In seed plants, the canonical role of 1-aminocyclopropane-1-carboxylic acid (ACC) is to serve as the precursor in the biosynthesis of the phytohormone ethylene, and indeed, ACC treatment is often used as a proxy for ethylene treatment. Increasing evidence suggests that ACC can also act independently of ethylene to regulate various aspects of plant growth and development. Here, we explore the effects of ACC on Arabidopsis thaliana root growth and the mechanisms by which it acts. ACC inhibits growth of the primary root in Arabidopsis seedlings when ethylene signaling is blocked, which becomes evident after 36 h of treatment with ACC. This reduced root growth is in part the result of suppressed cell proliferation in the root meristem resulting from altered expression of a key regulator of stem cell niche activity, WOX5. ACC also promotes lateral root (LR) development, in contrast to ethylene, which inhibits LR formation. Transcriptomic analysis of roots revealed no significant changes in gene expression after 45 min or 4 h of ACC treatment, but longer treatment times revealed a large number of differentially expressed genes, including the downregulation of the expression of a small group of phylogenetically related CLE peptides. Reduced expression of these group 1 CLEs in response to ACC leads to the activation of a transcription factor, LBD18, which promotes LR development. These results suggest that ACC acts to modulate multiple aspects of Arabidopsis root growth independently of ethylene via distinct transcriptional effects in the root meristem and LR precursor cells.

Land plants thrive in soils showing vastly different properties and environmental stresses1. Root systems can adapt to contrasting soil conditions and stresses, yet how their responses are programmed at the individual cell scale remains unclear. Using single-cell RNA sequencing and spatial transcriptomic approaches, we showed major expression changes in outer root cell types when comparing the single-cell transcriptomes of rice roots grown in gel versus soil conditions. These tissue-specific transcriptional responses are related to nutrient homeostasis, cell wall integrity and defence in response to heterogeneous soil versus homogeneous gel growth conditions. We also demonstrate how the model soil stress, termed compaction, triggers expression changes in cell wall remodelling and barrier formation in outer and inner root tissues, regulated by abscisic acid released from phloem cells. Our study reveals how root tissues communicate and adapt to contrasting soil conditions at single-cell resolution.

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 roots 1 , 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 model 3 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.

Abscission is the programmed separation of plant organs. It is widespread in the plant kingdom with important functions in development and environmental response. In Arabidopsis, abscission of floral organs (sepals, petals, and stamens) is controlled by two receptor-like protein kinases HAESA (HAE) and HAESA LIKE-2 (HSL2), which orchestrate the programmed dissolution of the abscission zone connecting floral organs to the developing fruit. In this work, we use single-cell RNA sequencing to characterize the core HAE/HSL2 abscission gene expression program. We identify the MAP KINASE PHOSPHATASE-1/MKP1 gene as a negative regulator of this pathway. MKP1 acts prior to activation of HAE/HSL2 signaling to establish a signaling threshold required for the initiation of abscission. Furthermore, we use single-cell data to identify genes expressed in two subpopulations of abscission zone cells: those proximal and those distal to the plane of separation. We identify INFLORESCENCE DEFICIENT IN ABSCISSION/IDA family genes, encoding activating ligands of HAE/HSL2, as enriched in distal abscission zone cells at the base of the abscising organs. We show how this expression pattern forms a biophysical circuit breaker whereby, when the organ is shed, the source of the IDA peptides is removed, leading to cessation of HAE/HSL2 signaling. Overall, this work provides insight into the multiple control mechanisms acting on the abscission-signaling pathway.

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.

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.

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.

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.

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.

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.

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.

Amundson’s letter (1) in response to our recent article (2) suggests that there are substantial challenges to the technologies that we envisioned being promising and/or needed to make the agricultural sector net negative. Aside from the difficulty in implementation, he makes no technical criticism of the findings. At the root of any disagreement between our article and these comments is the timeline over which change is necessary. Even if all other sectors reduce emissions to zero, the food sector will exceed the global carbon budget for 1.5 °C of warming in less than 40 y (3). Society cannot afford a slow approach to deep decarbonization. Nonetheless, we do not claim that innovation or adoption will be easy, nor is this transition inevitable. Our goal was to introduce technologies (some already commercially available and adopted) and call for changes in policy and investment to broaden deployment and make the transition a reality. We do not propose that any of the transitional states should be the goal of the sector. As Amundson (1) notes, agriculture has never been in steady state. Indeed, the agricultural system must be reimagined to increase its productivity to feed a growing population while meeting sustainability targets, which represents a dramatic paradigm shift. We chose a broad brush and a key metric—greenhouse gas emissions—for optimization of agricultural systems, as it fits current policy, incentives, and emerging markets. Amundson (1) highlights another interesting metric— elemental residence times in soils (4)—that could be a target for improving sustainability of agricultural systems. We are certain that using this metric will suggest additional technical thrusts for exploration. To alter the aphorism from George Box, “all predictions are wrong, some are useful,” the purpose of our manuscript (2) was to articulate a technical vision that provides entry points for a paradigm shift. We have heard from colleagues in government, industry, nonprofit, and academia that the article is spurring discussion about policies and research programs to foster innovation and implementation. By this metric, we believe that it has been useful. Amundson (1) notes that each innovation is a mini–Manhattan Project; the Manhattan Project succeeded in its goal in roughly 3 y. Mitigation of climate change is also likened to a moonshot, an effort that took 8 y. Given the consequences of failure, we hope that the climate challenge can be addressed on a decadal time scale. As Amundson concludes, we must do much work first. By laying out a plausible set of goals and identifying the areas for innovation, we aim to inspire that work.

We describe a protocol to perform fast and non-arbitrary quality control of sin-gle-cell RNA sequencing (scRNA-seq) raw data using scKB and COPILOT. scKB is a wrapper script of kallisto and bustools for accelerated alignment and tran-script 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).

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 sequenc-ing 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 compo-nents of cell-specific gene regulatory networks.