SHR and SCR coordinate root patterning and growth early in the cell cycle.

Precise control of cell division is essential for proper patterning and growth during the development of multicellular organisms. Coordination of formative divisions that generate new tissue patterns with proliferative divisions that promote growth is poorly understood. SHORTROOT (SHR) and SCARECROW (SCR) are transcription factors that are required for formative divisions in the stem cell niche of Arabidopsis roots1,2. Here we show that levels of SHR and SCR early in the cell cycle determine the orientation of the division plane, resulting in either formative or proliferative cell division. We used 4D quantitative, long-term and frequent (every 15 min for up to 48 h) light sheet and confocal microscopy to probe the dynamics of SHR and SCR in tandem within single cells of living roots. Directly controlling their dynamics with an SHR induction system enabled us to challenge an existing bistable model3 of the SHR-SCR gene-regulatory network and to identify key features that are essential for rescue of formative divisions in shr mutants. SHR and SCR kinetics do not align with the expected behaviour of a bistable system, and only low transient levels, present early in the cell cycle, are required for formative divisions. These results reveal an uncharacterized mechanism by which developmental regulators directly coordinate patterning and growth.

The rhizodynamics robot: Automated imaging system for studying long-term dynamic root growth.

The study of plant root growth in real time has been difficult to achieve in an automated, high-throughput, and systematic fashion. Dynamic imaging of plant roots is important in order to discover novel root growth behaviors and to deepen our understanding of how roots interact with their environments. We designed and implemented the Generating Rhizodynamic Observations Over Time (GROOT) robot, an automated, high-throughput imaging system that enables time-lapse imaging of 90 containers of plants and their roots growing in a clear gel medium over the duration of weeks to months. The system uses low-cost, widely available materials. As a proof of concept, we employed GROOT to collect images of root growth of Oryza sativa, Hudsonia montana, and multiple species of orchids including Platanthera integrilabia over six months. Beyond imaging plant roots, our system is highly customizable and can be used to collect time- lapse image data of different container sizes and configurations regardless of what is being imaged, making it applicable to many fields that require longitudinal time-lapse recording.

Cell-type-specific transcriptomics reveals that root hairs and endodermal barriers play important roles in beneficial plant-rhizobacterium interactions.

Growth- and health-promoting bacteria can boost crop productivity in a sustainable way. Pseudomonas simiae WCS417 is such a bacterium that efficiently colonizes roots, modifies the architecture of the root system to increase its size, and induces systemic resistance to make plants more resistant to pests and pathogens. Our previous work suggested that WCS417-induced phenotypes are controlled by root cell-type-specific mechanisms. However, it remains unclear how WCS417 affects these mechanisms. In this study, we transcriptionally profiled five Arabidopsis thaliana root cell types following WCS417 colonization. We found that the cortex and endodermis have the most differentially expressed genes, even though they are not in direct contact with this epiphytic bacterium. Many of these genes are associated with reduced cell wall biogenesis, and mutant analysis suggests that this downregulation facilitates WCS417-driven root architectural changes. Furthermore, we observed elevated expression of suberin biosynthesis genes and increased deposition of suberin in the endodermis of WCS417-colonized roots. Using an endodermal barrier mutant, we showed the importance of endodermal barrier integrity for optimal plant-beneficial bacterium association. Comparison of the transcriptome profiles in the two epidermal cell types that are in direct contact with WCS417-trichoblasts that form root hairs and atrichoblasts that do not-implies a difference in potential for defense gene activation. While both cell types respond to WCS417, trichoblasts displayed both higher basal and WCS417-dependent activation of defense-related genes compared with atrichoblasts. This suggests that root hairs may activate root immunity, a hypothesis that is supported by differential immune responses in root hair mutants. Taken together, these results highlight the strength of cell-type-specific transcriptional profiling to uncover "masked" biological mechanisms underlying beneficial plant-microbe associations.

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.

Brassinosteroid gene regulatory networks at cellular resolution in the Arabidopsis root.

