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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., Competing Interests: Declaration of interests The author declares no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of Interests The authors declare no competing interests.

A "ring" master of plant development and cellular genomics.

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., (© 2023. The Author(s).)

Different plant species within the grasses were parallel targets of domestication, giving rise to crops with distinct evolutionary histories and traits 1 . Key traits that distinguish these species are mediated by specialized cell types 2 . 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., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

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., Competing Interests: Declaration of interests A patent (application #63120640) has been filed related to this work on compositions and methods to enhance plant regeneration., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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.

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.

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., Competing Interests: SJ, AB, BC, NF, AF, SH, PK, ML, NP, SR, MS, PA, AA, CA, SB, JB, PD, LD, ME, SG, FG, KK, DK, SK, AM, NN, TO, MO, GP, EQ, RS, RU, JW, KV, RW, DE, KB, SR No competing interests declared, (© 2021, Plant Cell Atlas Consortium et al.)

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., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

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., (Copyright © 2020 Wang, Lu, delaBastide, Van Buren, Wang, Ghiban, Regulski, Drenkow, Xu, Ortiz-Ramirez, Marco, Goodwin, Dobin, Birnbaum, Jackson, Martienssen, McCombie, Micklos, Schatz, Ware and Gingeras.)

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., (© 2020 Elsevier Inc. All rights reserved.)

A key remit of the NSF-funded "Arabidopsis Research and Training for the 21 st 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.

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., Competing Interests: The authors declare that they have no competing interests.

The genus Selaginella resides in an early branch of the land plant lineage that possesses a vasculature and roots. The majority of the Selaginella root system is shoot borne and emerges through a distinctive structure known as the rhizophore, the organ identity of which has been a long-debated question. The rhizophore of Selaginella moellendorffii - a model for the lycophytes - shows plasticity to develop into a root or shoot up until 8 d after angle meristem emergence, after which it is committed to root fate. We subsequently use morphology and plasticity to define the stage of rhizophore identity. Transcriptomic analysis of the rhizophore during its plastic stage reveals that, despite some resemblance to the root meristem, rhizophore gene expression patterns are largely distinct from both shoot and root meristems. Based on this transcriptomic analysis and on historical anatomical work, we conclude that the rhizophore is a distinct organ with unique features., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

The growing scale and declining cost of single-cell RNA-sequencing (RNA-seq) now permit a repetition of cell sampling that increases the power to detect rare cell states, reconstruct developmental trajectories, and measure phenotype in new terms such as cellular variance. The characterization of anatomy and developmental dynamics has not had an equivalent breakthrough since groundbreaking advances in live fluorescent microscopy. The new resolution obtained by single-cell RNA-seq is a boon to genetics because the novel description of phenotype offers the opportunity to refine gene function and dissect pleiotropy. In addition, the recent pairing of high-throughput genetic perturbation with single-cell RNA-seq has made practical a scale of genetic screening not previously possible.

Maize is one the most widely cultivated crops worldwide and an important model system for the study of genetics and cytogenetics. Although the availability of a genome sequence has enabled new quantitative genomic studies, developing methods to isolate specific types of cells will enable useful approaches for transcriptomic analysis in the crop plant. Fluorescence-activated cell sorting (FACS) is a powerful technique for cell isolation and the study of transcriptional profiles from specific cell populations. The use of FACS on plant cells requires the generation of protoplasts by tissue digestion and cell wall removal. Although some protocols are available, they mainly focus on dicot species and obtaining sufficient protoplasts from inner tissue layers has been challenging in both monocots and dicots. Here, we report a new protocol that dramatically increases protoplast yield from maize for subsequent cell isolation by FACS. This protocol is efficient in generating protoplasts from root and shoot inner layers and can also be applied successfully to Arabidopsis thaliana. © 2018 by John Wiley & Sons, Inc., (© 2018 John Wiley & Sons, Inc.)

Plants have a high intrinsic capacity to regenerate from adult tissues, with the ability to reprogram adult cell fates. In contrast, epigenetic mechanisms have the potential to stabilize cell identity and maintain tissue organization. The question is whether epigenetic memory creates a barrier to reprogramming that needs to be erased or circumvented in plant regeneration. Early evidence suggests that, while chromatin dynamics impact gene expression in the meristem, a lasting constraint on cell fate is not established until late stages of plant cell differentiation. It is not yet clear whether the plasticity of plant cells arises from the ability of cells to erase identity memory or to deploy cells that may exhibit cellular specialization but still lack an epigenetic restriction on cell fate alteration.

