RESUMO
Clathrin-mediated endocytosis is an essential cellular internalization pathway involving the dynamic assembly of clathrin and accessory proteins to form membrane-bound vesicles. The evolutionarily ancient TSET-TPLATE complex (TPC) plays an essential, but ill-defined role in endocytosis in plants. Here we show that two highly disordered TPC subunits, AtEH1 and AtEH2, function as scaffolds to drive biomolecular condensation of the complex. These condensates specifically nucleate on the plasma membrane through interactions with anionic phospholipids, and facilitate the dynamic recruitment and assembly of clathrin, as well as early- and late-stage endocytic accessory proteins. Importantly, condensation promotes ordered clathrin assemblies. TPC-driven biomolecular condensation thereby facilitates dynamic protein assemblies throughout clathrin-mediated endocytosis. Furthermore, we show that a disordered region of AtEH1 controls the material properties of endocytic condensates in vivo. Alteration of these material properties disturbs the recruitment of accessory proteins, influences endocytosis dynamics and impairs plant responsiveness. Our findings reveal how collective interactions shape endocytosis.
Assuntos
Clatrina , Endocitose , Membrana Celular/metabolismo , Clatrina/metabolismoRESUMO
The nuclear TIR1/AFB-Aux/IAA auxin pathway plays a crucial role in regulating plant growth and development. Specifically, the IAA17/AXR3 protein participates in Arabidopsis thaliana root development, response to auxin and gravitropism. However, the mechanism by which AXR3 regulates cell elongation is not fully understood. We combined genetical and cell biological tools with transcriptomics and determination of auxin levels and employed live cell imaging and image analysis to address how the auxin response pathways influence the dynamics of root growth. We revealed that manipulations of the TIR1/AFB-Aux/IAA pathway rapidly modulate root cell elongation. While inducible overexpression of the AXR3-1 transcriptional inhibitor accelerated growth, overexpression of the dominant activator form of ARF5/MONOPTEROS inhibited growth. In parallel, AXR3-1 expression caused loss of auxin sensitivity, leading to transcriptional reprogramming, phytohormone signaling imbalance and increased levels of auxin. Furthermore, we demonstrated that AXR3-1 specifically perturbs nuclear auxin signaling, while the rapid auxin response remains functional. Our results shed light on the interplay between the nuclear and cytoplasmic auxin pathways in roots, revealing their partial independence but also the dominant role of the nuclear auxin pathway during the gravitropic response of Arabidopsis thaliana roots.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Raízes de Plantas/metabolismoRESUMO
The plant-signaling molecule auxin triggers fast and slow cellular responses across land plants and algae. The nuclear auxin pathway mediates gene expression and controls growth and development in land plants, but this pathway is absent from algal sister groups. Several components of rapid responses have been identified in Arabidopsis, but it is unknown if these are part of a conserved mechanism. We recently identified a fast, proteome-wide phosphorylation response to auxin. Here, we show that this response occurs across 5 land plant and algal species and converges on a core group of shared targets. We found conserved rapid physiological responses to auxin in the same species and identified rapidly accelerated fibrosarcoma (RAF)-like protein kinases as central mediators of auxin-triggered phosphorylation across species. Genetic analysis connects this kinase to both auxin-triggered protein phosphorylation and rapid cellular response, thus identifying an ancient mechanism for fast auxin responses in the green lineage.
Assuntos
Embriófitas , Transdução de Sinais , Arabidopsis/genética , Arabidopsis/metabolismo , Embriófitas/metabolismo , Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos/metabolismo , Fosforilação , Plantas/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Algas/metabolismoRESUMO
In flowering plants, two fertilization products develop within the limited space of the seed: the embryo and the surrounding nutritive endosperm. The final size of the endosperm is modulated by the degree of embryo growth. In Arabidopsis thaliana, the endosperm expands rapidly after fertilization, but later gets invaded by the embryo that occupies most of the seed volume at maturity, surrounded by a single remaining aleurone-like endosperm layer.1,2,3,4 Embryo invasion is facilitated by the endosperm-expressed bHLH-type transcription factor ZHOUPI, which promotes weakening of endosperm cell walls.5,6 Endosperm elimination in zou mutants is delayed, and embryo growth is severely affected; the endosperm finally collapses around the dwarf embryo, causing the shriveled appearance of mature zou seeds.5,6,7 However, whether ZHOUPI facilitates mechanical endosperm destruction by the invading embryo or whether an active programmed cell death (PCD) process causes endosperm elimination has been subject to debate.2,8 Here we show that developmental PCD controlled by multiple NAC transcription factors in the embryo-adjacent endosperm promotes gradual endosperm elimination. Misexpressing the NAC transcription factor KIRA1 in the entire endosperm caused total endosperm elimination, generating aleurone-less mature seeds. Conversely, dominant and recessive higher-order NAC mutants led to delayed endosperm elimination and impaired cell corpse clearance. Promoting PCD in the zhoupi mutant partially rescued its embryo growth defects, while the endosperm in a zhoupi nac higher-order mutant persisted until seed desiccation. These data suggest that a combination of cell wall weakening and PCD jointly facilitates embryo invasion by an active auto-elimination of endosperm cells.
