RESUMEN
Hormone-activated proteolysis is a recurring theme of plant hormone signaling mechanisms. In strigolactone signaling, the enzyme receptor DWARF14 (D14) and an F-box protein, MORE AXILLARY GROWTH2 (MAX2), mark SUPPRESSOR OF MAX2 1-LIKE (SMXL) family proteins SMXL6, SMXL7, and SMXL8 for rapid degradation. Removal of these transcriptional corepressors initiates downstream growth responses. The homologous proteins SMXL3, SMXL4, and SMXL5, however, are resistant to MAX2-mediated degradation. We discovered that the smxl4 smxl5 mutant has enhanced responses to strigolactone. SMXL5 attenuates strigolactone signaling by interfering with AtD14-SMXL7 interactions. SMXL5 interacts with AtD14 and SMXL7, providing two possible ways to inhibit SMXL7 degradation. SMXL5 function is partially dependent on an ethylene-responsive-element binding-factor-associated amphiphilic repression (EAR) motif, which typically mediates interactions with the TOPLESS family of transcriptional corepressors. However, we found that loss of the EAR motif reduces SMXL5-SMXL7 interactions and the attenuation of strigolactone signaling by SMXL5. We hypothesize that integration of SMXL5 into heteromeric SMXL complexes reduces the susceptibility of SMXL6/7/8 proteins to strigolactone-activated degradation and that the EAR motif promotes the formation or stability of these complexes. This mechanism may provide a way to spatially or temporally fine-tune strigolactone signaling through the regulation of SMXL5 expression or translation.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Compuestos Heterocíclicos con 3 Anillos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Lactonas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Co-Represoras/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
Plant vascular tissues are crucial for the long-distance transport of water, nutrients, and a multitude of signal molecules throughout the plant body and, therefore, central to plant growth and development. The intricate development of vascular tissues is orchestrated by unique populations of dedicated stem cells integrating endogenous as well as environmental cues. This review summarizes our current understanding of vascular-related stem cell biology and of vascular tissue differentiation. We present an overview of the molecular and cellular mechanisms governing the maintenance and fate determination of vascular stem cells and highlight the interplay between intrinsic and external cues. In this context, we emphasize the role of transcription factors, hormonal signaling, and epigenetic modifications. We also discuss emerging technologies and the large repertoire of cell types associated with vascular tissues, which have the potential to provide unprecedented insights into cellular specialization and anatomical adaptations to distinct ecological niches.
Asunto(s)
Diferenciación Celular , Células Madre , Células Madre/citología , Células Madre/fisiología , Células Madre/metabolismo , Epigénesis Genética , Haz Vascular de Plantas/crecimiento & desarrollo , Haz Vascular de Plantas/citología , Haz Vascular de Plantas/fisiología , Transducción de Señal , Plantas/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/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
Nuclei contain essential information for cell states, including chromatin and RNA profiles - features which are nowadays accessible using high-throughput sequencing applications. Here, we describe analytical pipelines including nucleus isolation from differentiated xylem tissues by fluorescence-activated nucleus sorting (FANS), as well as subsequent SMART-seq2-based transcriptome profiling and assay for transposase-accessible chromatin (ATAC)-seq-based chromatin analysis. Combined with tissue-specific expression of nuclear fluorescent reporters, these pipelines allow obtaining tissue-specific data on gene expression and on chromatin structure and are applicable for a large spectrum of cell types, tissues, and organs. Considering, however, the extreme degree of differentiation found in xylem cells with programmed cell death happening during vessel element formation and their role as a long-term depository for atmospheric CO2 in the form of wood, xylem cells represent intriguing and relevant objects for large-scale profilings of their cellular signatures.
Asunto(s)
Núcleo Celular , Cromatina , Cromatina/genética , Cromatina/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Transcriptoma , Secuenciación de Nucleótidos de Alto Rendimiento , Perfilación de la Expresión Génica , Xilema , Análisis de Secuencia de ADNRESUMEN
Inducible, tissue-specific gene expression is a potent tool to study gene regulatory networks as it allows spatially and temporally controlled genetic perturbations. To this end, we generated a toolkit that covers many cell types in the three main meristems: the root apical meristem, the shoot apical meristem, and the vascular cambium. The system is based on an extensive set of driver lines expressing a synthetic transcription factor under cell type-specific promoters. Induction leads to nuclear translocation of the transcription factor and expression of response elements under control of a cognate synthetic promoter. In addition, a fluorescent reporter incorporated in driver lines allows to monitor induction. All previously generated driver lines are available from the Nottingham Arabidopsis Stock Center. This protocol describes how users can create their own constructs compatible with the existing set of lines and as well as induction and imaging procedures.
