RESUMEN
Glutamate receptor-like channels (GLRs) play vital roles in various physiological processes in plants, such as wound response, stomatal aperture control, seed germination, root development, innate immune response, pollen tube growth, and morphogenesis. Despite the importance of GLRs, knowledge about their molecular organization is limited. Here we use X-ray crystallography and single-particle cryo-EM to solve structures of the Arabidopsis thaliana GLR3.4. Our structures reveal the tetrameric assembly of GLR3.4 subunits into a three-layer domain architecture, reminiscent of animal ionotropic glutamate receptors (iGluRs). However, the non-swapped arrangement between layers of GLR3.4 domains, binding of glutathione through S-glutathionylation of cysteine C205 inside the amino-terminal domain clamshell, unique symmetry, inter-domain interfaces, and ligand specificity distinguish GLR3.4 from representatives of the iGluR family and suggest distinct features of the GLR gating mechanism. Our work elaborates on the principles of GLR architecture and symmetry and provides a molecular template for deciphering GLR-dependent signaling mechanisms in plants.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Receptores de Glutamato/química , Receptores de Glutamato/metabolismo , Animales , Proteínas de Arabidopsis/genética , Sitios de Unión , Células COS , Calcio/metabolismo , Chlorocebus aethiops , Microscopía por Crioelectrón , Cristalografía por Rayos X , Cisteína/metabolismo , Glutatión/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Plantas Modificadas Genéticamente , Dominios Proteicos , Receptores de Glutamato/genéticaRESUMEN
Changes in cytosolic calcium (Ca2+) concentration are among the earliest reactions to a multitude of stress cues. While a plethora of Ca2+-permeable channels may generate distinct Ca2+ signatures and contribute to response specificities, the mechanisms by which Ca2+ signatures are decoded are poorly understood. Here, we developed a genetically encoded Förster resonance energy transfer (FRET)-based reporter that visualizes the conformational changes in Ca2+-dependent protein kinases (CDPKs/CPKs). We focused on two CDPKs with distinct Ca2+-sensitivities, highly Ca2+-sensitive Arabidopsis (Arabidopsis thaliana) AtCPK21 and rather Ca2+-insensitive AtCPK23, to report conformational changes accompanying kinase activation. In tobacco (Nicotiana tabacum) pollen tubes, which naturally display coordinated spatial and temporal Ca2+ fluctuations, CPK21-FRET, but not CPK23-FRET, reported oscillatory emission ratio changes mirroring cytosolic Ca2+ changes, pointing to the isoform-specific Ca2+-sensitivity and reversibility of the conformational change. In Arabidopsis guard cells, CPK21-FRET-monitored conformational dynamics suggest that CPK21 serves as a decoder of signal-specific Ca2+ signatures in response to abscisic acid and the flagellin peptide flg22. Based on these data, CDPK-FRET is a powerful approach for tackling real-time live-cell Ca2+ decoding in a multitude of plant developmental and stress responses.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Calcio/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , FlagelinaRESUMEN
Epigenetic inheritance is more widespread in plants than in mammals, in part because mammals erase epigenetic information by germline reprogramming. We sequenced the methylome of three haploid cell types from developing pollen: the sperm cell, the vegetative cell, and their precursor, the postmeiotic microspore, and found that unlike in mammals the plant germline retains CG and CHG DNA methylation. However, CHH methylation is lost from retrotransposons in microspores and sperm cells and restored by de novo DNA methyltransferase guided by 24 nt small interfering RNA, both in the vegetative nucleus and in the embryo after fertilization. In the vegetative nucleus, CG methylation is lost from targets of DEMETER (DME), REPRESSOR OF SILENCING 1 (ROS1), and their homologs, which include imprinted loci and recurrent epialleles that accumulate corresponding small RNA and are premethylated in sperm. Thus genome reprogramming in pollen contributes to epigenetic inheritance, transposon silencing, and imprinting, guided by small RNA.
