RESUMEN
A key question in plant biology is how oriented cell divisions are integrated with patterning mechanisms to generate organs with adequate cell type allocation. In the root vasculature, a gradient of miRNA165/6 controls the abundance of HD-ZIP III transcription factors, which in turn control cell fate and spatially restrict vascular cell proliferation to specific cells. Here, we show that vascular development requires the presence of ARGONAUTE10, which is thought to sequester miRNA165/6 and protect HD-ZIP III transcripts from degradation. Our results suggest that the miR165/6-AGO10-HDZIP III module acts by buffering cytokinin responses and restricting xylem differentiation. Mutants of AGO10 show faster growth rates and strongly enhanced survival under severe drought conditions. However, this superior performance is offset by markedly increased variation and phenotypic plasticity in sub-optimal carbon supply conditions. Thus, AGO10 is required for the control of formative cell division and coordination of robust cell fate specification of the vasculature, while altering its expression provides a means to adjust phenotypic plasticity.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Proteínas Argonautas , División Celular , Regulación de la Expresión Génica de las Plantas , MicroARNs , Raíces de Plantas , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/citología , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas Argonautas/metabolismo , Proteínas Argonautas/genética , División Celular/genética , Raíces de Plantas/citología , Raíces de Plantas/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/genética , MicroARNs/genética , MicroARNs/metabolismo , Diferenciación Celular , Xilema/citología , Xilema/metabolismo , Xilema/crecimiento & desarrollo , Xilema/genéticaRESUMEN
Single-cell and single-nucleus RNA-sequencing technologies capture the expression of plant genes at an unprecedented resolution. Therefore, these technologies are gaining traction in plant molecular and developmental biology for elucidating the transcriptional changes across cell types in a specific tissue or organ, upon treatments, in response to biotic and abiotic stresses, or between genotypes. Despite the rapidly accelerating use of these technologies, collective and standardized experimental and analytical procedures to support the acquisition of high-quality data sets are still missing. In this commentary, we discuss common challenges associated with the use of single-cell transcriptomics in plants and propose general guidelines to improve reproducibility, quality, comparability, and interpretation and to make the data readily available to the community in this fast-developing field of research.
Asunto(s)
Perfilación de la Expresión Génica , Plantas , Reproducibilidad de los Resultados , Plantas/genética , Estrés Fisiológico/genética , Almacenamiento y Recuperación de la InformaciónRESUMEN
Despite the importance of Nitric Oxide (NO) as signaling molecule in both plant and animal development, the regulatory mechanisms downstream of NO remain largely unclear. Here, we show that NO is involved in Arabidopsis shoot stem cell control via modifying expression and activity of ARGONAUTE 4 (AGO4), a core component of the RNA-directed DNA Methylation (RdDM) pathway. Mutations in components of the RdDM pathway cause meristematic defects, and reduce responses of the stem cell system to NO signaling. Importantly, we find that the stem cell inducing WUSCHEL transcription factor directly interacts with AGO4 in a NO dependent manner, explaining how these two signaling systems may converge to modify DNA methylation patterns. Taken together, our results reveal that NO signaling plays an important role in controlling plant stem cell homeostasis via the regulation of de novo DNA methylation.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Metilación de ADN/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Óxido Nítrico/metabolismo , Meristema/genética , Meristema/metabolismo , Arabidopsis/metabolismo , ARN/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
Plant growth is driven by apical meristems at the shoot and root growth points, which comprise continuously active stem cell populations. While many of the key factors involved in homeostasis of the shoot apical meristem (SAM) have been extensively studied under artificial constant growth conditions, only little is known how variations in the environment affect the underlying regulatory network. To shed light on the responses of the SAM to ambient temperature, we combined 3D live imaging of fluorescent reporter lines that allowed us to monitor the activity of two key regulators of stem cell homeostasis in the SAM namely CLAVATA3 (CLV3) and WUSCHEL (WUS), with computational image analysis to derive morphological and cellular parameters of the SAM. Whereas CLV3 expression marks the stem cell population, WUS promoter activity is confined to the organizing center (OC), the niche cells adjacent to the stem cells, hence allowing us to record on the two central cell populations of the SAM. Applying an integrated computational analysis of our data we found that variations in ambient temperature not only led to specific changes in spatial expression patterns of key regulators of SAM homeostasis, but also correlated with modifications in overall cellular organization and shoot meristem morphology.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Brotes de la Planta/metabolismo , Imagenología Tridimensional , Temperatura , Proteínas de Homeodominio/metabolismo , Células MadreRESUMEN
