RESUMEN
KEY MESSAGE: Interactor of WOX2, CDC48A, is crucial for early embryo patterning and shoot meristem stem cell initiation, but is not required for WOX2 protein turnover or subcellular localization. During Arabidopsis embryo patterning, the WUSCHEL HOMEOBOX 2 (WOX2) transcription factor is a major regulator of protoderm and shoot stem cell initiation. Loss of WOX2 function results in aberrant protodermal cell divisions and, redundantly with its paralogs WOX1, WOX3, and WOX5, compromised shoot meristem formation. To elucidate the molecular basis for WOX2 function, we searched for protein interactors by IP-MS/MS from WOX2-overexpression roots displaying reprogramming toward shoot-like cell fates. Here, we report that WOX2 directly interacts with the type II AAA ATPase molecular chaperone CELL DIVISION CYCLE 48A (CDC48A). We confirmed this interaction with bimolecular fluorescence complementation and co-immunoprecipitation and found that both proteins co-localize in the nucleus. We show that CDC48A loss of function results in protoderm and shoot meristem stem cell initiation defects similar to WOX2 loss of function. We also provide evidence that CDC48A promotes WOX2 activity independently of proteolysis or the regulation of nuclear localization, common mechanisms of CDC48A function in other processes. Our results point to a new role of CDC48A in potentiating WOX2 function during early embryo patterning.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Ciclo Celular , Regulación de la Expresión Génica de las Plantas , Proteínas de Homeodominio , Meristema , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/embriología , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/genética , Meristema/metabolismo , Meristema/genética , Meristema/embriología , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/genética , Semillas/metabolismo , Semillas/genética , Semillas/crecimiento & desarrollo , Plantas Modificadas Genéticamente , ATPasas Asociadas con Actividades Celulares Diversas , Factores de TranscripciónRESUMEN
In Arabidopsis thaliana, brassinosteroid (BR) signaling and stomatal development are connected through the SHAGGY/GSK3-like kinase BR INSENSITIVE2 (BIN2). BIN2 is a key negative regulator of BR signaling but it plays a dual role in stomatal development. BIN2 promotes or restricts stomatal asymmetric cell division (ACD) depending on its subcellular localization, which is regulated by the stomatal lineage-specific scaffold protein POLAR. BRs inactivate BIN2, but how they govern stomatal development remains unclear. Mapping the single-cell transcriptome of stomatal lineages after triggering BR signaling with either exogenous BRs or the specific BIN2 inhibitor, bikinin, revealed that the two modes of BR signaling activation generate spatiotemporally distinct transcriptional responses. We established that BIN2 is always sensitive to the inhibitor but, when in a complex with POLAR and its closest homolog POLAR-LIKE1, it becomes protected from BR-mediated inactivation. Subsequently, BR signaling in ACD precursors is attenuated, while it remains active in epidermal cells devoid of scaffolds and undergoing differentiation. Our study demonstrates how scaffold proteins contribute to cellular signal specificity of hormonal responses in plants.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Brasinoesteroides , División Celular Asimétrica , Glucógeno Sintasa Quinasa 3 , Transducción de Señal , Diferenciación Celular , Arabidopsis/genética , Proteínas Quinasas/genética , Proteínas de Arabidopsis/genéticaRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citología , Floema/citología , Floema/crecimiento & desarrollo , Raíces de Plantas/citología , Factores de Transcripción/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Diferenciación Celular , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Meristema/citología , Floema/genética , Floema/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , RNA-Seq , Transducción de Señal , Análisis de la Célula Individual , Factores de Transcripción/genética , TranscriptomaRESUMEN
During plant development, a precise balance of cytokinin is crucial for correct growth and patterning, but it remains unclear how this is achieved across different cell types and in the context of a growing organ. Here we show that in the root apical meristem, the TMO5/LHW complex increases active cytokinin levels via two cooperatively acting enzymes. By profiling the transcriptomic changes of increased cytokinin at single-cell level, we further show that this effect is counteracted by a tissue-specific increase in CYTOKININ OXIDASE 3 expression via direct activation of the mobile transcription factor SHORTROOT. In summary, we show that within the root meristem, xylem cells act as a local organizer of vascular development by non-autonomously regulating cytokinin levels in neighbouring procambium cells via sequential induction and repression modules.