Asunto(s)
Arabidopsis , Arabidopsis/genética , Cámbium , Colorantes , Factores de Transcripción/genética , Expresión GénicaRESUMEN
Plants produce the major part of terrestrial biomass and are long-term deposits of atmospheric carbon. This capacity is to a large extent due to radial growth of woody species - a process driven by cambium stem cells located in distinct niches of shoot and root axes. In the model species Arabidopsis thaliana, thousands of cells are produced by the cambium in radial orientation generating a complex organ anatomy enabling long-distance transport, mechanical support and protection against biotic and abiotic stressors. These complex organ dynamics make a comprehensive and unbiased analysis of radial growth challenging and asks for tools for automated quantification. Here, we combined the recently developed PlantSeg and MorphographX image analysis tools, to characterize tissue morphogenesis of the Arabidopsis hypocotyl. After sequential training of segmentation models on ovules, shoot apical meristems and adult hypocotyls using deep machine learning, followed by the training of cell type classification models, our pipeline segments complex images of transverse hypocotyl sections with high accuracy and classifies central hypocotyl cell types. By applying our pipeline on both wild type and phloem intercalated with xylem (pxy) mutants, we also show that this strategy faithfully detects major anatomical aberrations. Collectively, we conclude that our established pipeline is a powerful phenotyping tool comprehensively extracting cellular parameters and providing access to tissue topology during radial plant growth.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Desarrollo de la Planta , Arabidopsis/metabolismo , Meristema/metabolismo , Aprendizaje AutomáticoRESUMEN
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
Precise organization of growing structures is a fundamental process in developmental biology. In plants, radial growth is mediated by the cambium, a stem cell niche continuously producing wood (xylem) and bast (phloem) in a strictly bidirectional manner. While this process contributes large parts to terrestrial biomass, cambium dynamics eludes direct experimental access due to obstacles in live-cell imaging. Here, we present a cell-based computational model visualizing cambium activity and integrating the function of central cambium regulators. Performing iterative comparisons of plant and model anatomies, we conclude that the receptor-like kinase PXY and its ligand CLE41 are part of a minimal framework sufficient for instructing tissue organization. By integrating tissue-specific cell wall stiffness values, we moreover probe the influence of physical constraints on tissue geometry. Our model highlights the role of intercellular communication within the cambium and shows that a limited number of factors are sufficient to create radial growth by bidirectional tissue production.
Asunto(s)
Cámbium , Desarrollo de la Planta , Plantas , Xilema , Simulación por Computador , Regulación de la Expresión Génica de las Plantas , FloemaRESUMEN
Cellular heterogeneity in growth and differentiation results in organ patterning. Single-cell transcriptomics allows characterization of gene expression heterogeneity in developing organs at unprecedented resolution. However, the original physical location of the cell is lost during this methodology. To recover the original location of cells in the developing organ is essential to link gene activity with cellular identity and function in plants. Here, we propose a method to reconstruct genome-wide gene expression patterns of individual cells in a 3D flower meristem by combining single-nuclei RNA-seq with microcopy-based 3D spatial reconstruction. By this, gene expression differences among meristematic domains giving rise to different tissue and organ types can be determined. As a proof of principle, the method is used to trace the initiation of vascular identity within the floral meristem. Our work demonstrates the power of spatially reconstructed single cell transcriptome atlases to understand plant morphogenesis. The floral meristem 3D gene expression atlas can be accessed at http://threed-flower-meristem.herokuapp.com .
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Meristema , Flores , Expresión Génica , Proteínas de Plantas/genética , ARN , Análisis de Secuencia de ARNRESUMEN
Precise coordination of cell fate decisions is a hallmark of multicellular organisms. Especially in tissues with non-stereotypic anatomies, dynamic communication between developing cells is vital for ensuring functional tissue organization. Radial plant growth is driven by a plant stem cell niche known as vascular cambium, usually strictly producing secondary xylem (wood) inward and secondary phloem (bast) outward, two important structures serving as much-needed CO2 depositories and building materials. Because of its bidirectional nature and its developmental plasticity, the vascular cambium serves as an instructive paradigm for investigating principles of tissue patterning. Although genes and hormones involved in xylem and phloem formation have been identified, we have a yet incomplete picture of the initial steps of cell fate transitions of stem cell daughters into xylem and phloem progenitors. In this mini-review perspective, we describe two possible scenarios of cell fate decisions based on the current knowledge about gene regulatory networks and how cellular environments are established. In addition, we point out further possible research directions.