Spatial specificity of cell fate decisions is central for organismal development. The phloem tissue mediates long-distance transport of energy metabolites along plant bodies and is characterized by an exceptional degree of cellular specialization. How a phloem-specific developmental program is implemented is, however, unknown. Here we reveal that the ubiquitously expressed PHD-finger protein OBE3 forms a central module with the phloem-specific SMXL5 protein for establishing the phloem developmental program in Arabidopsis thaliana. By protein interaction studies and phloem-specific ATAC-seq analyses, we show that OBE3 and SMXL5 proteins form a complex in nuclei of phloem stem cells where they promote a phloem-specific chromatin profile. This profile allows expression of OPS, BRX, BAM3, and CVP2 genes acting as mediators of phloem differentiation. Our findings demonstrate that OBE3/SMXL5 protein complexes establish nuclear features essential for determining phloem cell fate and highlight how a combination of ubiquitous and local regulators generate specificity of developmental decisions in plants.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Floema/metabolismo , Arabidopsis/metabolismo , Proteínas de la Membrana/metabolismo , Diferenciación Celular , Regulación de la Expresión Génica de las PlantasRESUMEN
BACKGROUND: Root development and function have central roles in plant adaptation to the environment. The modification of root traits has additionally been a major driver of crop performance since the green revolution; however, the molecular underpinnings and the regulatory programmes defining root development and response to environmental stress remain largely unknown. Single-cell reconstruction of gene regulatory programmes provides an important tool to understand the cellular phenotypic variation in complex tissues and their response to endogenous and environmental stimuli. While single-cell transcriptomes of several plant organs have been elucidated, the underlying chromatin landscapes associated with cell type-specific gene expression remain largely unexplored. RESULTS: To comprehensively delineate chromatin accessibility during root development of an important crop, we applied single-cell ATAC-seq (scATAC-seq) to 46,758 cells from rice root tips under normal and heat stress conditions. Our data revealed cell type-specific accessibility variance across most of the major cell types and allowed us to identify sets of transcription factors which associate with accessible chromatin regions (ACRs). Using root hair differentiation as a model, we demonstrate that chromatin and gene expression dynamics during cell type differentiation correlate in pseudotime analyses. In addition to developmental trajectories, we describe chromatin responses to heat and identify cell type-specific accessibility changes to this key environmental stimulus. CONCLUSIONS: We report chromatin landscapes during rice root development at single-cell resolution. Our work provides a framework for the integrative analysis of regulatory dynamics in this important crop organ at single-cell resolution.
Asunto(s)
Meristema , Oryza , Cromatina/genética , Oryza/genéticaRESUMEN
Fluorescent imaging, especially in living tissue, has become a key method in modern life sciences, with the development of new tools for sample preparation, imaging, and data analysis continuously advancing our understanding of biological principles. Here, we present our strategy for in vivo imaging of the Arabidopsis shoot apical meristem (SAM), a central structure in plant development. We implement simplifications to previously published workflows and present a novel approach to subsequentially image the meristem from multiple angles at high resolution. This tool may represent a valuable resource for shoot meristem-centered research in general, but also for studies on plasmodesmata or intercellular connectivity within the SAM: via the analysis of fluorescently labeled plasmodesmata-localized proteins, via the tracing of fluorescent dyes, via analyzing the cell-to-cell mobility of fluorescently labeled proteins, but also via the analysis of morphological features of meristematic cells in mutants or upon perturbation of symplastic connectivity.
Asunto(s)
Arabidopsis , Meristema , Meristema/metabolismo , Microscopía Confocal/métodos , PlasmodesmosRESUMEN
Cell wall recalcitrance is a major constraint for the exploitation of lignocellulosic biomass as a renewable resource for energy and bio-based products. Transcriptional regulators of the lignin biosynthetic pathway represent promising targets for tailoring lignin content and composition in plant secondary cell walls. However, knowledge about the transcriptional regulation of lignin biosynthesis in lignocellulosic feedstocks, such as Miscanthus, is limited. In Miscanthus leaves, MsSCM1 and MsMYB103 are expressed at growth stages associated with lignification. The ectopic expression of MsSCM1 and MsMYB103 in N. benthamiana leaves was sufficient to trigger secondary cell wall deposition with distinct sugar and lignin compositions. Moreover, RNA-seq analysis revealed that the transcriptional responses to MsSCM1 and MsMYB103 overexpression showed an extensive overlap with the response to the NAC master transcription factor MsSND1, but were distinct from each other, underscoring the inherent complexity of secondary cell wall formation. Furthermore, conserved and previously described promoter elements as well as novel and specific motifs could be identified from the target genes of the three transcription factors. Together, MsSCM1 and MsMYB103 represent interesting targets for manipulations of lignin content and composition in Miscanthus towards a tailored biomass.