Brassinosteroids are plant steroid hormones that regulate diverse processes, such as cell division and cell elongation, through gene regulatory networks that vary in space and time. By using time series single-cell RNA sequencing to profile brassinosteroid-responsive gene expression specific to different cell types and developmental stages of the Arabidopsis root, we identified the elongating cortex as a site where brassinosteroids trigger a shift from proliferation to elongation associated with increased expression of cell wall-related genes. Our analysis revealed HOMEOBOX FROM ARABIDOPSIS THALIANA 7 (HAT7) and GT-2-LIKE 1 (GTL1) as brassinosteroid-responsive transcription factors that regulate cortex cell elongation. These results establish the cortex as a site of brassinosteroid-mediated growth and unveil a brassinosteroid signaling network regulating the transition from proliferation to elongation, which illuminates aspects of spatiotemporal hormone responses.

Plant physiology: The to-and-fro of hormone signals to respond to drought.

Xerobranching, a temporary suppression of root branching when water is limiting, is controlled by the plant hormone abscisic acid (ABA). A recently published study reveals how root branching is dynamically controlled by redistribution in opposite directions of ABA and auxin.

GRAS transcription factors regulate cell division planes in moss overriding the default rule.

Plant cells are surrounded by a cell wall and do not migrate, which makes the regulation of cell division orientation crucial for development. Regulatory mechanisms controlling cell division orientation may have contributed to the evolution of body organization in land plants. The GRAS family of transcription factors was transferred horizontally from soil bacteria to an algal common ancestor of land plants. SHORTROOT (SHR) and SCARECROW (SCR) genes in this family regulate formative periclinal cell divisions in the roots of flowering plants, but their roles in nonflowering plants and their evolution have not been studied in relation to body organization. Here, we show that SHR cell autonomously inhibits formative periclinal cell divisions indispensable for leaf vein formation in the moss Physcomitrium patens, and SHR expression is positively and negatively regulated by SCR and the GRAS member LATERAL SUPPRESSOR, respectively. While precursor cells of a leaf vein lacking SHR usually follow the geometry rule of dividing along the division plane with the minimum surface area, SHR overrides this rule and forces cells to divide nonpericlinally. Together, these results imply that these bacterially derived GRAS transcription factors were involved in the establishment of the genetic regulatory networks modulating cell division orientation in the common ancestor of land plants and were later adapted to function in flowering plant and moss lineages for their specific body organizations.

SCARECROW-LIKE28 modulates organ growth in Arabidopsis by controlling mitotic cell cycle exit, endoreplication, and cell expansion dynamics.

The processes that contribute to plant organ morphogenesis are spatial-temporally organized. Within the meristem, mitosis produces new cells that subsequently engage in cell expansion and differentiation programs. The latter is frequently accompanied by endoreplication, being an alternative cell cycle that replicates the DNA without nuclear division, causing a stepwise increase in somatic ploidy. Here, we show that the Arabidopsis SCL28 transcription factor promotes organ growth by modulating cell expansion dynamics in both root and leaf cells. Gene expression studies indicated that SCL28 regulates members of the SIAMESE/SIAMESE-RELATED (SIM/SMR) family, encoding cyclin-dependent kinase inhibitors with a role in promoting mitotic cell cycle (MCC) exit and endoreplication, both in response to developmental and environmental cues. Consistent with this role, mutants in SCL28 displayed reduced endoreplication, both in roots and leaves. We also found evidence indicating that SCL28 co-expresses with and regulates genes related to the biogenesis, assembly, and remodeling of the cytoskeleton and cell wall. Our results suggest that SCL28 controls, not only cell proliferation as reported previously but also cell expansion and differentiation by promoting MCC exit and endoreplication and by modulating aspects of the biogenesis, assembly, and remodeling of the cytoskeleton and cell wall.

Protocol for fast scRNA-seq raw data processing using scKB and non-arbitrary quality control with COPILOT.