RESUMEN
Plant secondary growth, which is the basis of wood formation, includes the production of secondary xylem, which is derived from meristematic cambium cells embedded in vascular tissue. Here, we identified an important role for the Arabidopsis thaliana (Arabidopsis) AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED 15 (AHL15) transcriptional regulator in controlling vascular cambium activity. The limited secondary xylem development in inflorescence stems of herbaceous Arabidopsis plants was significantly reduced in ahl15 loss-of-function mutants, whereas constitutive or vascular meristem-specific AHL15 overexpression produced woody inflorescence stems. AHL15 was required for enhanced secondary xylem formation in the woody suppressor of overexpression of constans 1 (soc1) fruitfull (ful) double loss-of-function mutant. Moreover, we found that AHL15 induces vascular cambium activity downstream of the repressing SOC1 and FUL transcription factors, most likely similar to how it enhances lateral branching by promoting biosynthesis of the hormone cytokinin. Our results uncover a novel pathway driving cambium development, in which AHL15 expression levels act in parallel to and are dependent on the well-established TDIF-PXY-WOX pathway to differentiate between herbaceous and woody stem growth.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cámbium/genética , Regulación de la Expresión Génica de las Plantas , Meristema/metabolismo , Xilema/metabolismoRESUMEN
Strigolactones (SLs) are a class of plant hormones that mediate biotic interactions and modulate developmental programs in response to endogenous and exogenous stimuli. However, a comprehensive view on the spatio-temporal pattern of SL signaling has not been established, and tools for a systematic in planta analysis do not exist. Here, we present Strigo-D2, a genetically encoded ratiometric SL signaling sensor that enables the examination of SL signaling distribution at cellular resolution and is capable of rapid response to altered SL levels in intact Arabidopsis (Arabidopsis thaliana) plants. By monitoring the abundance of a truncated and fluorescently labeled SUPPRESSOR OF MAX2 1-LIKE 6 (SMXL6) protein, a proteolytic target of the SL signaling machinery, we show that all cell types investigated have the capacity to respond to changes in SL levels but with very different dynamics. In particular, SL signaling is pronounced in vascular cells but low in guard cells and the meristematic region of the root. We also show that other hormones leave Strigo-D2 activity unchanged, indicating that initial SL signaling steps work in isolation from other hormonal signaling pathways. The specificity and spatio-temporal resolution of Strigo-D2 underline the value of the sensor for monitoring SL signaling in a broad range of biological contexts with highly instructive analytical depth.
Asunto(s)
Arabidopsis/crecimiento & desarrollo , Arabidopsis/genética , Arabidopsis/metabolismo , Compuestos Heterocíclicos con 3 Anillos/metabolismo , Lactonas/metabolismo , Meristema/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Transducción de Señal/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Meristema/genética , Meristema/crecimiento & desarrollo , Reguladores del Crecimiento de las Plantas/genética , Transducción de Señal/genéticaRESUMEN
Genome-wide gene expression maps with a high spatial resolution have substantially accelerated plant molecular science. However, the number of characterized tissues and growth stages is still small due to the limited accessibility of most tissues for protoplast isolation. Here, we provide gene expression profiles of the mature inflorescence stem of Arabidopsis thaliana covering a comprehensive set of distinct tissues. By combining fluorescence-activated nucleus sorting and laser-capture microdissection with next-generation RNA sequencing, we characterized the transcriptomes of xylem vessels, fibers, the proximal and distal cambium, phloem, phloem cap, pith, starch sheath, and epidermis cells. Our analyses classified more than 15,000 genes as being differentially expressed among different stem tissues and revealed known and novel tissue-specific cellular signatures. By determining overrepresented transcription factor binding regions in the promoters of differentially expressed genes, we identified candidate tissue-specific transcriptional regulators. Our datasets predict the expression profiles of an exceptional number of genes and allow hypotheses to be generated about the spatial organization of physiological processes. Moreover, we demonstrate that information about gene expression in a broad range of mature plant tissues can be established at high spatial resolution by nuclear mRNA profiling. Tissue-specific gene expression values can be accessed online at https://arabidopsis-stem.cos.uni-heidelberg.de/.