Asunto(s)
Arabidopsis/crecimiento & desarrollo , Citocininas , Raíces de Plantas/crecimiento & desarrollo , Proteínas de Arabidopsis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Oxidorreductasas , Transducción de Señal , TransactivadoresRESUMEN
The effects of brassinosteroid signaling on shoot and root development have been characterized in great detail but a simple consistent positive or negative impact on a basic cellular parameter was not identified. In this study, we combined digital 3D single-cell shape analysis and single-cell mRNA sequencing to characterize root meristems and mature root segments of brassinosteroid-blind mutants and wild type. The resultant datasets demonstrate that brassinosteroid signaling affects neither cell volume nor cell proliferation capacity. Instead, brassinosteroid signaling is essential for the precise orientation of cell division planes and the extent and timing of anisotropic cell expansion. Moreover, we found that the cell-aligning effects of brassinosteroid signaling can propagate to normalize the anatomy of both adjacent and distant brassinosteroid-blind cells through non-cell-autonomous functions, which are sufficient to restore growth vigor. Finally, single-cell transcriptome data discern directly brassinosteroid-responsive genes from genes that can react non-cell-autonomously and highlight arabinogalactans as sentinels of brassinosteroid-dependent anisotropic cell expansion.
Asunto(s)
Arabidopsis/citología , Arabidopsis/crecimiento & desarrollo , Brasinoesteroides/metabolismo , Diferenciación Celular/efectos de los fármacos , Raíces de Plantas/citología , Arabidopsis/metabolismo , Brasinoesteroides/farmacología , Regulación de la Expresión Génica de las Plantas , Meristema/crecimiento & desarrollo , Meristema/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Transcriptoma/efectos de los fármacosRESUMEN
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.
Asunto(s)
Análisis de la Célula Individual , Transcriptoma , Biología Computacional , Plantas/genética , Análisis de Secuencia de ARNRESUMEN
Optimal plant growth is hampered by deficiency of the essential macronutrient phosphate in most soils. Plant roots can, however, increase their root hair density to efficiently forage the soil for this immobile nutrient. By generating and exploiting a high-resolution single-cell gene expression atlas of Arabidopsis roots, we show an enrichment of TARGET OF MONOPTEROS 5/LONESOME HIGHWAY (TMO5/LHW) target gene responses in root hair cells. The TMO5/LHW heterodimer triggers biosynthesis of mobile cytokinin in vascular cells and increases root hair density during low-phosphate conditions by modifying both the length and cell fate of epidermal cells. Moreover, root hair responses in phosphate-deprived conditions are TMO5- and cytokinin-dependent. Cytokinin signaling links root hair responses in the epidermis to perception of phosphate depletion in vascular cells.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/crecimiento & desarrollo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Meristema/crecimiento & desarrollo , Floema/crecimiento & desarrollo , Fosfatos/deficiencia , Epidermis de la Planta/crecimiento & desarrollo , Transactivadores/fisiología , Xilema/crecimiento & desarrollo , Arabidopsis/citología , Arabidopsis/genética , Citocininas/biosíntesis , Citocininas/genética , Meristema/citología , Meristema/metabolismo , Floema/citología , Floema/metabolismo , Epidermis de la Planta/citología , Epidermis de la Planta/genética , Raíces de Plantas/citología , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Xilema/citología , Xilema/metabolismoRESUMEN
Cortical microtubule (MT) arrays play a critical role in plant cell shape determination by defining the direction of cell expansion. As plants continuously adapt to ever-changing environmental conditions, multiple environmental and developmental inputs need to be translated into changes of the MT cytoskeleton. Here, we identify and functionally characterize an auxin-inducible and MT-localized protein OsIQ67-DOMAIN14 (OsIQD14), which is highly expressed in rice seed hull cells. We show that while deficiency of OsIQD14 results in short and wide seeds and increases overall yield, overexpression leads to narrow and long seeds, caused by changed MT alignment. We further show that OsIQD14-mediated MT reordering is regulated by specifically affecting MT dynamics, and ectopic expression of OsIQD14 in Arabidopsis could change the cell shape both in pavement cells and in hypocotyl cells. Additionally, OsIQD14 activity is tightly controlled by calmodulin proteins, providing an alternative way to modify the OsIQD14 activity. Our results indicate that OsIQD14 acts as a key factor in regulating MT rearrangements in rice hull cells and hence the grain shape, and allows effective local cell shape manipulation to improve the rice yield trait.
