RESUMEN
Zinc (Zn) is an essential micronutrient but can be cytotoxic when present in excess. Plants have evolved mechanisms to tolerate Zn toxicity. To identify genetic loci responsible for natural variation of plant tolerance to Zn toxicity, we conduct genome-wide association studies for root growth responses to high Zn and identify 21 significant associated loci. Among these loci, we identify Trichome Birefringence (TBR) allelic variation determining root growth variation in high Zn conditions. Natural alleles of TBR determine TBR transcript and protein levels which affect pectin methylesterification in root cell walls. Together with previously published data showing that pectin methylesterification increase goes along with decreased Zn binding to cell walls in TBR mutants, our findings lead to a model in which TBR allelic variation enables Zn tolerance through modulating root cell wall pectin methylesterification. The role of TBR in Zn tolerance is conserved across dicot and monocot plant species.
Asunto(s)
Arabidopsis , Pared Celular , Regulación de la Expresión Génica de las Plantas , Pectinas , Raíces de Plantas , Zinc , Pared Celular/metabolismo , Pared Celular/efectos de los fármacos , Pared Celular/genética , Raíces de Plantas/metabolismo , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/genética , Zinc/metabolismo , Zinc/toxicidad , Pectinas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Esterificación , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Estudio de Asociación del Genoma Completo , Alelos , Variación GenéticaRESUMEN
Iron is critical during host-microorganism interactions1-4. Restriction of available iron by the host during infection is an important defence strategy, described as nutritional immunity5. However, this poses a conundrum for externally facing, absorptive tissues such as the gut epithelium or the plant root epidermis that generate environments that favour iron bioavailability. For example, plant roots acquire iron mostly from the soil and, when iron deficient, increase iron availability through mechanisms that include rhizosphere acidification and secretion of iron chelators6-9. Yet, the elevated iron bioavailability would also be beneficial for the growth of bacteria that threaten plant health. Here we report that microorganism-associated molecular patterns such as flagellin lead to suppression of root iron acquisition through a localized degradation of the systemic iron-deficiency signalling peptide Iron Man 1 (IMA1) in Arabidopsis thaliana. This response is also elicited when bacteria enter root tissues, but not when they dwell on the outer root surface. IMA1 itself has a role in modulating immunity in root and shoot, affecting the levels of root colonization and the resistance to a bacterial foliar pathogen. Our findings reveal an adaptive molecular mechanism of nutritional immunity that affects iron bioavailability and uptake, as well as immune responses.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Bacterias , Péptidos y Proteínas de Señalización Intracelular , Hierro , Moléculas de Patrón Molecular Asociado a Patógenos , Raíces de Plantas , Arabidopsis/inmunología , Arabidopsis/metabolismo , Arabidopsis/microbiología , Proteínas de Arabidopsis/metabolismo , Bacterias/inmunología , Bacterias/metabolismo , Flagelina/inmunología , Regulación de la Expresión Génica de las Plantas , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Hierro/metabolismo , Inmunidad de la Planta , Raíces de Plantas/inmunología , Raíces de Plantas/metabolismo , Raíces de Plantas/microbiología , Brotes de la Planta/inmunología , Brotes de la Planta/metabolismo , Brotes de la Planta/microbiología , Rizosfera , Moléculas de Patrón Molecular Asociado a Patógenos/inmunología , Moléculas de Patrón Molecular Asociado a Patógenos/metabolismoRESUMEN
Plants are sessile organisms that constantly adapt to their changing environment. The root is exposed to numerous environmental signals ranging from nutrients and water to microbial molecular patterns. These signals can trigger distinct responses including the rapid increase or decrease of root growth. Consequently, using root growth as a readout for signal perception can help decipher which external cues are perceived by roots, and how these signals are integrated. To date, studies measuring root growth responses using large numbers of roots have been limited by a lack of high-throughput image acquisition, poor scalability of analytical methods, or low spatiotemporal resolution. Here, we developed the Root Walker pipeline, which uses automated microscopes to acquire time-series images of many roots exposed to controlled treatments with high spatiotemporal resolution, in conjunction with fast and automated image analysis software. We demonstrate the power of Root Walker by quantifying root growth rate responses at different time and throughput scales upon treatment with natural auxin and two mitogen-associated protein kinase cascade inhibitors. We find a concentration-dependent root growth response to auxin and reveal the specificity of one MAPK inhibitor. We further demonstrate the ability of Root Walker to conduct genetic screens by performing a genome-wide association study on 260 accessions in under 2 weeks, revealing known and unknown root growth regulators. Root Walker promises to be a useful toolkit for the plant science community, allowing large-scale screening of root growth dynamics for a variety of purposes, including genetic screens for root sensing and root growth response mechanisms.