Plants often make the same organ in different development contexts. Roots are a quintessential example, with embryonic, primary, lateral, adventitious, and regenerative roots common to many plants. The cellular origins and early morphologies of different roots can vary greatly, but the adult structures can be remarkably similar. Recent studies have highlighted the diversity of mechanisms that can initiate roots while late patterning mechanisms are frequently shared. In the middle stages when patterning emerges, evidence shows that antagonistic auxin-cytokinin interactions regulate tissue patterns in root embryogenesis, vascular organization, and regeneration but it is not yet clear if a common ontogeny for the root body plan exists., (Copyright © 2016. Published by Elsevier Ltd.)

Plants form the basis of the food webs that sustain animal life. Exogenous factors, such as nutrients and sunlight, and endogenous factors, such as hormones, cooperate to control both the growth and the development of plants. We assessed how Arabidopsis thaliana integrated nutrient and hormone signaling pathways to control root growth and development by investigating the effects of combinatorial treatment with the nutrients nitrate and ammonium; the hormones auxin, cytokinin, and abscisic acid; and all binary combinations of these factors. We monitored and integrated short-term genome-wide changes in gene expression over hours and long-term effects on root development and architecture over several days. Our analysis revealed trends in nutrient and hormonal signal crosstalk and feedback, including responses that exhibited logic gate behavior, which means that they were triggered only when specific combinations of signals were present. From the data, we developed a multivariate network model comprising the signaling molecules, the early gene expression modulation, and the subsequent changes in root phenotypes. This multivariate network model pinpoints several genes that play key roles in the control of root development and may help understand how eukaryotes manage multifactorial signaling inputs., (Copyright © 2016, American Association for the Advancement of Science.)

The root meristem has a centrally located group of mitotically quiescent cells, to which current models assign a stem cell organizer function. However, evidence is emerging for decentralized control of stem cell activity, whereby self-renewing behavior emerges from the lack of cell displacement at the border of opposing differentiation gradients. We term this a "stagnation" model due to its reliance on passive mechanics. The position of stem cells is established by two opposing axes that reciprocally control each other's differentiation. Such broad tissue organization programs would allow plants, like some animal systems, to rapidly reconstitute stem cells from non-stem-cell tissues., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Plant roots can regenerate after excision of their tip, including the stem cell niche. To determine which developmental program mediates such repair, we applied a combination of lineage tracing, single-cell RNA sequencing, and marker analysis to test different models of tissue reassembly. We show that multiple cell types can reconstitute stem cells, demonstrating the latent potential of untreated plant cells. The transcriptome of regenerating cells prior to stem cell activation resembles that of an embryonic root progenitor. Regeneration defects are more severe in embryonic than in adult root mutants. Furthermore, the signaling domains of the hormones auxin and cytokinin mirror their embryonic dynamics and manipulation of both hormones alters the position of new tissues and stem cell niche markers. Our findings suggest that plant root regeneration follows, on a larger scale, the developmental stages of embryonic patterning and is guided by spatial information provided by complementary hormone domains., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Single-cell transcriptomics has been employed in a growing number of animal studies, but the technique has yet to be widely used in plants. Nonetheless, early studies indicate that single-cell RNA-seq protocols developed for animal cells produce informative datasets in plants. We argue that single-cell transcriptomics has the potential to provide a new perspective on plant problems, such as the nature of the stem cells or initials, the plasticity of plant cells, and the extent of localized cellular responses to environmental inputs. Single-cell experimental outputs require different analytical approaches compared with pooled cell profiles and new tools tailored to single-cell assays are being developed. Here, we highlight promising new single-cell profiling approaches, their limitations as applied to plants, and their potential to address fundamental questions in plant biology.