Assuntos
Arabidopsis , Endosperma , Endosperma/genética , Arabidopsis/genética , Fatores de Transcrição/genética , Morte Celular , Apoptose , Fatores de Transcrição Hélice-Alça-Hélice BásicosRESUMO
The phytohormone auxin triggers root growth inhibition within seconds via a non-transcriptional pathway. Among members of the TIR1/AFB auxin receptor family, AFB1 has a primary role in this rapid response. However, the unique features that confer this specific function have not been identified. Here we show that the N-terminal region of AFB1, including the F-box domain and residues that contribute to auxin binding, is essential and sufficient for its specific role in the rapid response. Substitution of the N-terminal region of AFB1 with that of TIR1 disrupts its distinct cytoplasm-enriched localization and activity in rapid root growth inhibition by auxin. Importantly, the N-terminal region of AFB1 is indispensable for auxin-triggered calcium influx, which is a prerequisite for rapid root growth inhibition. Furthermore, AFB1 negatively regulates lateral root formation and transcription of auxin-induced genes, suggesting that it plays an inhibitory role in canonical auxin signaling. These results suggest that AFB1 may buffer the transcriptional auxin response, whereas it regulates rapid changes in cell growth that contribute to root gravitropism.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas F-Box , Arabidopsis/metabolismo , Ácidos Indolacéticos/farmacologia , Ácidos Indolacéticos/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas F-Box/metabolismo , Raízes de Plantas/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Citosol/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
Plant roots navigate in the soil environment following the gravity vector. Cell divisions in the meristem and rapid cell growth in the elongation zone propel the root tips through the soil. Actively elongating cells acidify their apoplast to enable cell wall extension by the activity of plasma membrane AHA H+-ATPases. The phytohormone auxin, central regulator of gravitropic response and root development, inhibits root cell growth, likely by rising the pH of the apoplast. However, the role of auxin in the regulation of the apoplastic pH gradient along the root tip is unclear. Here, we show, by using an improved method for visualization and quantification of root surface pH, that the Arabidopsis thaliana root surface pH shows distinct acidic and alkaline zones, which are not primarily determined by the activity of AHA H+-ATPases. Instead, the distinct domain of alkaline pH in the root transition zone is controlled by a rapid auxin response module, consisting of the AUX1 auxin influx carrier, the AFB1 auxin co-receptor, and the CNCG14 calcium channel. We demonstrate that the rapid auxin response pathway is required for an efficient navigation of the root tip.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Raízes de Plantas , Arabidopsis/metabolismo , Ácidos Indolacéticos/metabolismo , Concentração de Íons de Hidrogênio , Solo , Adenosina Trifosfatases/metabolismo , Regulação da Expressão Gênica de Plantas , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismoRESUMO
In vivo microscopy of plants with high-frequency imaging allows observation and characterization of the dynamic responses of plants to stimuli. It provides access to responses that could not be observed by imaging at a given time point. Such methods are particularly suitable for the observation of fast cellular events such as membrane potential changes. Classical measurement of membrane potential by probe impaling gives quantitative and precise measurements. However, it is invasive, requires specialized equipment, and only allows measurement of one cell at a time. To circumvent some of these limitations, we developed a method to relatively quantify membrane potential variations in Arabidopsis thaliana roots using the fluorescence of the voltage reporter DISBAC2(3). In this protocol, we describe how to prepare experiments for agar media and microfluidics, and we detail the image analysis. We take an example of the rapid plasma membrane depolarization induced by the phytohormone auxin to illustrate the method. Relative membrane potential measurements using DISBAC2(3) fluorescence increase the spatio-temporal resolution of the measurements and are non-invasive and suitable for live imaging of growing roots. Studying membrane potential with a more flexible method allows to efficiently combine mature electrophysiology literature and new molecular knowledge to achieve a better understanding of plant behaviors. Key features Non-invasive method to relatively quantify membrane potential in plant roots. Method suitable for imaging seedlings root in agar or liquid medium. Straightforward quantification.