Asunto(s)
Estudio de Asociación del Genoma Completo , Raíces de Plantas , Raíces de Plantas/metabolismo , Ácidos Indolacéticos/metabolismo , Transducción de Señal , Procesamiento de Imagen Asistido por Computador/métodosRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Proteínas F-Box , Arabidopsis/metabolismo , Ácidos Indolacéticos/farmacología , Ácidos Indolacéticos/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas F-Box/metabolismo , Raíces de Plantas/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Citosol/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
Brassinosteroids (BRs) are steroidal phytohormones that are essential for plant growth, development and adaptation to environmental stresses. BRs act in a dose-dependent manner and do not travel over long distances; hence, BR homeostasis maintenance is critical for their function. Biosynthesis of bioactive BRs relies on the cell-to-cell movement of hormone precursors. However, the mechanism of the short-distance BR transport is unknown, and its contribution to the control of endogenous BR levels remains unexplored. Here we demonstrate that plasmodesmata (PD) mediate the passage of BRs between neighboring cells. Intracellular BR content, in turn, is capable of modulating PD permeability to optimize its own mobility, thereby manipulating BR biosynthesis and signaling. Our work uncovers a thus far unknown mode of steroid transport in eukaryotes and exposes an additional layer of BR homeostasis regulation in plants.
Asunto(s)
Proteínas de Arabidopsis , Brasinoesteroides , Plasmodesmos/metabolismo , Reguladores del Crecimiento de las Plantas , Plantas/metabolismo , Hormonas , Regulación de la Expresión Génica de las Plantas , Proteínas de Arabidopsis/metabolismoRESUMEN
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.
RESUMEN
Iron is critical for host-pathogen interactions. While pathogens seek to scavenge iron to spread, the host aims at decreasing iron availability to reduce pathogen virulence. Thus, iron sensing and homeostasis are of particular importance to prevent host infection and part of nutritional immunity. While the link between iron homeostasis and immunity pathways is well established in plants, how iron levels are sensed and integrated with immune response pathways remains unknown. Here we report a receptor kinase SRF3, with a role in coordinating root growth, iron homeostasis and immunity pathways via regulation of callose synthases. These processes are modulated by iron levels and rely on SRF3 extracellular and kinase domains which tune its accumulation and partitioning at the cell surface. Mimicking bacterial elicitation with the flagellin peptide flg22 phenocopies SRF3 regulation upon low iron levels and subsequent SRF3-dependent responses. We propose that SRF3 is part of nutritional immunity responses involved in sensing external iron levels.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Flagelina/metabolismo , Hierro/metabolismo , Proteínas Quinasas/metabolismoRESUMEN
A main characteristic of sphingolipids is the presence of a very long chain fatty acid (VLCFA) whose function in cellular processes is not yet fully understood. VLCFAs of sphingolipids are involved in the intracellular traffic to the vacuole and the maturation of early endosomes into late endosomes is one of the major pathways for vacuolar traffic. Additionally, the anionic phospholipid phosphatidylinositol-3-phosphate (PtdIns (3)P or PI3P) is involved in protein sorting and recruitment of small GTPase effectors at late endosomes/multivesicular bodies (MVBs) during vacuolar trafficking. In contrast to animal cells, PI3P mainly localizes to late endosomes in plant cells and to a minor extent to a discrete sub-domain of the plant's early endosome (EE)/trans-Golgi network (TGN) where the endosomal maturation occurs. However, the mechanisms that control the relative levels of PI3P between TGN and MVBs are unknown. Using metazachlor, an inhibitor of VLCFA synthesis, we found that VLCFAs are involved in the TGN/MVB distribution of PI3P. This effect is independent from either synthesis of PI3P by PI3-kinase or degradation of PI(3,5)P2 into PI3P by the SUPPRESSOR OF ACTIN1 (SAC1) phosphatase. Using high-resolution live cell imaging microscopy, we detected transient associations between TGNs and MVBs but VLCFAs are not involved in those interactions. Nonetheless, our results suggest that PI3P might be transferable from TGN to MVBs and that VLCFAs act in this process.
