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Regenerative organisms such as plants must have specific signals that respond to damage and instruct remnant tissue to undergo repair. A recent paper identifies a long-sought candidate for the signal that links injury to regenerative programs.
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Regeneração , Regeneração/fisiologia , Transdução de Sinais , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Desenvolvimento Vegetal/fisiologiaRESUMO
The plasticity of plant cells underlies their wide capacity to regenerate, with increasing evidence in plants and animals implicating cell cycle dynamics in cellular reprogramming. To investigate the cell cycle during cellular reprogramming, we developed a comprehensive set of cell cycle phase markers in the Arabidopsis root. Using single-cell RNA-seq profiles and live imaging during regeneration, we found that a subset of cells near an ablation injury dramatically increases division rate by truncating G1. Cells in G1 undergo a transient nuclear peak of glutathione (GSH) prior to coordinated entry into S phase followed by rapid divisions and cellular reprogramming. A symplastic block of the ground tissue impairs regeneration, which is rescued by exogenous GSH. We propose a model in which GSH from the outer tissues is released upon injury licensing an exit from G1 near the wound to induce rapid cell division and reprogramming.
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The C4 photosynthetic pathway provided a major advantage to plants growing in hot, dry environments, including the ancestors of our most productive crops. Two traits were essential for the evolution of this pathway: increased vein density and the functionalization of bundle sheath cells for photosynthesis. Although GRAS transcriptional regulators, including SHORT ROOT (SHR), have been implicated in mediating leaf patterning in both C3 and C4 species, little is known about what controls the specialized features of the cells that mediate C4 metabolism and physiology. We show in the model monocot, Setaria viridis, that SHR regulates components of multiple cell identities, including chloroplast biogenesis and photosynthetic gene expression in bundle sheath cells, a central feature of C4 plants. Furthermore, we found that it also contributes to the two-cell compartmentalization of the characteristic four-carbon shuttle pathway. Disruption of SHR function clearly reduced photosynthetic capacity and seed yield in mutant plants under heat stress. Together, these results show how cell identities are remodeled by SHR to host the suite of traits characteristic of C4 regulation, which are a main engineering target in non-C4 crops to improve climate resilience.
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A "ring" master of plant development and cellular genomics.
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Genômica , Desenvolvimento Vegetal , Desenvolvimento Vegetal/genética , Genômica/históriaRESUMO
BACKGROUND: In the past few years, there has been an explosion in single-cell transcriptomics datasets, yet in vivo confirmation of these datasets is hampered in plants due to lack of robust validation methods. Likewise, modeling of plant development is hampered by paucity of spatial gene expression data. RNA fluorescence in situ hybridization (FISH) enables investigation of gene expression in the context of tissue type. Despite development of FISH methods for plants, easy and reliable whole mount FISH protocols have not yet been reported. RESULTS: We adapt a 3-day whole mount RNA-FISH method for plant species based on a combination of prior protocols that employs hybridization chain reaction (HCR), which amplifies the probe signal in an antibody-free manner. Our whole mount HCR RNA-FISH method shows expected spatial signals with low background for gene transcripts with known spatial expression patterns in Arabidopsis inflorescences and monocot roots. It allows simultaneous detection of three transcripts in 3D. We also show that HCR RNA-FISH can be combined with endogenous fluorescent protein detection and with our improved immunohistochemistry (IHC) protocol. CONCLUSIONS: The whole mount HCR RNA-FISH and IHC methods allow easy investigation of 3D spatial gene expression patterns in entire plant tissues.
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Different plant species within the grasses were parallel targets of domestication, giving rise to crops with distinct evolutionary histories and traits1. Key traits that distinguish these species are mediated by specialized cell types2. Here we compare the transcriptomes of root cells in three grass species-Zea mays, Sorghum bicolor and Setaria viridis. We show that single-cell and single-nucleus RNA sequencing provide complementary readouts of cell identity in dicots and monocots, warranting a combined analysis. Cell types were mapped across species to identify robust, orthologous marker genes. The comparative cellular analysis shows that the transcriptomes of some cell types diverged more rapidly than those of others-driven, in part, by recruitment of gene modules from other cell types. The data also show that a recent whole-genome duplication provides a rich source of new, highly localized gene expression domains that favour fast-evolving cell types. Together, the cell-by-cell comparative analysis shows how fine-scale cellular profiling can extract conserved modules from a pan transcriptome and provide insight on the evolution of cells that mediate key functions in crops.