Asunto(s)
Lignina/biosíntesis , Proteínas de Plantas/metabolismo , Poaceae/metabolismo , Transducción de Señal/genética , Factores de Transcripción/metabolismo , Biomasa , Pared Celular/metabolismo , Regulación de la Expresión Génica de las Plantas , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Poaceae/genética , Poaceae/crecimiento & desarrollo , Regiones Promotoras Genéticas/genética , RNA-Seq/métodos , Factores de Transcripción/genética , Transcriptoma/genéticaRESUMEN
Embryogenesis of flowering plants is initiated by polarization of the zygote, a prerequisite for correct axis formation in the embryo. The daughter cells of the asymmetric zygote division form the pro-embryo and the mostly extra-embryonic suspensor.1 The suspensor plays a pivotal role in nutrient and hormone transport and rapid growth of the embryo.2,3 Zygote polarization is controlled by a MITOGEN-ACTIVATING PROTEIN (MAP) kinase signaling pathway including the MAPKK kinase (MAP3K) YODA (YDA)4 and the upstream membrane-associated proteins BRASINOSTEROID SIGNALING KINASE 1 (BSK1) and BSK2.5,6 Furthermore, suspensor development is controlled by cysteine-rich peptides of the EMBRYO SURROUNDING FACTOR 1 (ESF1) family.7 While they act genetically upstream of YDA, the corresponding receptor to perceive these potential ligands is unknown. In other developmental processes, such as stomata development, YDA activity is controlled by receptor kinases of the ERECTA family (ERf).8-12 While the receptor kinases upstream of BSK1/2 in the embryo have so far not been identified,1 YDA is in part activated by the sperm cell-derived BSK family member SHORT SUSPENSOR (SSP) that represents a naturally occurring, constitutively active variant of BSK1.5,13 It has been speculated that SSP might be a paternal component of a parental tug-of-war controlling resource allocation toward the embryo.2,13 Here, we show that in addition to SSP, the receptor kinase ERECTA plays a crucial role in zygote polarization as a maternally contributed part of the embryonic YDA pathway. We conclude that two independent parental contributions initiate zygote polarization and control embryo development.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Quinasas Quinasa Quinasa PAM/metabolismo , Proteínas Quinasas/metabolismo , Semillas/metabolismo , Cigoto/metabolismoRESUMEN
Auxin represents one of the most potent and most versatile hormonal signals in the plant kingdom. Built on a simple core of only a few dedicated components, the auxin signaling system plays important roles for diverse aspects of plant development, physiology, and defense. Key to the diversity of context-dependent functional outputs generated by cells in response to this small molecule are gene duplication events and sub-functionalization of signaling components on the one hand, and a deep embedding of the auxin signaling system into complex regulatory networks on the other hand. Together, these evolutionary innovations provide the mechanisms to allow each cell to display a highly specific auxin response that suits its individual requirements. In this review, we discuss the regulatory networks connecting auxin with a large number of diverse pathways at all relevant levels of the signaling system ranging from biosynthesis to transcriptional response.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Redes Reguladoras de Genes , Ácidos Indolacéticos/metabolismo , Plantas/metabolismo , Genoma de Planta , Homeostasis , Plantas/genética , Transducción de SeñalRESUMEN
There are two main types of root systems in flowering plants, namely taproot systems of dicots and fibrous root systems found in monocots. Despite this fundamental split, our current knowledge of cellular and molecular mechanism driving root development is mainly based on studies of the dicot model Arabidopsis. However, the world major crops are monocots and little is known about the transcriptional programs underlying cell-type specification in this clade. Here, we report the transcriptomes of more than 20 000 single cells derived from root tips of two agronomically important rice cultivars. Using combined computational and experimental analyses we were able to robustly identify most of the major cell types and define novel cell-type-specific marker genes for both cultivars. Importantly, we found divergent cell types associated with specific regulatory programs, including phytohormone biosynthesis, signaling, and response, which were well conserved between the two rice cultivars. In addition, we detected substantial differences between the cell-type transcript profiles of Arabidopsis and rice. These species-specific features emphasize the importance of analyzing tissues across diverse model species, including rice. Taken together, our study provides insight into the transcriptomic landscape of major cell types of rice root tip at single-cell resolution and opens new avenues to study cell-type specification, function, and evolution in plants.