Asunto(s)
Arabidopsis/metabolismo , Endosomas/metabolismo , Ácidos Grasos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Vías Biosintéticas , Esfingolípidos/metabolismo , Red trans-Golgi/metabolismoRESUMEN
Lipids are major components of membranes with pleiotropic roles and interconnected metabolism, so experimentally addressing the primary function of individual lipid species in vivo can be difficult. Genetic approaches are particularly challenging to interpret due to compensatory mechanisms and indirect effects. Here, we describe a fast inducible approach to complement the phenotypes of Arabidopsis lipid mutants through exogenous treatment with the depleted lipid, followed by live confocal imaging to observe genetically encoded lipid sensors in wild-type and mutant root tissues. For complete details on the use and execution of this protocol, please refer to Platre et al. (2018).
Asunto(s)
Arabidopsis/crecimiento & desarrollo , Lípidos/análisis , Lisofosfolípidos/farmacología , Mutación , Plantones/efectos de los fármacos , Arabidopsis/genética , Genes de Plantas , Microscopía Confocal , Raíces de Plantas/metabolismo , Plantones/metabolismoRESUMEN
The lipid composition of organelles acts as a landmark to define membrane identity and specify subcellular function. Phosphoinositides are anionic lipids acting in protein sorting and trafficking at the trans-Golgi network (TGN). In animal cells, sphingolipids control the turnover of phosphoinositides through lipid exchange mechanisms at endoplasmic reticulum/TGN contact sites. In this study, we discover a mechanism for how sphingolipids mediate phosphoinositide homeostasis at the TGN in plant cells. Using multiple approaches, we show that a reduction of the acyl-chain length of sphingolipids results in an increased level of phosphatidylinositol-4-phosphate (PtdIns(4)P or PI4P) at the TGN but not of other lipids usually coupled to PI4P during exchange mechanisms. We show that sphingolipids mediate Phospholipase C (PLC)-driven consumption of PI4P at the TGN rather than local PI4P synthesis and that this mechanism is involved in the polar sorting of the auxin efflux carrier PIN2 at the TGN. Together, our data identify a mode of action of sphingolipids in lipid interplay at the TGN during protein sorting.
Asunto(s)
Fosfatidilinositoles/metabolismo , Esfingolípidos/metabolismo , Red trans-Golgi/metabolismo , Animales , Retículo Endoplásmico/metabolismo , Humanos , Fosfatidilinositoles/genética , Esfingolípidos/genética , Fosfolipasas de Tipo C/metabolismo , Red trans-Golgi/genéticaRESUMEN
Super-resolution microscopy techniques have pushed the limit of optical imaging to unprecedented spatial resolutions. However, one of the frontiers in nanoscopy is its application to intact living organisms. Here we describe the implementation and application of super-resolution single-particle tracking photoactivated localization microscopy (sptPALM) to probe single-molecule dynamics of membrane proteins in live roots of the model plant Arabidopsis thaliana. We first discuss the advantages and limitations of sptPALM for studying the diffusion properties of membrane proteins and compare this to fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS). We describe the technical details for handling and imaging the samples for sptPALM, with a particular emphasis on the specificity of imaging plant cells, such as their thick cell walls or high degree of autofluorescence. We then provide a practical guide from data collection to image analyses. In particular, we introduce our sptPALM_viewer software and describe how to install and use it for analyzing sptPALM experiments. Finally, we report an R statistical analysis pipeline to analyze and compare sptPALM experiments. Altogether, this protocol should enable plant researchers to perform sptPALM using a benchmarked reproducible protocol. Routinely, the procedure takes 3-4 h of imaging followed by 3-4 d of image processing and data analysis.