Asunto(s)
Arabidopsis/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Inflorescencia/genética , Tallos de la Planta/genética , Arabidopsis/fisiología , Sitios de Unión , Núcleo Celular/metabolismo , Bases de Datos Genéticas , Proteínas Fluorescentes Verdes/metabolismo , Especificidad de Órganos/genética , Floema/metabolismo , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , RNA-Seq , Especificidad de la Especie , Factores de Transcripción/metabolismo , Transgenes , Madera/genéticaRESUMEN
The phloem tissue is the main conduit for sugars in plants, and its anatomy has to be tightly controled to ensure its functionality. A new study indicates the involvement of receptor-based intercellular signaling in the coordination of cell fate determination within the phloem tissue.
Asunto(s)
Floema , Plantas , AzúcaresRESUMEN
As a pre-requisite for constant growth, plants produce vascular tissues at different sites within their post-embryonic body. Interestingly, the formation of vascular tissues during longitudinal and radial expansion of shoot and root axes differs fundamentally with respect to its anatomical configuration. This raises the question to which level regulatory mechanisms of vascular tissue formation are shared throughout plant development. Here, we show that, similar to primary phloem formation during longitudinal growth, the cambium-based formation of secondary phloem depends on the function of SUPPRESSOR OF MAX2 1-LIKE (SMXL) genes. In particular, local SMXL5 deficiency results in the absence of secondary phloem. Moreover, the additional disruption of SMXL4 activity increases tissue production in the cambium region without secondary phloem being formed. Using promoter-reporter lines, we observed that SMXL4 and SMXL5 activities are associated with different stages of secondary phloem formation in the Arabidopsis stem. Based on genome-wide transcriptional profiling and expression analyses of phloem-related markers, we concluded that early steps of phloem formation are impaired in smxl4;smxl5 double mutants and that the additional cambium-derived cells fail to establish phloem-related features. Our results showed that molecular mechanisms determining primary and secondary phloem formation share important properties, but differ slightly with SMXL5 playing a more dominant role in the formation of secondary phloem.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Floema/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Péptidos y Proteínas de Señalización Intracelular/genética , Células Madre/metabolismoRESUMEN
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
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.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Clonación Molecular/métodos , Transactivadores/metabolismo , Factores de Transcripción/genética , Animales , Arabidopsis/metabolismo , Pared Celular/metabolismo , Regulación de la Expresión Génica de las Plantas , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , Ratas , Receptores de Glucocorticoides/metabolismoRESUMEN
The cambium is a plant-borne stem cell system producing wood and bast, two distinct types of vascular tissues, in strictly opposite directions. Thereby, the cambium contributes substantially to terrestrial biomass accumulation and represents the basis for the formation of large plant bodies. Although the bidirectional mode of tissue production by a common stem cell pool holds interesting implications for developmental biology, functional domains of the cambium, and their interaction remained poorly defined for decades. Here, we summarize recent findings on domain organization of the cambium and discuss potential mechanisms important for its bipartite organization. By highlighting the conceptual implication for stem cell biology, we integrate our understanding of cambium regulation into a larger context.
Asunto(s)
Cámbium , Regulación de la Expresión Génica de las Plantas , Células Vegetales , Plantas , Células MadreRESUMEN
Developmental plasticity, defined as the capacity to respond to changing environmental conditions, is an inherent feature of plant growth. Recent studies have brought the phloem tissue, the quintessential conduit for energy metabolites and inter-organ communication, into focus as an instructive developmental system. Those studies have clarified long-standing questions about essential aspects of phloem development and function, such as the pressure flow hypothesis, mechanisms of phloem unloading, and source-sink relationships. Interestingly, plants with impaired phloem development show characteristic changes in body architecture, thereby highlighting the capacity of the phloem to integrate environmental cues and to fine-tune plant development. Therefore, understanding the plasticity of phloem development provides scenarios of how environmental stimuli are translated into differential plant growth. In this Review, we summarize novel insights into how phloem identity is established and how phloem cells fulfil their core function as transport units. Moreover, we discuss possible interfaces between phloem physiology and development as sites for mediating the plastic growth mode of plants.