Asunto(s)
Oryza/genética , Raíces de Plantas/genética , Regulación de la Expresión Génica de las Plantas/genética , Oryza/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Raíces de Plantas/metabolismo , RNA-SeqRESUMEN
Stem cells in plants constantly supply daughter cells to form new organs and are expected to safeguard the integrity of the cells from biological invasion. Here, we show how stem cells of the Arabidopsis shoot apical meristem and their nascent daughter cells suppress infection by cucumber mosaic virus (CMV). The stem cell regulator WUSCHEL responds to CMV infection and represses virus accumulation in the meristem central and peripheral zones. WUSCHEL inhibits viral protein synthesis by repressing the expression of plant S-adenosyl-l-methionine-dependent methyltransferases, which are involved in ribosomal RNA processing and ribosome stability. Our results reveal a conserved strategy in plants to protect stem cells against viral intrusion and provide a molecular basis for WUSCHEL-mediated broad-spectrum innate antiviral immunity in plants.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/inmunología , Arabidopsis/virología , Cucumovirus , Proteínas de Homeodominio/fisiología , Inmunidad Innata , Enfermedades de las Plantas/virología , Inmunidad de la Planta , Proteínas de Arabidopsis/genética , Proteínas de Homeodominio/genética , Meristema/citología , Meristema/inmunología , Meristema/virología , Metiltransferasas/metabolismo , ARN Ribosómico/metabolismo , Células Madre/inmunología , Células Madre/virologíaRESUMEN
Coordination of fate transition and cell division is crucial to maintain the plant architecture and to achieve efficient production of plant organs. In this paper, we analysed the stem cell dynamics at the shoot apical meristem (SAM) that is one of the plant stem cells locations. We designed a mathematical model to elucidate the impact of hormonal signaling on the fate transition rates between different zones corresponding to slowly dividing stem cells and fast dividing transit amplifying cells. The model is based on a simplified two-dimensional disc geometry of the SAM and accounts for a continuous displacement towards the periphery of cells produced in the central zone. Coupling growth and hormonal signaling results in a nonlinear system of reaction-diffusion equations on a growing domain with the growth rate depending on the model components. The model is tested by simulating perturbations in the level of key transcription factors that maintain SAM homeostasis. The model provides new insights on how the transcription factor HECATE is integrated in the regulatory network that governs stem cell differentiation.
Asunto(s)
Diferenciación Celular/fisiología , Modelos Biológicos , Células Vegetales , Reguladores del Crecimiento de las Plantas/fisiología , Transducción de Señal/fisiología , Biología Computacional , Simulación por Computador , Meristema/citología , Meristema/crecimiento & desarrollo , Meristema/metabolismo , Meristema/fisiología , Células Vegetales/metabolismo , Células Vegetales/fisiologíaRESUMEN
Quantitative analysis of plant and animal morphogenesis requires accurate segmentation of individual cells in volumetric images of growing organs. In the last years, deep learning has provided robust automated algorithms that approach human performance, with applications to bio-image analysis now starting to emerge. Here, we present PlantSeg, a pipeline for volumetric segmentation of plant tissues into cells. PlantSeg employs a convolutional neural network to predict cell boundaries and graph partitioning to segment cells based on the neural network predictions. PlantSeg was trained on fixed and live plant organs imaged with confocal and light sheet microscopes. PlantSeg delivers accurate results and generalizes well across different tissues, scales, acquisition settings even on non plant samples. We present results of PlantSeg applications in diverse developmental contexts. PlantSeg is free and open-source, with both a command line and a user-friendly graphical interface.