We describe a protocol to perform fast and non-arbitrary quality control of single-cell RNA sequencing (scRNA-seq) raw data using scKB and COPILOT. scKB is a wrapper script of kallisto and bustools for accelerated alignment and transcript count matrix generation, which runs significantly faster than the popular tool Cell Ranger. COPILOT then offers non-arbitrary background noise removal by comparing distributions of low-quality and high-quality cells. Together, this protocol streamlines the processing workflow and provides an easy entry for new scRNA-seq users. For complete details on the use and execution of this protocol, please refer to Shahan et al. (2022).

Single-cell genomics revolutionizes plant development studies across scales.

Understanding the development of tissues, organs and entire organisms through the lens of single-cell genomics has revolutionized developmental biology. Although single-cell transcriptomics has been pioneered in animal systems, from an experimental perspective, plant development holds some distinct advantages: cells do not migrate in relation to one another, and new organ formation (of leaves, roots, flowers, etc.) continues post-embryonically from persistent stem cell populations known as meristems. For a time, plant studies lagged behind animal or cell culture-based, single-cell approaches, largely owing to the difficulty in dissociating plant cells from their rigid cell walls. Recent intensive development of single-cell and single-nucleus isolation techniques across plant species has opened up a wide range of experimental approaches. This has produced a rapidly expanding diversity of information across tissue types and species, concomitant with the creative development of methods. In this brief Spotlight, we highlight some of the technical developments and how they have led to profiling single-cell genomics in various plant organs. We also emphasize the contribution of single-cell genomics in revealing developmental trajectories among different cell types within plant organs. Furthermore, we present efforts toward comparative analysis of tissues and organs at a single-cell level. Single-cell genomics is beginning to generate comprehensive information relating to how plant organs emerge from stem cell populations.

A single-cell Arabidopsis root atlas reveals developmental trajectories in wild-type and cell identity mutants.

In all multicellular organisms, transcriptional networks orchestrate organ development. The Arabidopsis root, with its simple structure and indeterminate growth, is an ideal model for investigating the spatiotemporal transcriptional signatures underlying developmental trajectories. To map gene expression dynamics across root cell types and developmental time, we built a comprehensive, organ-scale atlas at single-cell resolution. In addition to estimating developmental progressions in pseudotime, we employed the mathematical concept of optimal transport to infer developmental trajectories and identify their underlying regulators. To demonstrate the utility of the atlas to interpret new datasets, we profiled mutants for two key transcriptional regulators at single-cell resolution, shortroot and scarecrow. We report transcriptomic and in vivo evidence for tissue trans-differentiation underlying a mixed cell identity phenotype in scarecrow. Our results support the atlas as a rich community resource for unraveling the transcriptional programs that specify and maintain cell identity to regulate spatiotemporal organ development.

Spatiotemporal analysis identifies ABF2 and ABF3 as key hubs of endodermal response to nitrate.

Nitrate is a nutrient and a potent signal that impacts global gene expression in plants. However, the regulatory factors controlling temporal and cell type-specific nitrate responses remain largely unknown. We assayed nitrate-responsive transcriptome changes in five major root cell types of the Arabidopsis thaliana root as a function of time. We found that gene-expression response to nitrate is dynamic and highly localized and predicted cell type-specific transcription factor (TF)-target interactions. Among cell types, the endodermis stands out as having the largest and most connected nitrate-regulatory gene network. ABF2 and ABF3 are major hubs for transcriptional responses in the endodermis cell layer. We experimentally validated TF-target interactions for ABF2 and ABF3 by chromatin immunoprecipitation followed by sequencing and a cell-based system to detect TF regulation genome-wide. Validated targets of ABF2 and ABF3 account for more than 50% of the nitrate-responsive transcriptome in the endodermis. Moreover, ABF2 and ABF3 are involved in nitrate-induced lateral root growth. Our approach offers an unprecedented spatiotemporal resolution of the root response to nitrate and identifies important components of cell-specific gene regulatory networks.

Capturing in-field root system dynamics with RootTracker.