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Arabidopsis/genética , Metilación de ADN , Epigénesis Genética , Polen/genética , ARN de Planta/genética , ARN Interferente Pequeño/genética , Animales , Arabidopsis/crecimiento & desarrollo , Elementos Transponibles de ADN , Mamíferos/genética , ARN de Planta/metabolismo , ARN Interferente Pequeño/metabolismo , Semillas/genética , Semillas/metabolismoRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Perception of biotic and abiotic stresses often leads to stomatal closure in plants1,2. Rapid influx of calcium ions (Ca2+) across the plasma membrane has an important role in this response, but the identity of the Ca2+ channels involved has remained elusive3,4. Here we report that the Arabidopsis thaliana Ca2+-permeable channel OSCA1.3 controls stomatal closure during immune signalling. OSCA1.3 is rapidly phosphorylated upon perception of pathogen-associated molecular patterns (PAMPs). Biochemical and quantitative phosphoproteomics analyses reveal that the immune receptor-associated cytosolic kinase BIK1 interacts with and phosphorylates the N-terminal cytosolic loop of OSCA1.3 within minutes of treatment with the peptidic PAMP flg22, which is derived from bacterial flagellin. Genetic and electrophysiological data reveal that OSCA1.3 is permeable to Ca2+, and that BIK1-mediated phosphorylation on its N terminus increases this channel activity. Notably, OSCA1.3 and its phosphorylation by BIK1 are critical for stomatal closure during immune signalling, and OSCA1.3 does not regulate stomatal closure upon perception of abscisic acid-a plant hormone associated with abiotic stresses. This study thus identifies a plant Ca2+ channel and its activation mechanisms underlying stomatal closure during immune signalling, and suggests specificity in Ca2+ influx mechanisms in response to different stresses.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/inmunología , Arabidopsis/metabolismo , Canales de Calcio/metabolismo , Calcio/metabolismo , Inmunidad de la Planta , Estomas de Plantas/inmunología , Estomas de Plantas/metabolismo , Ácido Abscísico/metabolismo , Moléculas de Patrón Molecular Asociado a Patógenos/inmunología , Moléculas de Patrón Molecular Asociado a Patógenos/metabolismo , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de SeñalRESUMEN
The mutagenic activity of transposable elements (TEs) is suppressed by epigenetic silencing and small interfering RNAs (siRNAs), especially in gametes that could transmit transposed elements to the next generation. In pollen from the model plant Arabidopsis, we show that TEs are unexpectedly reactivated and transpose, but only in the pollen vegetative nucleus, which accompanies the sperm cells but does not provide DNA to the fertilized zygote. TE expression coincides with downregulation of the heterochromatin remodeler decrease in DNA methylation 1 and of many TE siRNAs. However, 21 nucleotide siRNAs from Athila retrotransposons are generated and accumulate in pollen and sperm, suggesting that siRNA from TEs activated in the vegetative nucleus can target silencing in gametes. We propose a conserved role for reprogramming in germline companion cells, such as nurse cells in insects and vegetative nuclei in plants, to reveal intact TEs in the genome and regulate their activity in gametes.
Asunto(s)
Arabidopsis/genética , Epigénesis Genética , Polen/genética , Interferencia de ARN , Arabidopsis/metabolismo , Metilación de ADN , Elementos Transponibles de ADN , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Polen/metabolismoRESUMEN
Nitric oxide (NO) is a key signaling molecule that regulates diverse biological processes in both animals and plants, including important roles in male gamete physiology. In plants, NO is generated in pollen tubes (PTs) and affects intracellular responses through the modulation of Ca2+ signaling, actin organization, vesicle trafficking and cell wall deposition, bearing consequences in pollen-stigma interactions and PT guidance. In contrast, the NO-responsive proteins that mediate these responses remain elusive. Here, we show that PTs of Arabidopsis thaliana mutants impaired in the pollen-specific DIACYLGLYCEROL KINASE4 (DGK4) grow slower and become partially insensitive to NO-dependent growth inhibition and re-orientation responses. Recombinant DGK4 protein yields NO-responsive spectral and catalytic changes in vitro that are compatible with a role in NO perception and signaling in PTs. In addition to the expected phosphatidic acid-producing kinase activity, DGK4 recombinant protein also revealed guanylyl cyclase activity, as inferred by sequence analysis. Our results are compatible with a role for the fast-diffusible NO gas in signaling and cell-cell communication via the modulation of DGK4 activity during the progamic phase of angiosperm reproduction.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Diacilglicerol Quinasa/metabolismo , Fertilización/fisiología , Óxido Nítrico/metabolismo , Tubo Polínico/enzimología , Tubo Polínico/fisiología , Secuencia de Aminoácidos , Proteínas de Arabidopsis/química , Biocatálisis , Diacilglicerol Quinasa/química , Tubo Polínico/crecimiento & desarrolloRESUMEN
Glutamate receptors are well characterized channels that mediate cell-to-cell communication during neurotransmission in animals, but their functional role in organisms without a nervous system remains unclear. In plants, genes of the GLUTAMATE RECEPTOR-LIKE (GLR) family have been implicated in defence against pathogens, reproduction, control of stomata aperture and light signal transduction. However, the large number of GLR genes present in angiosperm genomes (20 to 70) has prevented the observation of strong phenotypes in loss-of-function mutants. Here we show that in the basal land plant Physcomitrella patens, mutation of the GLR genes GLR1 and GLR2 causes failure of sperm cells to target the female reproductive organs. In addition, we show that GLR genes encode non-selective Ca2+-permeable channels that can regulate cytoplasmic Ca2+ and are needed to induce the expression of a BELL1-like transcription factor essential for zygote development. Our work reveals functions for GLR channels in sperm chemotaxis and transcriptional regulation. Sperm chemotaxis is essential for fertilization in both animals and early land plants such as bryophytes and pteridophytes. Therefore, our results suggest that ionotropic glutamate receptors may have been conserved throughout plant evolution to mediate cell-to-cell communication during sexual reproduction.