Polyhedral-shaped plant cells have faces, corners, and edges that can have different material properties. As Kirchhelle et al. (2016) now show, RAB-A5c reveals a trafficking compartment that localizes to the edges where two cell walls meet, with a potential role in mediating local wall stiffness., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Under saline conditions, higher plants restrict the accumulation of chloride ions (Cl(-)) in the shoot by regulating their transfer from the root symplast into the xylem-associated apoplast. To identify molecular mechanisms underpinning this phenomenon, we undertook a transcriptional screen of salt stressed Arabidopsis (Arabidopsis thaliana) roots. Microarrays, quantitative RT-PCR, and promoter-GUS fusions identified a candidate gene involved in Cl(-) xylem loading from the Nitrate transporter 1/Peptide Transporter family (NPF2.4). This gene was highly expressed in the root stele compared to the cortex, and its expression decreased after exposure to NaCl or abscisic acid. NPF2.4 fused to fluorescent proteins, expressed either transiently or stably, was targeted to the plasma membrane. Electrophysiological analysis of NPF2.4 in Xenopus laevis oocytes suggested that NPF2.4 catalyzed passive Cl(-) efflux out of cells and was much less permeable to NO3(-). Shoot Cl(-) accumulation was decreased following NPF2.4 artificial microRNA knockdown, whereas it was increased by overexpression of NPF2.4. Taken together, these results suggest that NPF2.4 is involved in long-distance transport of Cl(-) in plants, playing a role in the loading and the regulation of Cl(-) loading into the xylem of Arabidopsis roots during salinity stress., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Developmental transitions can be described in terms of morphology and the roles of individual genes, but also in terms of global transcriptional and epigenetic changes. Temporal dissections of transcriptome changes, however, are rare for intact, developing tissues. We used RNA sequencing and microarray platforms to quantify gene expression from labeled cells isolated by fluorescence-activated cell sorting to generate cell-type-specific transcriptomes during development of an adult stem-cell lineage in the Arabidopsis leaf. We show that regulatory modules in this early lineage link cell types that had previously been considered to be under separate control and provide evidence for recruitment of individual members of gene families for different developmental decisions. Because stomata are physiologically important and because stomatal lineage cells exhibit exemplary division, cell fate, and cell signaling behaviors, this dataset serves as a valuable resource for further investigations of fundamental developmental processes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The definition of cell identity is a central problem in biology. While single-cell RNA-seq provides a wealth of information regarding cell states, better methods are needed to map their identity, especially during developmental transitions. Here, we use repositories of cell type-specific transcriptomes to quantify identities from single-cell RNA-seq profiles, accurately classifying cells from Arabidopsis root tips and human glioblastoma tumors. We apply our approach to single cells captured from regenerating roots following tip excision. Our technique exposes a previously uncharacterized transient collapse of identity distant from the injury site, demonstrating the biological relevance of a quantitative cell identity index.

Many cell populations have been labeled using stable reporters containing a fluorescent protein. These same marker lines can be used to capture specific cell types or marked cell populations after a brief enzymatic digestion to dissociate tissues. Here, we describe the use of Fluorescence Activated Cell Sorting (FACS) to isolate a rare population of cells marked with Green Fluorescent Protein (GFP). The FACS is a powerful selection tool that can be used to isolate high-quality RNA while selecting for fluorescence, cell size, and other properties.

The dynamic nature of gene regulatory networks allows cells to rapidly respond to environmental change. However, the underlying temporal connections are missed, even in kinetic studies, as transcription factor (TF) binding within at least one time point is required to identify primary targets. The TF-regulated but unbound genes are dismissed as secondary targets. Instead, we report that these genes comprise transient TF-target interactions most relevant to rapid signal transduction. We temporally perturbed a master TF (Basic Leucine Zipper 1, bZIP1) and the nitrogen (N) signal it transduces and integrated TF regulation and binding data from the same cell samples. Our enabling approach could identify primary TF targets based solely on gene regulation, in the absence of TF binding. We uncovered three classes of primary TF targets: (i) poised (TF-bound but not TF-regulated), (ii) stable (TF-bound and TF-regulated), and (iii) transient (TF-regulated but not TF-bound), the largest class. Unexpectedly, the transient bZIP1 targets are uniquely relevant to rapid N signaling in planta, enriched in dynamic N-responsive genes, and regulated by TF and N signal interactions. These transient targets include early N responders nitrate transporter 2.1 and NIN-like protein 3, bound by bZIP1 at 1-5 min, but not at later time points following TF perturbation. Moreover, promoters of these transient targets are uniquely enriched with cis-regulatory motifs coinherited with bZIP1 binding sites, suggesting a recruitment role for bZIP1. This transient mode of TF action supports a classic, but forgotten, "hit-and-run" transcription model, which enables a "catalyst TF" to activate a large set of targets within minutes of signal perturbation.

We present a lab that enables students to test the role of genes involved in the regulation of lateral roots growth in the model plant Arabidopsis thaliana. Here, students design an experiment that follows the effects of the hormone auxin on the stimulation of genes involved in the formation of lateral root initials. These genes, known as lateral organ boundary domain containing protein (LBD) genes, are upregulated in the presence of auxin as part of a multistep molecular and biochemically controlled pathway. Depending on which LBD gene is tested, and the stage of root development, expression patterns are localized in a discrete and punctate fashion at the site of lateral root initials (LBD33), or reveal a broader localization pattern (LBD16). Students view expression using the reporter gene GUS (beta-glucuronidase). Before GUS staining, students view root growth in a "pseudo-aseptic" agar-based environment that allows complete visualization of whole root development to determine the proper stage to test molecular expression., (Copyright © 2014 by The International Union of Biochemistry and Molecular Biology.)