Optimizing root system architecture offers a promising approach to developing stress tolerant cultivars in the face of climate change, as root systems are critical for water and nutrient uptake as well as mechanical stability. However, breeding for optimal root system architecture has been hindered by the difficulty in measuring root growth in the field. Here, we describe the RootTracker, a technology that employs impedance touch sensors to monitor in-field root growth over time. Configured in a cylindrical, window shutter-like fashion around a planted seed, 264 electrodes are individually charged multiple times over the course of an experiment. Signature changes in the measured capacitance and resistance readings indicate when a root has touched or grown close to an electrode. Using the RootTracker, we have measured root system dynamics of commercial maize (Zea mays) hybrids growing in both typical Midwest field conditions and under different irrigation regimes. We observed rapid responses of root growth to water deficits and found evidence for a "priming response" in which an early water deficit causes more and deeper roots to grow at later time periods. Genotypic variation among hybrid maize lines in their root growth in response to drought indicated a potential to breed for root systems adapted for different environments. Thus, the RootTracker is able to capture changes in root growth over time in response to environmental perturbations.

Plant immune system activation is necessary for efficient root colonization by auxin-secreting beneficial bacteria.

Although plant roots encounter a plethora of microorganisms in the surrounding soil, at the rhizosphere, plants exert selective forces on their bacterial colonizers. Unlike immune recognition of pathogenic bacteria, the mechanisms by which beneficial bacteria are selected and how they interact with the plant immune system are not well understood. To better understand this process, we studied the interaction of auxin-producing Bacillus velezensis FZB42 with Arabidopsis roots and found that activation of the plant immune system is necessary for efficient bacterial colonization and auxin secretion. A feedback loop is established in which bacterial colonization triggers an immune reaction and production of reactive oxygen species, which, in turn, stimulate auxin production by the bacteria. Auxin promotes bacterial survival and efficient root colonization, allowing the bacteria to inhibit fungal infection and promote plant health. Thus, a feedback loop between bacteria and the plant immune system promotes the fitness of both partners.

Phage-Resistant Bacteria Reveal a Role for Potassium in Root Colonization.

Bacteriophage predation is an important factor in bacterial community dynamics and evolution. Phage-bacterium interaction has mainly been studied in lab cultures, while dynamics in natural habitats, and especially in the plant root niche, are underexplored. To better understand this process, we characterized infection of the soil bacterium Bacillus subtilis NCBI 3610 by the lytic phage SPO1 during growth in LB medium and compared it to root colonization. Resistance in vitro was primarily through modification of the phage receptor. However, this type of resistance reduced the ability to colonize the root. From a line that survived phage infection while retaining the ability to colonize the root, we identified a new phage resistance mechanism involving potassium (K+) ion influx modulation and enhanced biofilm formation. Furthermore, we show that potassium serves as a stimulator of root colonization among diverse growth-promoting bacilli species, with implications for plant health. IMPORTANCE Bacteriophage predation is an important factor in bacterial community dynamics and evolution. Phage-bacterium interaction has mainly been studied in lab cultures, while dynamics in natural habitats, and especially in the plant root niche, are underexplored. To better understand this process, we characterized infection of the soil bacterium Bacillus subtilis NCBI 3610 by the lytic phage SPO1 during growth in LB medium and compared it to root colonization. Resistance in vitro was primarily through modification of the phage receptor. However, this type of resistance reduced the ability to colonize the root. From a line that survived phage infection while retaining the ability to colonize the root, we identified a new phage resistance mechanism involving potassium (K+) ion influx modulation and enhanced biofilm formation. Furthermore, we show that potassium serves as a stimulator of root colonization among diverse growth-promoting bacilli species, with implications for plant health.

A plant lipocalin promotes retinal-mediated oscillatory lateral root initiation.

In Arabidopsis, de novo organogenesis of lateral roots is patterned by an oscillatory mechanism called the root clock, which is dependent on unidentified metabolites. To determine whether retinoids regulate the root clock, we used a chemical reporter for retinaldehyde (retinal)­binding proteins. We found that retinal binding precedes the root clock and predicts sites of lateral root organogenesis. Application of retinal increased root clock oscillations and promoted lateral root formation. Expression of an Arabidopsis protein with homology to vertebrate retinoid-binding proteins, TEMPERATURE INDUCED LIPOCALIN (TIL), oscillates in the region of retinal binding to the reporter, confers retinal-binding activity in a heterologous system, and, when mutated, decreases retinal sensitivity. These results demonstrate a role for retinal and its binding partner in lateral root organogenesis.