RESUMO
The phytohormone auxin triggers root growth inhibition within seconds via a non-transcriptional pathway. Among members of the TIR1/AFBs auxin receptor family, AFB1 has a primary role in this rapid response. However, the unique features that confer this specific function have not been identified. Here we show that the N-terminal region of AFB1, including the F-box domain and residues that contribute to auxin binding, are essential and sufficient for its specific role in the rapid response. Substitution of the N-terminal region of AFB1 with that of TIR1 disrupts its distinct cytoplasm-enriched localization and activity in rapid root growth inhibition. Importantly, the N-terminal region of AFB1 is indispensable for auxin-triggered calcium influx which is a prerequisite for rapid root growth inhibition. Furthermore, AFB1 negatively regulates lateral root formation and transcription of auxin-induced genes, suggesting that it plays an inhibitory role in canonical auxin signaling. These results suggest that AFB1 may buffer the transcriptional auxin response while it regulates rapid changes in cell growth that contribute to root gravitropism.
RESUMO
Genomic imprinting promotes differential expression of parental alleles in the endosperm of flowering plants and is regulated by epigenetic modification such as DNA methylation and histone tail modifications in chromatin. After fertilization, the endosperm develops through a syncytial stage before it cellularizes and becomes a nutrient source for the growing embryo. Regional compartmentalization has been shown both in early and late endosperm development, and different transcriptional domains suggest divergent spatial and temporal regional functions. The analysis of the role of parent-of-origin allelic expression in the endosperm as a whole and the investigation of domain-specific functions have been hampered by the inaccessibility of the tissue for high-throughput transcriptome analyses and contamination from surrounding tissue. Here, we used fluorescence-activated nuclear sorting (FANS) of nuclear targeted GFP fluorescent genetic markers to capture parental-specific allelic expression from different developmental stages and specific endosperm domains. This approach allowed us to successfully identify differential genomic imprinting with temporal and spatial resolution. We used a systematic approach to report temporal regulation of imprinted genes in the endosperm, as well as region-specific imprinting in endosperm domains. Analysis of our data identified loci that are spatially differentially imprinted in one domain of the endosperm, while biparentally expressed in other domains. These findings suggest that the regulation of genomic imprinting is dynamic and challenge the canonical mechanisms for genomic imprinting.
Assuntos
Metilação de DNA , Endosperma , Endosperma/genética , Endosperma/metabolismo , Alelos , Metilação de DNA/genética , Impressão Genômica/genética , Epigênese Genética , Regulação da Expressão Gênica de PlantasRESUMO
Potassium ion (K+) plays a critical role as an essential electrolyte in all biological systems. Genetically-encoded fluorescent K+ biosensors are promising tools to further improve our understanding of K+-dependent processes under normal and pathological conditions. Here, we report the crystal structure of a previously reported genetically-encoded fluorescent K+ biosensor, GINKO1, in the K+-bound state. Using structure-guided optimization and directed evolution, we have engineered an improved K+ biosensor, designated GINKO2, with higher sensitivity and specificity. We have demonstrated the utility of GINKO2 for in vivo detection and imaging of K+ dynamics in multiple model organisms, including bacteria, plants, and mice.
Assuntos
Técnicas Biossensoriais , Transferência Ressonante de Energia de Fluorescência , Animais , Técnicas Biossensoriais/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Íons , Camundongos , PotássioRESUMO
The ability of plants to sense and orient their root growth towards gravity is studied in many laboratories. It is known that manual analysis of image data is subjected to human bias. Several semi-automated tools are available for analysing images from flatbed scanners, but there is no solution to automatically measure root bending angle over time for vertical-stage microscopy images. To address these problems, we developed ACORBA, which is an automated software that can measure root bending angle over time from vertical-stage microscope and flatbed scanner images. ACORBA also has a semi-automated mode for camera or stereomicroscope images. It represents a flexible approach based on both traditional image processing and deep machine learning segmentation to measure root angle progression over time. As the software is automated, it limits human interactions and is reproducible. ACORBA will support the plant biologist community by reducing labour and increasing reproducibility of image analysis of root gravitropism.