Asunto(s)
Proteínas de Arabidopsis , Oryza , Citoesqueleto , Proteínas Asociadas a Microtúbulos , Microtúbulos , Oryza/genéticaRESUMEN
Land plants reproduce sexually by developing an embryo from a fertilized egg cell. However, embryos can also be formed from other cell types in many plant species. Thus, a key question is how embryo identity in plants is controlled, and how this process is modified during nonzygotic embryogenesis. The Arabidopsis (Arabidopsis thaliana) zygote divides to produce an embryonic lineage and an extra-embryonic suspensor. Yet, normally quiescent suspensor cells can develop a second embryo when the initial embryo is damaged, or when response to the signaling molecule auxin is locally blocked. Here we used auxin-dependent suspensor embryogenesis as a model to determine transcriptome changes during embryonic reprogramming. We found that reprogramming is complex and accompanied by large transcriptomic changes before anatomical changes. This analysis revealed a strong enrichment for genes encoding components of auxin homeostasis and response among misregulated genes. Strikingly, deregulation among multiple auxin-related gene families converged upon the re-establishment of cellular auxin levels or response. This finding points to a remarkable degree of feedback regulation to create resilience in the auxin response during embryo development. Starting from the transcriptome of auxin-deregulated embryos, we identified an auxin-dependent basic Helix Loop Helix transcription factor network that mediates the activity of this hormone in suppressing embryo development from the suspensor.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Ácidos Indolacéticos/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Semillas/metabolismo , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Plantas Modificadas Genéticamente/genética , Semillas/genética , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
In the version of this Resource originally published, the author information was incorrect. Jos R. Wendrich should have had a present address: Department of Plant Biotechnology and Bioinformatics and VIB Center for Plant Systems Biology, Ghent University, Technologiepark 927, 9052 Ghent, Belgium. Mark Boekschoten and Guido J. Hooiveld should have been affiliated to the Nutrition, Metabolism and Genomics Group, Division of Human Nutrition, Wageningen University, 6708 WE Wageningen, The Netherlands. In addition, the version of Supplementary Table 5 originally published with this Resource was not the intended final version and included inaccurate citations to the display items of the Resource, and the file format and extension did not match. These errors have now been corrected in all versions of the Resource.
RESUMEN
During early plant embryogenesis, precursors for all major tissues and stem cells are formed. While several components of the regulatory framework are known, how cell fates are instructed by genome-wide transcriptional activity remains unanswered-in part because of difficulties in capturing transcriptome changes at cellular resolution. Here, we have adapted a two-component transgenic labelling system to purify cell-type-specific nuclear RNA and generate a transcriptome atlas of early Arabidopsis embryo development, with a focus on root stem cell niche formation. We validated the dataset through gene expression analysis, and show that gene activity shifts in a spatio-temporal manner, probably signifying transcriptional reprogramming, to induce developmental processes reflecting cell states and state transitions. This atlas provides the most comprehensive tissue- and cell-specific description of genome-wide gene activity in the early plant embryo, and serves as a valuable resource for understanding the genetic control of early plant development.
Asunto(s)
Arabidopsis/genética , Semillas/genética , Transcriptoma , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Técnicas Genéticas , Células Vegetales , Raíces de Plantas/citología , Raíces de Plantas/genética , Plantas Modificadas Genéticamente , Coloración y Etiquetado/métodosRESUMEN
In plants, apical meristems allow continuous growth along the body axis. Within the root apical meristem, a group of slowly dividing quiescent center cells is thought to limit stem cell activity to directly neighboring cells, thus endowing them with unique properties, distinct from displaced daughters. This binary identity of the stem cells stands in apparent contradiction to the more gradual changes in cell division potential and differentiation that occur as cells move further away from the quiescent center. To address this paradox and to infer molecular organization of the root meristem, we used a whole-genome approach to determine dominant transcriptional patterns along root ontogeny zones. We found that the prevalent patterns are expressed in two opposing gradients. One is characterized by genes associated with development, the other enriched in differentiation genes. We confirmed these transcript gradients, and demonstrate that these translate to gradients in protein accumulation and gradual changes in cellular properties. We also show that gradients are genetically controlled through multiple pathways. Based on these findings, we propose that cells in the Arabidopsis root meristem gradually transition from stem cell activity toward differentiation.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/citología , Meristema/citología , Raíces de Plantas/citología , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Citometría de Flujo/métodos , Regulación de la Expresión Génica de las Plantas , Proteínas Fluorescentes Verdes/genética , Meristema/genética , Células Vegetales , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Plantas Modificadas GenéticamenteRESUMEN
Individual proteins often function as part of a protein complex. The identification of interacting proteins is therefore vital to understand the biological role and function of the studied protein. Here we describe a method for the in vivo identification of nuclear, cytoplasmic, and membrane-associated protein complexes from plant tissues using a strategy of immunoprecipitation followed by tandem mass spectrometry. By performing quantitative mass spectrometry measurements on biological triplicates, relative abundance of proteins in GFP-tagged complexes compared to background controls can be statistically evaluated to identify high-confidence interactors. We detail the entire workflow of this approach.
