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  1. Abstract 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.


    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.


    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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  2. 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 (maize), Sorghum bicolor (sorghum), and Setaria viridis (Setaria). We first show that single-cell and single-nucleus RNA-seq provide complementary readouts of cell identity in both 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 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 favor 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 shed light on the evolution of cells that mediate key functions in crops. 
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    Free, publicly-accessible full text available May 25, 2024
  3. 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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  4. 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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  5. Abstract

    A key remit of theNSF‐funded “Arabidopsis Research and Training for the 21stCentury” (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 usingArabidopsis thalianacan 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 secondART‐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 speciesArabidopsis thalianaand other valuable plant systems.

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