RESUMO
The membrane potential reflects the difference between cytoplasmic and apoplastic electrical potentials and is essential for cellular operation. The application of the phytohormone auxin (3-indoleacetic acid (IAA)) causes instantaneous membrane depolarization in various cell types1-6, making depolarization a hallmark of IAA-induced rapid responses. In root hairs, depolarization requires functional IAA transport and TIR1-AFB signalling5, but its physiological importance is not understood. Specifically in roots, auxin triggers rapid growth inhibition7-9 (RGI), a process required for gravitropic bending. RGI is initiated by the TIR1-AFB co-receptors, with the AFB1 paralogue playing a crucial role10,11. The nature of the underlying rapid signalling is unknown, as well as the molecular machinery executing it. Even though the growth and depolarization responses to auxin show remarkable similarities, the importance of membrane depolarization for root growth inhibition and gravitropism is unclear. Here, by combining the DISBAC2(3) voltage sensor with microfluidics and vertical-stage microscopy, we show that rapid auxin-induced membrane depolarization tightly correlates with RGI. Rapid depolarization and RGI require the AFB1 auxin co-receptor. Finally, AFB1 is essential for the rapid formation of the membrane depolarization gradient across the gravistimulated root. These results clarify the role of AFB1 as the central receptor for rapid auxin responses.
Assuntos
Proteínas de Arabidopsis/metabolismo , Membrana Celular/efeitos dos fármacos , Gravitropismo/efeitos dos fármacos , Ácidos Indolacéticos/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Raízes de Plantas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas , Variação Genética , Genótipo , Potenciais da Membrana/fisiologia , Plantas Geneticamente Modificadas/metabolismoRESUMO
Brassinosteroid (BR) hormones are indispensable for root growth and control both cell division and cell elongation through the establishment of an increasing signalling gradient along the longitudinal root axis. Because of their limited mobility, the importance of BR distribution in achieving a signalling maximum is largely overlooked. Expression pattern analysis of all known BR biosynthetic enzymes revealed that not all cells in the Arabidopsis thaliana root possess full biosynthetic machinery, and that completion of biosynthesis relies on cell-to-cell movement of hormone precursors. We demonstrate that BR biosynthesis is largely restricted to the root elongation zone, where it overlaps with BR signalling maxima. Moreover, optimal root growth requires hormone concentrations to be low in the meristem and high in the root elongation zone, attributable to increased biosynthesis. Our finding that spatiotemporal regulation of hormone synthesis results in local hormone accumulation provides a paradigm for hormone-driven organ growth in the absence of long-distance hormone transport in plants.
Assuntos
Brassinosteroides/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Brassinosteroides/biossíntese , Regulação da Expressão Gênica de Plantas , Meristema/metabolismo , Redes e Vias Metabólicas , Reguladores de Crescimento de Plantas/fisiologia , Raízes de Plantas/metabolismoRESUMO
Polar subcellular localization of the PIN exporters of the phytohormone auxin is a key determinant of directional, intercellular auxin transport and thus a central topic of both plant cell and developmental biology. Arabidopsis mutants lacking PID, a kinase that phosphorylates PINs, or the MAB4/MEL proteins of unknown molecular function display PIN polarity defects and phenocopy pin mutants, but mechanistic insights into how these factors convey PIN polarity are missing. Here, by combining protein biochemistry with quantitative live-cell imaging, we demonstrate that PINs, MAB4/MELs, and AGC kinases interact in the same complex at the plasma membrane. MAB4/MELs are recruited to the plasma membrane by the PINs and in concert with the AGC kinases maintain PIN polarity through limiting lateral diffusion-based escape of PINs from the polar domain. The PIN-MAB4/MEL-PID protein complex has self-reinforcing properties thanks to positive feedback between AGC kinase-mediated PIN phosphorylation and MAB4/MEL recruitment. We thus uncover the molecular mechanism by which AGC kinases and MAB4/MEL proteins regulate PIN localization and plant development.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Polaridade Celular , Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos , Proteínas de Membrana Transportadoras/genética , Células Vegetais/metabolismo , Raízes de Plantas/metabolismoRESUMO
Auxin regulates the transcription of auxin-responsive genes by the TIR1/AFBs-Aux/IAA-ARF signaling pathway, and in this way facilitates plant growth and development. However, rapid, nontranscriptional responses to auxin that cannot be explained by this pathway have been reported. In this review, we focus on several examples of rapid auxin responses: (1) the triggering of changes in plasma membrane potential in various plant species and tissues, (2) inhibition of root growth, which also correlates with membrane potential changes, cytosolic Ca2+ spikes, and a rise of apoplastic pH, (3) the influence on endomembrane trafficking of PIN proteins and other membrane cargoes, and (4) activation of ROPs (Rho of plants) and their downstream effectors such as the cytoskeleton or vesicle trafficking. In most cases, the signaling pathway triggering the response is poorly understood. A role for the TIR1/AFBs in rapid root growth regulation is emerging, as well as the involvement of transmembrane kinases (TMKs) in the activation of ROPs. We discuss similarities and differences among these rapid responses and focus on their physiological significance, which remains an enigma in most cases.