Asunto(s)
Proteínas de Plantas/metabolismo , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Inmunoprecipitación/métodos , Proteínas de la Membrana/metabolismo , Espectrometría de Masas en Tándem/métodosRESUMEN
KEY MESSAGE: We describe a novel set of domain-specific markers that can be used in genetic studies, and we used two examples to show loss of stem cells in a monopteros background. Multicellular organisms can be defined by their ability to establish distinct cell identities, and it is therefore of critical importance to distinguish cell types. One step that leads to cell identity specification is activation of unique sets of transcripts. This property is often exploited in order to infer cell identity; the availability of good domain-specific marker lines is, however, poor in the Arabidopsis embryo. Here we describe a novel set of domain-specific marker lines that can be used in Arabidopsis (embryo) research. Based on transcriptomic data, we selected 12 genes for expression analysis, and according to the observed expression domain during embryogenesis, we divided them into four categories (1-ground tissue; 2-root stem cell; 3-shoot apical meristem; 4-post-embryonic). We additionally show the use of two markers from the "stem cell" category in a genetic study, where we use the absence of the markers to infer developmental defects in the monopteros mutant background. Finally, in order to judge whether the established marker lines also play a role in normal development, we generated loss-of-function resources. None of the analyzed T-DNA insertion, artificial microRNA, or misexpression lines showed any apparent phenotypic difference from wild type, indicating that these genes are not nonredundantly required for development, but also suggesting that marker activation can be considered an output of the patterning process. This set of domain-specific marker lines is therefore a valuable addition to the currently available markers and will help to move toward a generic set of tissue identity markers.
Asunto(s)
Antígenos de Diferenciación/genética , Arabidopsis/embriología , Arabidopsis/genética , Arabidopsis/citología , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Meristema , Raíces de Plantas/citología , Brotes de la Planta/citología , Semillas/citología , Semillas/crecimiento & desarrollo , Células Madre/citología , Células Madre/metabolismoRESUMEN
Molecular cloning is a vital step in much of today's plant biological research. Particularly, when a species is amenable to transgenic manipulation, cloning enables detailed study of gene and protein function in vivo. Therefore, accurate, consistent, and efficient cloning methods have the potential to accelerate biological research. Traditional restriction-enzyme/ligase-based strategies are often inefficient, while novel alternative methods can be less economical. We have recently optimized a method for Ligation-Independent Cloning (LIC) that is both efficient and economical. We have developed a large set of LIC-compatible plasmids for application in plant research. These include dedicated vectors for gene expression analysis, protein localization studies, and protein misexpression. We describe a detailed protocol that allows the reliable generation of plant transformation-ready constructs from PCR fragments in 2-3 days.
Asunto(s)
Clonación Molecular/métodos , Plantas/genética , Vectores Genéticos/genética , Plásmidos/genética , Reacción en Cadena de la Polimerasa/métodosRESUMEN
Coordination of cell division and pattern formation is central to tissue and organ development, particularly in plants where walls prevent cell migration. Auxin and cytokinin are both critical for division and patterning, but it is unknown how these hormones converge upon tissue development. We identify a genetic network that reinforces an early embryonic bias in auxin distribution to create a local, nonresponding cytokinin source within the root vascular tissue. Experimental and theoretical evidence shows that these cells act as a tissue organizer by positioning the domain of oriented cell divisions. We further demonstrate that the auxin-cytokinin interaction acts as a spatial incoherent feed-forward loop, which is essential to generate distinct hormonal response zones, thus establishing a stable pattern within a growing vascular tissue.
Asunto(s)
Arabidopsis/crecimiento & desarrollo , Tipificación del Cuerpo/fisiología , Ácidos Indolacéticos/metabolismo , Haz Vascular de Plantas/crecimiento & desarrollo , Aminohidrolasas , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Tipificación del Cuerpo/efectos de los fármacos , Tipificación del Cuerpo/genética , División Celular/genética , División Celular/fisiología , Citocinas/biosíntesis , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Redes Reguladoras de Genes , Ácidos Indolacéticos/farmacología , Proteínas Nucleares/genética , Haz Vascular de Plantas/efectos de los fármacos , Transactivadores/metabolismoRESUMEN
Four basic ingredients of morphogenesis, oriented cell division and expansion, cell-cell communication and cell fate specification allow plant cells to develop into a wide variety of organismal architectures. A central question in plant biology is how these cellular processes are regulated and orchestrated. Here, we present the advantages of the early Arabidopsis embryo as a model for studying the control of morphogenesis. All ingredients of morphogenesis converge during embryogenesis, and the highly predictable nature of embryo development offers unprecedented opportunities for understanding their regulation in time and space. In this review we describe the morphogenetic principles underlying embryo patterning and discuss recent advances in their regulation. Morphogenesis is under tight transcriptional control and most genes that were identified as important regulators of embryo patterning encode transcription factors or components of signaling pathways. There exists, therefore, a large gap between the transcriptional control of embryo morphogenesis and the cellular execution. We describe the first such connections, and propose future directions that should help bridge this gap and generate comprehensive understanding of the control of morphogenesis.