Asunto(s)
Arabidopsis/anatomía & histología , Imagenología Tridimensional/métodos , Células Vegetales , Programas Informáticos , Arabidopsis/citología , Redes Neurales de la ComputaciónRESUMEN
Stem cells are one of the foundational evolutionary novelties that allowed the independent emergence of multicellularity in the plant and animal lineages. In plants, the homeodomain (HD) transcription factor WUSCHEL (WUS) is essential for the maintenance of stem cells in the shoot apical meristem. WUS has been reported to bind to diverse DNA motifs and to act as transcriptional activator and repressor. However, the mechanisms underlying this remarkable behavior have remained unclear. Here, we quantitatively delineate WUS binding to three divergent DNA motifs and resolve the relevant structural underpinnings. We show that WUS exhibits a strong binding preference for TGAA repeat sequences, while retaining the ability to weakly bind to TAAT elements. This behavior is attributable to the formation of dimers through interactions of specific residues in the HD that stabilize WUS DNA interaction. Our results provide a mechanistic basis for dissecting WUS dependent regulatory networks in plant stem cell control.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Homeodominio/química , Proteínas de Homeodominio/metabolismo , Motivos de Nucleótidos/genética , Arabidopsis/química , Arabidopsis/genética , Proteínas de Arabidopsis/genética , ADN/metabolismo , Dimerización , Proteínas de Homeodominio/genética , Brotes de la Planta/genética , Unión Proteica , Secuencias Repetitivas de Ácidos Nucleicos/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Positional information is essential for coordinating the development of multicellular organisms. In plants, positional information provided by the hormone auxin regulates rhythmic organ production at the shoot apex, but the spatio-temporal dynamics of auxin gradients is unknown. We used quantitative imaging to demonstrate that auxin carries high-definition graded information not only in space but also in time. We show that, during organogenesis, temporal patterns of auxin arise from rhythmic centrifugal waves of high auxin travelling through the tissue faster than growth. We further demonstrate that temporal integration of auxin concentration is required to trigger the auxin-dependent transcription associated with organogenesis. This provides a mechanism to temporally differentiate sites of organ initiation and exemplifies how spatio-temporal positional information can be used to create rhythmicity.
Plants, like animals and many other multicellular organisms, control their body architecture by creating organized patterns of cells. These patterns are generally defined by signal molecules whose levels differ across the tissue and change over time. This tells the cells where they are located in the tissue and therefore helps them know what tasks to perform. A plant hormone called auxin is one such signal molecule and it controls when and where plants produce new leaves and flowers. Over time, this process gives rise to the dashing arrangements of spiraling organs exhibited by many plant species. The leaves and flowers form from a relatively small group of cells at the tip of a growing stem known as the shoot apical meristem. Auxin accumulates at precise locations within the shoot apical meristem before cells activate the genes required to make a new leaf or flower. However, the precise role of auxin in forming these new organs remained unclear because the tools to observe the process in enough detail were lacking. Galvan-Ampudia, Cerutti et al. have now developed new microscopy and computational approaches to observe auxin in a small plant known as Arabidopsis thaliana. This showed that dozens of shoot apical meristems exhibited very similar patterns of auxin. Images taken over a period of several hours showed that the locations where auxin accumulated were not fixed on a group of cells but instead shifted away from the center of the shoot apical meristems faster than the tissue grew. This suggested the cells experience rapidly changing levels of auxin. Further experiments revealed that the cells needed to be exposed to a high level of auxin over time to activate genes required to form an organ. This mechanism sheds a new light on how auxin regulates when and where plants make new leaves and flowers. The tools developed by Galvan-Ampudia, Cerutti et al. could be used to study the role of auxin in other plant tissues, and to investigate how plants regulate the response to other plant hormones.
Asunto(s)
Arabidopsis/metabolismo , Ácidos Indolacéticos/metabolismo , Organogénesis de las Plantas , Reguladores del Crecimiento de las Plantas/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Técnicas Biosensibles , Regulación de la Expresión Génica de las Plantas , Genes Reporteros , Microscopía Confocal , Organogénesis de las Plantas/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Factores de Tiempo , Transcripción GenéticaRESUMEN
In multicellular organisms, not all cells are created equal. Instead, organismal complexity is achieved by specialisation and division of labour between distinct cell types. Therefore, the organism depends on the presence, correct proportion and function of all cell types. It follows that early development is geared towards setting up the basic body plan and to specify cell lineages. Since plants employ a post-embryonic mode of development, the continuous growth and addition of new organs require a source of new cells, as well as a strict regulation of cellular composition throughout the entire life-cycle. To meet these demands, evolution has brought about complex regulatory systems to maintain and control continuously active stem cell systems. Here, we review recent work on the mechanisms of non cell-autonomous control of shoot stem cells in the model plant Arabidopsis thaliana with a strong focus on the cell-to-cell mobility and function of the WUSCHEL homeodomain transcription factor.
