (vertical) extraction, became an ideal for many authors, since molars were the most difficult teeth to remove. However, extraction instruments available at that time, caused pronounced damage to the alveolar bone and gingiva. For many authors and clinicians, vertical extraction was the only way to meet this challenge. This approach to tooth removal, while viable, came to an end when anatomically shaped forceps, adapted to the different morphologies of the various teeth were introduced, thereby changing 19th century dentistry with a new standard for tooth removal.

The AFB1 auxin receptor controls the cytoplasmic auxin response pathway in Arabidopsis thaliana.

The phytohormone auxin triggers root growth inhibition within seconds via a non-transcriptional pathway. Among members of the TIR1/AFBs auxin receptor family, AFB1 has a primary role in this rapid response. However, the unique features that confer this specific function have not been identified. Here we show that the N-terminal region of AFB1, including the F-box domain and residues that contribute to auxin binding, are essential and sufficient for its specific role in the rapid response. Substitution of the N-terminal region of AFB1 with that of TIR1 disrupts its distinct cytoplasm-enriched localization and activity in rapid root growth inhibition. Importantly, the N-terminal region of AFB1 is indispensable for auxin-triggered calcium influx which is a prerequisite for rapid root growth inhibition. Furthermore, AFB1 negatively regulates lateral root formation and transcription of auxin-induced genes, suggesting that it plays an inhibitory role in canonical auxin signaling. These results suggest that AFB1 may buffer the transcriptional auxin response while it regulates rapid changes in cell growth that contribute to root gravitropism.

Climate change challenges, plant science solutions.

Climate change is a defining challenge of the 21st century, and this decade is a critical time for action to mitigate the worst effects on human populations and ecosystems. Plant science can play an important role in developing crops with enhanced resilience to harsh conditions (e.g. heat, drought, salt stress, flooding, disease outbreaks) and engineering efficient carbon-capturing and carbon-sequestering plants. Here, we present examples of research being conducted in these areas and discuss challenges and open questions as a call to action for the plant science community.

The receptor kinase SRF3 coordinates iron-level and flagellin dependent defense and growth responses in plants.

Iron is critical for host-pathogen interactions. While pathogens seek to scavenge iron to spread, the host aims at decreasing iron availability to reduce pathogen virulence. Thus, iron sensing and homeostasis are of particular importance to prevent host infection and part of nutritional immunity. While the link between iron homeostasis and immunity pathways is well established in plants, how iron levels are sensed and integrated with immune response pathways remains unknown. Here we report a receptor kinase SRF3, with a role in coordinating root growth, iron homeostasis and immunity pathways via regulation of callose synthases. These processes are modulated by iron levels and rely on SRF3 extracellular and kinase domains which tune its accumulation and partitioning at the cell surface. Mimicking bacterial elicitation with the flagellin peptide flg22 phenocopies SRF3 regulation upon low iron levels and subsequent SRF3-dependent responses. We propose that SRF3 is part of nutritional immunity responses involved in sensing external iron levels.

Removing systemic barriers to equity, diversity, and inclusion: Report of the 2019 Plant Science Research Network workshop "Inclusivity in the Plant Sciences".

A future in which scientific discoveries are valued and trusted by the general public cannot be achieved without greater inclusion and participation of diverse communities. To envision a path towards this future, in January 2019 a diverse group of researchers, educators, students, and administrators gathered to hear and share personal perspectives on equity, diversity, and inclusion (EDI) in the plant sciences. From these broad perspectives, the group developed strategies and identified tactics to facilitate and support EDI within and beyond the plant science community. The workshop leveraged scenario planning and the richness of its participants to develop recommendations aimed at promoting systemic change at the institutional level through the actions of scientific societies, universities, and individuals and through new funding models to support research and training. While these initiatives were formulated specifically for the plant science community, they can also serve as a model to advance EDI in other disciplines. The proposed actions are thematically broad, integrating into discovery, applied and translational science, requiring and embracing multidisciplinarity, and giving voice to previously unheard perspectives. We offer a vision of barrier-free access to participation in science, and a plant science community that reflects the diversity of our rapidly changing nation, and supports and invests in the training and well-being of all its members. The relevance and robustness of our recommendations has been tested by dramatic and global events since the workshop. The time to act upon them is now.