Phenotypic plasticity is presumed to be involved in adaptive change toward species diversification. We thus examined how candidate genes underlying natural variation across populations might also mediate plasticity within an individual. Our implementation of an integrative "plasticity space" approach revealed that the root plasticity of a single Arabidopsis accession exposed to distinct environments broadly recapitulates the natural variation "space." Genome-wide association mapping identified the known gene PHOSPHATE 1 (PHO1) and other genes such as Root System Architecture 1 (RSA1) associated with differences in root allometry, a highly plastic trait capturing the distribution of lateral roots along the primary axis. The response of mutants in the Columbia-0 background suggests their involvement in signaling key modulators of root development including auxin, abscisic acid, and nitrate. Moreover, genotype-by-environment interactions for the PHO1 and RSA1 genes in Columbia-0 phenocopy the root allometry of other natural variants. This finding supports a role for plasticity responses in phenotypic evolution in natural environments.

The architecture of plant roots affects essential functions including nutrient and water uptake, soil anchorage, and symbiotic interactions. Root architecture comprises many features that arise from the growth of the primary and lateral roots. These root features are dictated by the genetic background but are also highly responsive to the environment. Thus, root system architecture (RSA) represents an important and complex trait that is highly variable, affected by genotype × environment interactions, and relevant to survival/performance. Quantification of RSA in Arabidopsis (Arabidopsis thaliana) using plate-based tissue culture is a very common and relatively rapid assay, but quantifying RSA represents an experimental bottleneck when it comes to medium- or high-throughput approaches used in mutant or genotype screens. Here, we present RootScape, a landmark-based allometric method for rapid phenotyping of RSA using Arabidopsis as a case study. Using the software AAMToolbox, we created a 20-point landmark model that captures RSA as one integrated trait and used this model to quantify changes in the RSA of Arabidopsis (Columbia) wild-type plants grown under different hormone treatments. Principal component analysis was used to compare RootScape with conventional methods designed to measure root architecture. This analysis showed that RootScape efficiently captured nearly all the variation in root architecture detected by measuring individual root traits and is 5 to 10 times faster than conventional scoring. We validated RootScape by quantifying the plasticity of RSA in several mutant lines affected in hormone signaling. The RootScape analysis recapitulated previous results that described complex phenotypes in the mutants and identified novel gene × environment interactions.

Plant shoots display indeterminate growth, while their evolutionary decedents, the leaves, are determinate. Determinate leaf growth is conditioned by the CIN-TCP transcription factors, which promote leaf maturation and are negatively regulated by miR319 in leaf primordia. Here we show that CIN-TCPs reduce leaf sensitivity to cytokinin (CK), a phytohormone implicated in inhibition of differentiation in the shoot. We identify the SWI/SNF chromatin remodeling ATPase BRAHMA (BRM) as a genetic mediator of CIN-TCP activities and CK responses. An interactome screen further revealed that SWI/SNF complex components including BRM preferentially interacted with basic-helix-loop-helix (bHLH) transcription factors and the bHLH-related CIN-TCPs. Indeed, TCP4 and BRM interacted in planta. Both TCP4 and BRM bound the promoter of an inhibitor of CK responses, ARR16, and induced its expression. Reconstituting ARR16 levels in leaves with reduced CIN-TCP activity restored normal growth. Thus, CIN-TCP and BRM together promote determinate leaf growth by stage-specific modification of CK responses., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Plant development is remarkably plastic but how precisely can the plant customize its form to specific environments? When the plant adjusts its development to different environments, related traits can change in a coordinated fashion, such that two traits co-vary across many genotypes. Alternatively, traits can vary independently, such that a change in one trait has little predictive value for the change in a second trait. To characterize such "tunability" in developmental plasticity, we carried out a detailed phenotypic characterization of complex root traits among 96 accessions of the model Arabidopsis thaliana in two nitrogen environments. The results revealed a surprising level of independence in the control of traits to environment - a highly tunable form of plasticity. We mapped genetic architecture of plasticity using genome-wide association studies and further used gene expression analysis to narrow down gene candidates in mapped regions. Mutants in genes implicated by association and expression analysis showed precise defects in the predicted traits in the predicted environment, corroborating the independent control of plasticity traits. The overall results suggest that there is a pool of genetic variability in plants that controls traits in specific environments, with opportunity to tune crop plants to a given environment., Competing Interests: The authors have declared that no competing interests exist.