Asunto(s)
Arabidopsis/crecimiento & desarrollo , Meristema/citología , Brotes de la Planta/citología , Células Madre/citología , Proteínas de Arabidopsis , Regulación de la Expresión Génica de las Plantas , Proteínas de HomeodominioRESUMEN
To maintain the balance between long-term stem cell self-renewal and differentiation, dynamic signals need to be translated into spatially precise and temporally stable gene expression states. In the apical plant stem cell system, local accumulation of the small, highly mobile phytohormone auxin triggers differentiation while at the same time, pluripotent stem cells are maintained throughout the entire life-cycle. We find that stem cells are resistant to auxin mediated differentiation, but require low levels of signaling for their maintenance. We demonstrate that the WUSCHEL transcription factor confers this behavior by rheostatically controlling the auxin signaling and response pathway. Finally, we show that WUSCHEL acts via regulation of histone acetylation at target loci, including those with functions in the auxin pathway. Our results reveal an important mechanism that allows cells to differentially translate a potent and highly dynamic developmental signal into stable cell behavior with high spatial precision and temporal robustness.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Diferenciación Celular , Autorrenovación de las Células , Proteínas de Homeodominio/metabolismo , Ácidos Indolacéticos/metabolismo , Meristema/metabolismo , Células Madre Pluripotentes/metabolismo , Proliferación Celular , Meristema/citología , Brotes de la Planta , Plantas Modificadas Genéticamente , Células Madre Pluripotentes/citología , Transducción de SeñalRESUMEN
Complex multicellular organisms evolved on Earth in an oxygen-rich atmosphere1; their tissues, including stem-cell niches, require continuous oxygen provision for efficient energy metabolism2. Notably, the maintenance of the pluripotent state of animal stem cells requires hypoxic conditions, whereas higher oxygen tension promotes cell differentiation3. Here we demonstrate, using a combination of genetic reporters and in vivo oxygen measurements, that plant shoot meristems develop embedded in a low-oxygen niche, and that hypoxic conditions are required to regulate the production of new leaves. We show that hypoxia localized to the shoot meristem inhibits the proteolysis of an N-degron-pathway4,5 substrate known as LITTLE ZIPPER 2 (ZPR2)-which evolved to control the activity of the class-III homeodomain-leucine zipper transcription factors6-8-and thereby regulates the activity of shoot meristems. Our results reveal oxygen as a diffusible signal that is involved in the control of stem-cell activity in plants grown under aerobic conditions, which suggests that the spatially distinct distribution of oxygen affects plant development. In molecular terms, this signal is translated into transcriptional regulation by the N-degron pathway, thereby linking the control of metabolic activity to the regulation of development in plants.
Asunto(s)
Arabidopsis/crecimiento & desarrollo , Hipoxia de la Célula , Meristema/crecimiento & desarrollo , Oxígeno/metabolismo , Aerobiosis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Meristema/genética , Meristema/metabolismo , Desarrollo de la Planta , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/metabolismo , Proteolisis , Células Madre/citología , Dedos de ZincRESUMEN
Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the flexible and efficient GreenGate cloning system with the proven and benchmarked LhGR system (here termed GR-LhG4) for the inducible expression, we have generated a set of transgenic Arabidopsis lines that can drive the expression of an effector cassette in a range of specific cell types in the three main plant meristems. To this end, we chose the previously developed GR-LhG4 system based on a chimeric transcription factor and a cognate pOp-type promoter ensuring tight control over a wide range of expression levels. In addition, to visualize the expression domain where the synthetic transcription factor is active, an ER-localized mTurquoise2 fluorescent reporter under control of the pOp4 or pOp6 promoter is encoded in driver lines. Here, we describe the steps necessary to generate a driver or effector line and demonstrate how cell type specific expression can be induced and followed in the shoot apical meristem, the root apical meristem and the cambium of Arabidopsis. By using several or all driver lines, the context specific effect of expressing one or multiple factors (effectors) under control of the synthetic pOp promoter can be assessed rapidly, for example in F1 plants of a cross between one effector and multiple driver lines. This approach is exemplified by the ectopic expression of VND7, a NAC transcription factor capable of inducing ectopic secondary cell wall deposition in a cell autonomous manner.