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Produtos Agrícolas , Setaria (Planta) , Sorghum , Transcriptoma , Zea mays , Sequência de Bases , Regulação da Expressão Gênica de Plantas/genética , Sorghum/citologia , Sorghum/genética , Transcriptoma/genética , Zea mays/citologia , Zea mays/genética , Setaria (Planta)/citologia , Setaria (Planta)/genética , Raízes de Plantas/citologia , Análise da Expressão Gênica de Célula Única , Análise de Sequência de RNA , Produtos Agrícolas/citologia , Produtos Agrícolas/genética , Evolução MolecularRESUMO
The Plant Cell Atlas (PCA) community hosted a virtual symposium on December 9 and 10, 2021 on single cell and spatial omics technologies. The conference gathered almost 500 academic, industry, and government leaders to identify the needs and directions of the PCA community and to explore how establishing a data synthesis center would address these needs and accelerate progress. This report details the presentations and discussions focused on the possibility of a data synthesis center for a PCA and the expected impacts of such a center on advancing science and technology globally. Community discussions focused on topics such as data analysis tools and annotation standards; computational expertise and cyber-infrastructure; modes of community organization and engagement; methods for ensuring a broad reach in the PCA community; recruitment, training, and nurturing of new talent; and the overall impact of the PCA initiative. These targeted discussions facilitated dialogue among the participants to gauge whether PCA might be a vehicle for formulating a data synthesis center. The conversations also explored how online tools can be leveraged to help broaden the reach of the PCA (i.e., online contests, virtual networking, and social media stakeholder engagement) and decrease costs of conducting research (e.g., virtual REU opportunities). Major recommendations for the future of the PCA included establishing standards, creating dashboards for easy and intuitive access to data, and engaging with a broad community of stakeholders. The discussions also identified the following as being essential to the PCA's success: identifying homologous cell-type markers and their biocuration, publishing datasets and computational pipelines, utilizing online tools for communication (such as Slack), and user-friendly data visualization and data sharing. In conclusion, the development of a data synthesis center will help the PCA community achieve these goals by providing a centralized repository for existing and new data, a platform for sharing tools, and new analytical approaches through collaborative, multidisciplinary efforts. A data synthesis center will help the PCA reach milestones, such as community-supported data evaluation metrics, accelerating plant research necessary for human and environmental health.
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Wounding is a trigger for both regeneration and defense in plants, but it is not clear whether the two responses are linked by common activation or regulated as trade-offs. Although plant glutamate-receptor-like proteins (GLRs) are known to mediate defense responses, here, we implicate GLRs in regeneration through dynamic changes in chromatin and transcription in reprogramming cells near wound sites. We show that genetic and pharmacological inhibition of GLR activity increases regeneration efficiency in multiple organ repair systems in Arabidopsis and maize. We show that the GLRs work through salicylic acid (SA) signaling in their effects on regeneration, and mutants in the SA receptor NPR1 are hyper-regenerative and partially resistant to GLR perturbation. These findings reveal a conserved mechanism that regulates a trade-off between defense and regeneration, and they also offer a strategy to improve regeneration in agriculture and conservation.
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Regulação da Expressão Gênica de Plantas/genética , Receptores de Glutamato/metabolismo , Regeneração/fisiologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cromatina/metabolismo , Plantas/metabolismo , Receptores de Glutamato/genética , Transdução de Sinais/fisiologiaRESUMO
Most plant roots have multiple cortex layers that make up the bulk of the organ and play key roles in physiology, such as flood tolerance and symbiosis. However, little is known about the formation of cortical layers outside of the highly reduced anatomy of Arabidopsis. Here, we used single-cell RNA sequencing to rapidly generate a cell-resolution map of the maize root, revealing an alternative configuration of the tissue formative transcription factor SHORT-ROOT (SHR) adjacent to an expanded cortex. We show that maize SHR protein is hypermobile, moving at least eight cell layers into the cortex. Higher-order SHR mutants in both maize and Setaria have reduced numbers of cortical layers, showing that the SHR pathway controls expansion of cortical tissue to elaborate anatomical complexity.