In plants, changes in local auxin concentrations can trigger a range of developmental processes as distinct tissues respond differently to the same auxin stimulus. However, little is known about how auxin is interpreted by individual cell types. We performed a transcriptomic analysis of responses to auxin within four distinct tissues of the Arabidopsis thaliana root and demonstrate that different cell types show competence for discrete responses. The majority of auxin-responsive genes displayed a spatial bias in their induction or repression. The novel data set was used to examine how auxin influences tissue-specific transcriptional regulation of cell-identity markers. Additionally, the data were used in combination with spatial expression maps of the root to plot a transcriptomic auxin-response gradient across the apical and basal meristem. The readout revealed a strong correlation for thousands of genes between the relative response to auxin and expression along the longitudinal axis of the root. This data set and comparative analysis provide a transcriptome-level spatial breakdown of the response to auxin within an organ where this hormone mediates many aspects of development.

For the nematode Caenorhabditis elegans, automated selection of animals of specific genotypes from a mixed pool has become essential for genetic interaction or chemical screens. To date, such selection has been accomplished using specialized instruments. However, access to such dedicated equipment is not common. Here we describe live animal fluorescence-activated cell sorting (laFACS), a protocol for automatic selection of live first larval stage (L1) animals using a standard FACS system. We show that FACS can be used for the precise identification of GFP-expressing and non-GFP-expressing subpopulations and can accomplish high-speed sorting of live animals. We have routinely collected 100,000 or more homozygotes from a mixed starting population within 2 h, and with greater than 99% purity. The sorted animals continue to develop normally, making this protocol ideally suited for the isolation of terminal mutants for use in genetic interaction or chemical genetic screens.

Fluorescence-activated cell sorting (FACS) provides a rapid means of isolating large numbers of fluorescently tagged cells from a heterogeneous mixture of cells. Collections of transgenic plants with cell type-specific expression of fluorescent marker genes such as green fluorescent protein (GFP) are ideally suited for FACS-assisted studies of individual cell types. Here we describe the use of Arabidopsis and rice enhancer trap lines with tissue-specific GFP expression patterns in the root to isolate specific cell types of root tissues using FACS. Additionally, protocols are provided to impose a ramped salinity stress for 48 h prior to cell sorting.

As sessile organisms, root plasticity enables plants to forage for and acquire nutrients in a fluctuating underground environment. Here, we use genetic and genomic approaches in a "split-root" framework--in which physically isolated root systems of the same plant are challenged with different nitrogen (N) environments--to investigate how systemic signaling affects genome-wide reprogramming and root development. The integration of transcriptome and root phenotypes enables us to identify distinct mechanisms underlying "N economy" (i.e., N supply and demand) of plants as a system. Under nitrate-limited conditions, plant roots adopt an "active-foraging strategy", characterized by lateral root outgrowth and a shared pattern of transcriptome reprogramming, in response to either local or distal nitrate deprivation. By contrast, in nitrate-replete conditions, plant roots adopt a "dormant strategy", characterized by a repression of lateral root outgrowth and a shared pattern of transcriptome reprogramming, in response to either local or distal nitrate supply. Sentinel genes responding to systemic N signaling identified by genome-wide comparisons of heterogeneous vs. homogeneous split-root N treatments were used to probe systemic N responses in Arabidopsis mutants impaired in nitrate reduction and hormone synthesis and also in decapitated plants. This combined analysis identified genetically distinct systemic signaling underlying plant N economy: (i) N supply, corresponding to a long-distance systemic signaling triggered by nitrate sensing; and (ii) N demand, experimental support for the transitive closure of a previously inferred nitrate-cytokinin shoot-root relay system that reports the nitrate demand of the whole plant, promoting a compensatory root growth in nitrate-rich patches of heterogeneous soil.

Global gene expression measurements are increasingly obtained as a function of cell type, spatial position within a tissue and other biologically meaningful coordinates. Such data should enable quantitative analysis of the cell-type specificity of gene expression, but such analyses can often be confounded by the presence of noise. We introduce a specificity measure Spec that quantifies the information in a gene's complete expression profile regarding any given cell type, and an uncertainty measure dSpec, which measures the effect of noise on specificity. Using global gene expression data from the mouse brain, plant root and human white blood cells, we show that Spec identifies genes with variable expression levels that are nonetheless highly specific of particular cell types. When samples from different individuals are used, dSpec measures genes' transcriptional plasticity in each cell type. Our approach is broadly applicable to mapped gene expression measurements in stem cell biology, developmental biology, cancer biology and biomarker identification. As an example of such applications, we show that Spec identifies a new class of biomarkers, which exhibit variable expression without compromising specificity. The approach provides a unifying theoretical framework for quantifying specificity in the presence of noise, which is widely applicable across diverse biological systems.