Novel technologies for emission reduction complement conservation agriculture to achieve negative emissions from row-crop production.

Plants remove carbon dioxide from the atmosphere through photosynthesis. Because agriculture's productivity is based on this process, a combination of technologies to reduce emissions and enhance soil carbon storage can allow this sector to achieve net negative emissions while maintaining high productivity. Unfortunately, current row-crop agricultural practice generates about 5% of greenhouse gas emissions in the United States and European Union. To reduce these emissions, significant effort has been focused on changing farm management practices to maximize soil carbon. In contrast, the potential to reduce emissions has largely been neglected. Through a combination of innovations in digital agriculture, crop and microbial genetics, and electrification, we estimate that a 71% (1,744 kg CO2e/ha) reduction in greenhouse gas emissions from row crop agriculture is possible within the next 15 y. Importantly, emission reduction can lower the barrier to broad adoption by proceeding through multiple stages with meaningful improvements that gradually facilitate the transition to net negative practices. Emerging voluntary and regulatory ecosystems services markets will incentivize progress along this transition pathway and guide public and private investments toward technology development. In the difficult quest for net negative emissions, all tools, including emission reduction and soil carbon storage, must be developed to allow agriculture to maintain its critical societal function of provisioning society while, at the same time, generating environmental benefits.

Single-cell analysis of cell identity in the Arabidopsis root apical meristem: insights and opportunities.

A fundamental question in developmental biology is how the progeny of stem cells become differentiated tissues. The Arabidopsis root is a tractable model to address this question due to its simple organization and defined cell lineages. In particular, the zone of dividing cells at the root tip-the root apical meristem-presents an opportunity to map the gene regulatory networks underlying stem cell niche maintenance, tissue patterning, and cell identity acquisition. To identify molecular regulators of these processes, studies over the last 20 years employed global profiling of gene expression patterns. However, these technologies are prone to information loss due to averaging gene expression signatures over multiple cell types and/or developmental stages. Recently developed high-throughput methods to profile gene expression at single-cell resolution have been successfully applied to plants. Here, we review insights from the first published single-cell mRNA sequencing and chromatin accessibility datasets generated from Arabidopsis roots. These studies successfully reconstruct developmental trajectories, phenotype cell identity mutants at unprecedented resolution, and reveal cell type-specific responses to environmental stimuli. The experimental insight gained from Arabidopsis paves the way to profile roots from additional species.

VAP-RELATED SUPPRESSORS OF TOO MANY MOUTHS (VST) family proteins are regulators of root system architecture.

Root system architecture (RSA) is a key factor in the efficiency of nutrient capture and water uptake in plants. Understanding the genetic control of RSA will be useful in minimizing fertilizer and water usage in agricultural cropping systems. Using a hydroponic screen and a gel-based imaging system, we identified a rice (Oryza sativa) gene, VAP-RELATED SUPPRESSOR OF TOO MANY MOUTHS1 (OsVST1), which plays a key role in controlling RSA. This gene encodes a homolog of the VAP-RELATED SUPPRESSORS OF TOO MANY MOUTHS (VST) proteins in Arabidopsis (Arabidopsis thaliana), which promote signaling in stomata by mediating plasma membrane-endoplasmic reticulum contacts. OsVST1 mutants have shorter primary roots, decreased root meristem size, and a more compact RSA. We show that the Arabidopsis VST triple mutants have similar phenotypes, with reduced primary root growth and smaller root meristems. Expression of OsVST1 largely complements the short root length and reduced plant height in the Arabidopsis triple mutant, supporting conservation of function between rice and Arabidopsis VST proteins. In a field trial, mutations in OsVST1 did not adversely affect grain yield, suggesting that modulation of this gene could be used as a way to optimize RSA without an inherent yield penalty.