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Proteínas de Plantas/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/metabolismo , Setaria (Planta)/metabolismo , Fatores de Transcrição/metabolismo , Zea mays/metabolismo , Citometria de Fluxo , Genoma de Planta , Proteínas de Plantas/genética , Raízes de Plantas/genética , RNA-Seq , Setaria (Planta)/citologia , Setaria (Planta)/genética , Análise de Célula Única , Fatores de Transcrição/genética , Transcrição Gênica , Zea mays/citologia , Zea mays/genéticaRESUMO
In the plant meristem, tissue-wide maturation gradients are coordinated with specialized cell networks to establish various developmental phases required for indeterminate growth. Here, we used single-cell transcriptomics to reconstruct the protophloem developmental trajectory from the birth of cell progenitors to terminal differentiation in the Arabidopsis thaliana root. PHLOEM EARLY DNA-BINDING-WITH-ONE-FINGER (PEAR) transcription factors mediate lineage bifurcation by activating guanosine triphosphatase signaling and prime a transcriptional differentiation program. This program is initially repressed by a meristem-wide gradient of PLETHORA transcription factors. Only the dissipation of PLETHORA gradient permits activation of the differentiation program that involves mutual inhibition of early versus late meristem regulators. Thus, for phloem development, broad maturation gradients interface with cell-type-specific transcriptional regulators to stage cellular differentiation.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Floema/citologia , Floema/crescimento & desenvolvimento , Raízes de Plantas/citologia , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Diferenciação Celular , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Meristema/citologia , Floema/genética , Floema/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , RNA-Seq , Transdução de Sinais , Análise de Célula Única , Fatores de Transcrição/genética , TranscriptomaRESUMO
With growing populations and pressing environmental problems, future economies will be increasingly plant-based. Now is the time to reimagine plant science as a critical component of fundamental science, agriculture, environmental stewardship, energy, technology and healthcare. This effort requires a conceptual and technological framework to identify and map all cell types, and to comprehensively annotate the localization and organization of molecules at cellular and tissue levels. This framework, called the Plant Cell Atlas (PCA), will be critical for understanding and engineering plant development, physiology and environmental responses. A workshop was convened to discuss the purpose and utility of such an initiative, resulting in a roadmap that acknowledges the current knowledge gaps and technical challenges, and underscores how the PCA initiative can help to overcome them.
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Células Vegetais , Agricultura , Chlamydomonas reinhardtii , Cloroplastos , Biologia Computacional , Processamento de Imagem Assistida por Computador , Células Vegetais/fisiologia , Desenvolvimento Vegetal , Plantas/classificação , Plantas/genética , Zea maysRESUMO
Postembryonic organogenesis is critical for plant development. Underground, lateral roots (LRs) form the bulk of mature root systems, yet the ontogeny of the LR primordium (LRP) is not clear. In this study, we performed the single-cell RNA sequencing through the first four stages of LR formation in Arabidopsis. Our analysis led to a model in which a single group of precursor cells, with a cell identity different from their pericycle origins, rapidly reprograms and splits into a mixed ground tissue/stem cell niche fate and a vascular precursor fate. The ground tissue and stem cell niche fates soon separate and a subset of more specialized vascular cells form sucrose transporting phloem cells that appear to connect to the primary root. We did not detect cells resembling epidermis or root cap, suggesting that outer tissues may form later, preceding LR emergence. At this stage, some remaining initial precursor cells form the primordium flanks, while the rest create a reservoir of pluripotent cells that are able to replace the LR if damaged. Laser ablation of the central and lateral LRP regions showed that remaining cells restart the sequence of tissue initiation to form a LR. Collectively, our study reveals an ontological hierarchy for LR formation with an early and sequential split of main root tissues and stem cells.