Asunto(s)
Proteínas de la Membrana/metabolismo , Microscopía Fluorescente/métodos , Imagen Individual de Molécula/métodos , Arabidopsis/metabolismo , Difusión , Recuperación de Fluorescencia tras Fotoblanqueo/métodos , Proteínas de la Membrana/aislamiento & purificación , Imagen Óptica/métodos , Células Vegetales/química , Plantas/química , Plantas/metabolismo , Espectrometría de Fluorescencia/métodosRESUMEN
Plants are sessile organisms which adapt to their everchanging environment. The root is buried is the soil and continuously explores its surroundings. Indeed, while growing downwards to anchor the plants in the ground, it has to avoid obstacles and seek for nutrients and water. This seeking mechanism depends on the root perception of gravity. Through differential growth, the root is able to align according to the gravity vector. The growth is regulated at the cellular level by an increase of the plant hormone auxin, which activates the small Rho Guanine triphosphatase (Rho GTPase) of plant 6 (ROP6) at the plasma membrane to inhibit endocytosis and trigger cytoskeleton reorganization. Through a collaborative work, four French laboratories addressed the question of ROP6 membrane dynamics upon gravistimulation. Based on cellular biology, biochemistry and super resolution imaging approaches, they discovered that ROP6 is organized into nanoclusters at the plasma membrane of plant cells in response to auxin. The stabilization of ROP6 in these nanoclusters is required for signaling and thus the regulation of gravitropic bending. The formation of these nanoclusters is dependent upon the membrane lipid phosphatidylserine, which directly interact with ROP6. Using a genetic toolkit, the authors uncovered that phosphatidylserine is rate limiting for the ROP6-dependent nanocluster formation, which in turn tunes the cellular read outs. This work, not only explain the fine mechanism of the root response to gravity from the developmental level to the nanoscale but also provide a valuable insight towards the understanding of small GTPase signaling in eukaryotic system.
Les plantes sont des organismes sessiles qui nécessitent de s'adapter constamment à leur environnement. La racine qui se trouve enfouie dans la terre doit explorer le sol pour remplir ses fonctions. En effet, elle doit être capable de pousser vers le bas pour donner un point d'ancrage mais aussi de capturer l'eau et les nutriments tout en évitant les obstacles. Ce jeu d'exploration est possible du fait que la racine est capable de percevoir la gravité. C'est par un mécanisme de croissance différentielle qu'elle adapte sa croissance en fonction du vecteur de gravité. Au niveau cellulaire, la croissance est régulée par l'augmentation de l'hormone auxine qui active au niveau de la membrane plasmique la Rho Guanine triphosphatase (Rho GTPase) of plant 6 (ROP6) et déclenche l'inhibition de l'endocytose et réorganise le cytosquelette. À travers un travail collaboratif, quatre équipes françaises se sont attelées à étudier la dynamique membranaire de ROP6 lors de la réponse à la gravité. Alliant des expertises de biologie cellulaire, de biochimie et d'imagerie de super résolution, elles ont mis en lumière la nécessité de ROP6 à s'organiser en nanoclusters via des interactions électrostatiques avec un lipide, la phosphatidylsérine, pour promouvoir sa fonction. De plus, par une approche génétique, ils ont démontré que la signalisation dépendante de ROP6 était proportionnellement corrélée à la quantité de phosphatidylsérine présente à la membrane plasmique, via la modulation du nombre de nanoclusters. Ces travaux mettent non seulement en avant le mécanisme précis permettant l'adaptation de la racine à son environnement mais représentent aussi une avancée majeure à la compréhension générale de la régulation des GTPases chez les eucaryotes.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Unión al GTP Monoméricas , Gravitación , Ácidos Indolacéticos , Proteínas de Unión al GTP Monoméricas/metabolismo , Raíces de PlantasRESUMEN
Plants are able to orient their growth according to gravity, which ultimately controls both shoot and root architecture.1 Gravitropism is a dynamic process whereby gravistimulation induces the asymmetric distribution of the plant hormone auxin, leading to asymmetric growth, organ bending, and subsequent reset of auxin distribution back to the original pre-gravistimulation situation.1-3 Differential auxin accumulation during the gravitropic response depends on the activity of polarly localized PIN-FORMED (PIN) auxin-efflux carriers.1-4 In particular, the timing of this dynamic response is regulated by PIN2,5,6 but the underlying molecular mechanisms are poorly understood. Here, we show that MEMBRANE ASSOCIATED KINASE REGULATOR2 (MAKR2) controls the pace of the root gravitropic response. We found that MAKR2 is required for the PIN2 asymmetry during gravitropism by acting as a negative regulator of the cell-surface signaling mediated by the receptor-like kinase TRANSMEMBRANE KINASE1 (TMK1).2,7-10 Furthermore, we show that the MAKR2 inhibitory effect on TMK1 signaling is antagonized by auxin itself, which triggers rapid MAKR2 membrane dissociation in a TMK1-dependent manner. Our findings suggest that the timing of the root gravitropic response is orchestrated by the reversible inhibition of the TMK1 signaling pathway at the cell surface.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Gravitropismo/fisiología , Ácidos Indolacéticos/metabolismo , Proteínas de la Membrana/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Arabidopsis/antagonistas & inhibidores , Proteínas de Arabidopsis/genética , Mutación con Ganancia de Función , Gravitación , Mutación con Pérdida de Función , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Plantas Modificadas Genéticamente , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal/fisiologíaRESUMEN