Assuntos
Ácidos Indolacéticos/metabolismo , Plantas/metabolismo , Cálcio/metabolismo , Endocitose , Proteínas de Ligação ao GTP/metabolismo , Potenciais da Membrana , Proteínas de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Receptores de Superfície Celular/metabolismoRESUMO
Auxin is a major plant growth regulator, but current models on auxin perception and signaling cannot explain the whole plethora of auxin effects, in particular those associated with rapid responses. A possible candidate for a component of additional auxin perception mechanisms is the AUXIN BINDING PROTEIN 1 (ABP1), whose function in planta remains unclear. Here we combined expression analysis with gain- and loss-of-function approaches to analyze the role of ABP1 in plant development. ABP1 shows a broad expression largely overlapping with, but not regulated by, transcriptional auxin response activity. Furthermore, ABP1 activity is not essential for the transcriptional auxin signaling. Genetic in planta analysis revealed that abp1 loss-of-function mutants show largely normal development with minor defects in bolting. On the other hand, ABP1 gain-of-function alleles show a broad range of growth and developmental defects, including root and hypocotyl growth and bending, lateral root and leaf development, bolting, as well as response to heat stress. At the cellular level, ABP1 gain-of-function leads to impaired auxin effect on PIN polar distribution and affects BFA-sensitive PIN intracellular aggregation. The gain-of-function analysis suggests a broad, but still mechanistically unclear involvement of ABP1 in plant development, possibly masked in abp1 loss-of-function mutants by a functional redundancy.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/crescimento & desenvolvimento , Proteínas de Plantas/fisiologia , Receptores de Superfície Celular/fisiologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Hipocótilo/crescimento & desenvolvimento , Hipocótilo/metabolismo , Ácidos Indolacéticos/metabolismo , Microscopia Confocal , Reguladores de Crescimento de Plantas/metabolismo , Reguladores de Crescimento de Plantas/fisiologia , Proteínas de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Protoplastos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Superfície Celular/metabolismoRESUMO
Cell polarity is crucial for the coordinated development of all multicellular organisms. In plants, this is exemplified by the PIN-FORMED (PIN) efflux carriers of the phytohormone auxin: The polar subcellular localization of the PINs is instructive to the directional intercellular auxin transport, and thus to a plethora of auxin-regulated growth and developmental processes. Despite its importance, the regulation of PIN polar subcellular localization remains poorly understood. Here, we have employed advanced live-cell imaging techniques to study the roles of microtubules and actin microfilaments in the establishment of apical polar localization of PIN2 in the epidermis of the Arabidopsis root meristem. We report that apical PIN2 polarity requires neither intact actin microfilaments nor microtubules, suggesting that the primary spatial cue for polar PIN distribution is likely independent of cytoskeleton-guided endomembrane trafficking.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Citoesqueleto/metabolismo , Arabidopsis/citologia , Espaço Intracelular/metabolismo , Transporte ProteicoRESUMO
Cell polarity, manifested by the localization of proteins to distinct polar plasma membrane domains, is a key prerequisite of multicellular life. In plants, PIN auxin transporters are prominent polarity markers crucial for a plethora of developmental processes. Cell polarity mechanisms in plants are distinct from other eukaryotes and still largely elusive. In particular, how the cell polarities are propagated and maintained following cell division remains unknown. Plant cytokinesis is orchestrated by the cell plate-a transient centrifugally growing endomembrane compartment ultimately forming the cross wall1. Trafficking of polar membrane proteins is typically redirected to the cell plate, and these will consequently have opposite polarity in at least one of the daughter cells2-5. Here, we provide mechanistic insights into post-cytokinetic re-establishment of cell polarity as manifested by the apical, polar localization of PIN2. We show that the apical domain is defined in a cell-intrinsic manner and that re-establishment of PIN2 localization to this domain requires de novo protein secretion and endocytosis, but not basal-to-apical transcytosis. Furthermore, we identify a PINOID-related kinase WAG1, which phosphorylates PIN2 in vitro6 and is transcriptionally upregulated specifically in dividing cells, as a crucial regulator of post-cytokinetic PIN2 polarity re-establishment.