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Arabidopsis/crescimento & desenvolvimento , Organogênese Vegetal/genética , Desenvolvimento Vegetal/genética , Raízes de Plantas/crescimento & desenvolvimento , Arabidopsis/citologia , Raízes de Plantas/citologia , Análise de Sequência de RNA , Células-Tronco/citologiaRESUMO
Single-cell approaches are quickly changing our view on biological systems by increasing the spatiotemporal resolution of our analyses to the level of the individual cell. The field of plant biology has fully embraced single-cell transcriptomics and is rapidly expanding the portfolio of available technologies and applications. In this review, we give an overview of the main advances in plant single-cell transcriptomics over the past few years and provide the reader with an accessible guideline covering all steps, from sample preparation to data analysis. We end by offering a glimpse of how these technologies will shape and accelerate plant-specific research in the near future.
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Análise de Célula Única , Transcriptoma , Biologia Computacional , Plantas/genética , Análise de Sequência de RNARESUMO
The societal challenges posed by a growing human population and climate change necessitate technical advances in plant science. Plant research makes vital contributions to society by advancing technologies that improve agricultural food production, biological energy capture and conversion, and human health. However, the plant biology community lacks a comprehensive understanding of molecular machinery, including their locations within cells, distributions and variations among different cell types, and real-time dynamics. Fortunately, rapid advances in molecular methods, imaging, proteomics, and metabolomics made in the last decade afford unprecedented opportunities to develop a molecular-level map of plant cells with high temporal and spatial resolution. The Plant Cell Atlas (PCA) initiative aims to generate a resource that will provide fresh insight into poorly understood aspects of plant cell structure and organization and enable the discovery of new cellular compartments and features. The PCA will be a community resource (www.plantcellatlas.org/) that describes the state of various plant cell types and integrates high-resolution spatio-temporal information of nucleic acids, proteins, and metabolites within plant cells. This first PCA initiative workshop convened scientists passionate about developing a comprehensive PCA to brainstorm about the state of the field, recent advances, the development of tools, and the future directions of this initiative. The workshop featured invited talks to share initial data, along with broader ideas for the PCA. Additionally, breakout sessions were organized around topics including the conceptual goals of the PCA, technical challenges, and community wants and needs. These activities connected scientists with diverse expertise and sparked important discussions about how to leverage and extend leading-edge technologies and develop new techniques. A major outcome of the workshop was that the community wishes to redefine concepts of plant cell types and tissues quantitatively. A long-term goal is to delineate all molecules within the cell at high spatio-temporal resolution, obtain information about interacting molecular networks, and identify the contribution of these networks to development of the organism as a whole. As a first step, we wish to create comprehensive cellular and subcellular biomolecular maps of transcripts, proteins, and metabolites, track the dynamic interactions of these molecules intra- and intercellularly, discern complete states and transitions of specialized cell types, and integrate these disparate data points to generate testable models of cellular function. Ultimately, the PCA initiative will have a substantial positive impact by empowering a broad, diverse group of scientists to forge exciting paths in the field of plant science, facilitating connections with interested stakeholders beyond the scientific community, and enabling new agricultural technologies for a sustainable future.
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MaizeCODE is a project aimed at identifying and analyzing functional elements in the maize genome. In its initial phase, MaizeCODE assayed up to five tissues from four maize strains (B73, NC350, W22, TIL11) by RNA-Seq, Chip-Seq, RAMPAGE, and small RNA sequencing. To facilitate reproducible science and provide both human and machine access to the MaizeCODE data, we enhanced SciApps, a cloud-based portal, for analysis and distribution of both raw data and analysis results. Based on the SciApps workflow platform, we generated new components to support the complete cycle of MaizeCODE data management. These include publicly accessible scientific workflows for the reproducible and shareable analysis of various functional data, a RESTful API for batch processing and distribution of data and metadata, a searchable data page that lists each MaizeCODE experiment as a reproducible workflow, and integrated JBrowse genome browser tracks linked with workflows and metadata. The SciApps portal is a flexible platform that allows the integration of new analysis tools, workflows, and genomic data from multiple projects. Through metadata and a ready-to-compute cloud-based platform, the portal experience improves access to the MaizeCODE data and facilitates its analysis.