Temperature is one of the most impactful environmental factors to which plants adjust their growth and development. Although the regulation of temperature signaling has been extensively investigated for the aerial part of plants, much less is known and understood about how roots sense and modulate their growth in response to fluctuating temperatures. Here, we found that shoot and root growth responses to high ambient temperature are coordinated during early seedling development in Arabidopsis A shoot signaling module that includes HY5, the phytochromes and the PIFs exerts a central function in coupling these growth responses and maintaining auxin levels in the root. In addition to the HY5/PIF-dependent shoot module, a regulatory axis composed of auxin biosynthesis and auxin perception factors controls root responses to high ambient temperature. Taken together, our findings show that shoot and root developmental responses to temperature are tightly coupled during thermomorphogenesis and suggest that roots integrate energy signals with local hormonal inputs.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Morfogénesis/genética , Organogénesis de las Plantas/genética , Arabidopsis/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas/genética , Ácidos Indolacéticos/metabolismo , Fitocromo/genética , Brotes de la Planta/genética , Brotes de la Planta/crecimiento & desarrollo , Transducción de SeñalRESUMEN
In the course of their growth and development, plants have to constantly perceive and react to their environment. This is achieved in cells by the coordination of complex combinatorial signaling networks. However, how signal integration and specificity are achieved in this context is unknown. With a focus on the hyperosmotic stimulus, we use live super-resolution light imaging methods to demonstrate that a Rho GTPase, Rho-of-Plant 6 (ROP6), forms stimuli-dependent nanodomains within the plasma membrane (PM). These nanodomains are necessary and sufficient to transduce production of reactive oxygen species (ROS) that act as secondary messengers and trigger several plant adaptive responses to osmotic constraints. Furthermore, osmotic signal triggers interaction between ROP6 and two NADPH oxidases that subsequently generate ROS. ROP6 nanoclustering is also needed for cell surface auxin signaling, but short-time auxin treatment does not induce ROS accumulation. We show that auxin-induced ROP6 nanodomains, unlike osmotically driven ROP6 clusters, do not recruit the NADPH oxidase, RBOHD. Together, our results suggest that Rho GTPase nano-partitioning at the PM ensures signal specificity downstream of independent stimuli.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Proteínas de Unión al GTP Monoméricas/metabolismo , Presión Osmótica/fisiología , Adaptación Fisiológica , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Ácidos Indolacéticos/metabolismo , Proteínas de Unión al GTP Monoméricas/genética , NADPH Oxidasas/metabolismo , Ósmosis/fisiología , Raíces de Plantas/citología , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Rho guanosine triphosphatases (GTPases) are master regulators of cell signaling, but how they are regulated depending on the cellular context is unclear. We found that the phospholipid phosphatidylserine acts as a developmentally controlled lipid rheostat that tunes Rho GTPase signaling in Arabidopsis Live superresolution single-molecule imaging revealed that the protein Rho of Plants 6 (ROP6) is stabilized by phosphatidylserine into plasma membrane nanodomains, which are required for auxin signaling. Our experiments also revealed that the plasma membrane phosphatidylserine content varies during plant root development and that the level of phosphatidylserine modulates the quantity of ROP6 nanoclusters induced by auxin and hence downstream signaling, including regulation of endocytosis and gravitropism. Our work shows that variations in phosphatidylserine levels are a physiological process that may be leveraged to regulate small GTPase signaling during development.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Arabidopsis/crecimiento & desarrollo , Proteínas de Unión al GTP Monoméricas/metabolismo , Fosfatidilserinas/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Membrana Celular/química , Membrana Celular/metabolismo , Endocitosis/genética , Regulación de la Expresión Génica de las Plantas , Gravitropismo/genética , Ácidos Indolacéticos/metabolismo , Proteínas de Unión al GTP Monoméricas/genética , Fosfatidilserinas/farmacología , Raíces de Plantas/enzimología , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Transducción de Señal , Imagen Individual de MoléculaRESUMEN