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The root meristem-one of the plant's centers of continuous growth-is a conveyer belt in which cells of different identities are pushed through gradients along the root's longitudinal axis. An auxin gradient has long been implicated in controlling the progression of cell states in the root meristem. Recent work has shown that a PLETHORA (PLT) protein transcription factor gradient, which is under a delayed auxin response, has a dose-dependent effect on the differentiation state of cells. The direct effect of auxin concentration on differential transcriptional outputs remains unclear. Genomic and other analyses of regulatory sequences show that auxin responses are likely controlled by combinatorial inputs from transcription factors outside the core auxin signaling pathway. The passage through the meristem exposes cells to varying positional signals that could help them interpret auxin inputs independent of gradient effects. One open question is whether cells process information from the changes in the gradient over time as they move through the auxin gradient.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Ácidos Indolacéticos/farmacologia , Meristema/fisiologia , Raízes de Plantas/fisiologia , Arabidopsis/citologia , Arabidopsis/efeitos dos fármacos , Proteínas de Arabidopsis/genética , Diferenciação Celular , Meristema/citologia , Meristema/efeitos dos fármacos , Reguladores de Crescimento de Plantas/farmacologia , Raízes de Plantas/citologia , Raízes de Plantas/efeitos dos fármacosRESUMO
A key remit of the NSF-funded "Arabidopsis Research and Training for the 21st Century" (ART-21) Research Coordination Network has been to convene a series of workshops with community members to explore issues concerning research and training in plant biology, including the role that research using Arabidopsis thaliana can play in addressing those issues. A first workshop focused on training needs for bioinformatic and computational approaches in plant biology was held in 2016, and recommendations from that workshop have been published (Friesner et al., Plant Physiology, 175, 2017, 1499). In this white paper, we provide a summary of the discussions and insights arising from the second ART-21 workshop. The second workshop focused on experimental aspects of omics data acquisition and analysis and involved a broad spectrum of participants from academics and industry, ranging from graduate students through post-doctorates, early career and established investigators. Our hope is that this article will inspire beginning and established scientists, corporations, and funding agencies to pursue directions in research and training identified by this workshop, capitalizing on the reference species Arabidopsis thaliana and other valuable plant systems.
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BACKGROUND: Characterizing the behaviors of dynamic systems requires capturing them with high temporal and spatial resolution. Owing to its transparency and genetic tractability, the Arabidopsis thaliana root lends itself well to live imaging when combined with cell and tissue-specific fluorescent reporters. We developed a novel 4D imaging method that utilizes simple confocal microscopy and readily available components to track cell divisions in the root stem cell niche and surrounding region for up to 1 week. RESULTS: Using this method, we performed a direct measurement of cell division intervals within and around the root stem cell niche. The results reveal a short, steep gradient of cell division rates in proximal stem cells, with progressively more rapid cell division rates from quiescent center (QC), to cells in direct contact with the QC (initials), to their immediate daughters, after which division rates appear to become more homogeneous. CONCLUSIONS: These results provide a baseline to study how perturbations in signaling could affect cell division patterns in the root meristem. This new setup further allows us to finely analyze meristematic cell division rates that lead to patterning.
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Enormous societal challenges, such as feeding and providing energy for a growing population in a dramatically changing climate, necessitate technological advances in plant science. Plant cells are fundamental organizational units that mediate the production, transport, and storage of our primary food sources, and they sequester a significant proportion of the world's carbon. New technologies allow comprehensive descriptions of cells that could accelerate research across fields of plant science. Complementary to the efforts towards understanding the cellular diversity in human brain and immune systems, a Plant Cell Atlas (PCA) that maps molecular machineries to cellular and subcellular domains, follows their dynamic movements, and describes their interactions would accelerate discovery in plant science and help to solve imminent societal problems.