Membrane surface charge is critical for the transient, yet specific recruitment of proteins with polybasic regions to certain organelles. In eukaryotes, the plasma membrane (PM) is the most electronegative compartment of the cell, which specifies its identity. As such, membrane electrostatics is a central parameter in signaling, intracellular trafficking, and polarity. Here, we explore which are the lipids that control membrane electrostatics using plants as a model. We show that phosphatidylinositol-4-phosphate (PI4P), phosphatidic acidic (PA), and phosphatidylserine (PS) are separately required to generate the electrostatic signature of the plant PM. In addition, we reveal the existence of an electrostatic territory that is organized as a gradient along the endocytic pathway and is controlled by PS/PI4P combination. Altogether, we propose that combinatorial lipid composition of the cytosolic leaflet of organelles not only defines the electrostatic territory but also distinguishes different functional compartments within this territory by specifying their varying surface charges.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Membrana Celular/metabolismo , Ácidos Fosfatidicos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilserinas/metabolismo , Electricidad Estática , Arabidopsis/crecimiento & desarrollo , Orgánulos , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: In plants, the shoot apical meristem (SAM) has two main functions, involving the production of all aerial organs on the one hand and self-maintenance on the other, allowing the production of organs during the entire post-embryonic life of the plant. Transcription factors, microRNA, hormones, peptides and forces have been involved in meristem function. Whereas phosphatidylinositol phosphates (PIPs) have been involved in almost all biological functions, including stem cell maintenance and organogenesis in animals, the processes in meristem biology to which PIPs contribute still need to be delineated. RESULTS: Using biosensors for PI4P and PI(4,5)P2, the two most abundant PIPs at the plasma membrane, we reveal that meristem functions are associated with a stereotypical PIP tissue-scale pattern, with PI(4,5)P2 always displaying a more clear-cut pattern than PI4P. Using clavata3 and pin-formed1 mutants, we show that stem cell maintenance is associated with reduced levels of PIPs. In contrast, high PIP levels are signatures for organ-meristem boundaries. Interestingly, this pattern echoes that of cortical microtubules and stress anisotropy at the meristem. Using ablations and pharmacological approaches, we further show that PIP levels can be increased when the tensile stress pattern is altered. Conversely, we find that katanin mutant meristems, with increased isotropy of microtubule arrays and slower response to mechanical perturbations, exhibit reduced PIP gradients within the SAM. Comparable PIP pattern defects were observed in phospholipase A3ß overexpressor lines, which largely phenocopy katanin mutants at the whole plant level. CONCLUSIONS: Using phospholipid biosensors, we identified a stereotypical PIP accumulation pattern in the SAM that negatively correlates with stem cell maintenance and positively correlates with organ-boundary establishment. While other cues are very likely to contribute to the final PIP pattern, we provide evidence that the patterns of PIP, cortical microtubules and mechanical stress are positively correlated, suggesting that the PIP pattern, and its reproducibility, relies at least in part on the mechanical status of the SAM.
Asunto(s)
Proteínas de Arabidopsis/biosíntesis , Arabidopsis/metabolismo , Meristema/metabolismo , Fosfatos de Fosfatidilinositol/biosíntesis , Tallos de la Planta/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Arabidopsis/química , Arabidopsis/genética , Proteínas de Arabidopsis/análisis , Proteínas de Arabidopsis/genética , Técnicas Biosensibles/métodos , Meristema/química , Meristema/genética , Fosfatos de Fosfatidilinositol/análisis , Fosfatos de Fosfatidilinositol/genética , Tallos de la Planta/química , Tallos de la Planta/genética , Plantas Modificadas Genéticamente/química , Plantas Modificadas Genéticamente/genéticaRESUMEN
Plasma Membrane is the primary structure for adjusting to ever changing conditions. PM sub-compartmentalization in domains is thought to orchestrate signaling. Yet, mechanisms governing membrane organization are mostly uncharacterized. The plant-specific REMORINs are proteins regulating hormonal crosstalk and host invasion. REMs are the best-characterized nanodomain markers via an uncharacterized moiety called REMORIN C-terminal Anchor. By coupling biophysical methods, super-resolution microscopy and physiology, we decipher an original mechanism regulating the dynamic and organization of nanodomains. We showed that targeting of REMORIN is independent of the COP-II-dependent secretory pathway and mediated by PI4P and sterol. REM-CA is an unconventional lipid-binding motif that confers nanodomain organization. Analyses of REM-CA mutants by single particle tracking demonstrate that mobility and supramolecular organization are critical for immunity. This study provides a unique mechanistic insight into how the tight control of spatial segregation is critical in the definition of PM domain necessary to support biological function.