Asunto(s)
Bryopsida/metabolismo , Quimiotaxis , Receptores Ionotrópicos de Glutamato/metabolismo , Bryopsida/embriología , Bryopsida/genética , Calcio/metabolismo , Comunicación Celular/genética , Quimiotaxis/genética , Regulación de la Expresión Génica , Genes Esenciales , Mutación , Receptores Ionotrópicos de Glutamato/genética , Reproducción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Cigoto/metabolismoRESUMEN
Whereas the role of calcium ions (Ca2+ ) in plant signaling is well studied, the physiological significance of pH-changes remains largely undefined. Here we developed CapHensor, an optimized dual-reporter for simultaneous Ca2+ and pH ratio-imaging and studied signaling events in pollen tubes (PTs), guard cells (GCs), and mesophyll cells (MCs). Monitoring spatio-temporal relationships between membrane voltage, Ca2+ - and pH-dynamics revealed interconnections previously not described. In tobacco PTs, we demonstrated Ca2+ -dynamics lag behind pH-dynamics during oscillatory growth, and pH correlates more with growth than Ca2+ . In GCs, we demonstrated abscisic acid (ABA) to initiate stomatal closure via rapid cytosolic alkalization followed by Ca2+ elevation. Preventing the alkalization blocked GC ABA-responses and even opened stomata in the presence of ABA, disclosing an important pH-dependent GC signaling node. In MCs, a flg22-induced membrane depolarization preceded Ca2+ -increases and cytosolic acidification by c. 2 min, suggesting a Ca2+ /pH-independent early pathogen signaling step. Imaging Ca2+ and pH resolved similar cytosol and nuclear signals and demonstrated flg22, but not ABA and hydrogen peroxide to initiate rapid membrane voltage-, Ca2+ - and pH-responses. We propose close interrelation in Ca2+ - and pH-signaling that is cell type- and stimulus-specific and the pH having crucial roles in regulating PT growth and stomata movement.
Asunto(s)
Calcio , Nicotiana/fisiología , Estomas de Plantas/fisiología , Transducción de Señal , Ácido Abscísico , Citosol/metabolismo , Concentración de Iones de HidrógenoRESUMEN
We investigated the molecular basis and physiological implications of anion transport during pollen tube (PT) growth in Arabidopsis thaliana (Col-0). Patch-clamp whole-cell configuration analysis of pollen grain protoplasts revealed three subpopulations of anionic currents differentially regulated by cytoplasmic calcium ([Ca2+ ]cyt ). We investigated the pollen-expressed proteins AtSLAH3, AtALMT12, AtTMEM16 and AtCCC as the putative anion transporters responsible for these currents. AtCCC-GFP was observed at the shank and AtSLAH3-GFP at the tip and shank of the PT plasma membrane. Both are likely to carry the majority of anion current at negative potentials, as extracellular anionic fluxes measured at the tip of PTs with an anion vibrating probe were significantly lower in slah3-/- and ccc-/- mutants, but unaffected in almt12-/- and tmem16-/- . We further characterised the effect of pH and GABA by patch clamp. Strong regulation by extracellular pH was observed in the wild-type, but not in tmem16-/- . Our results are compatible with AtTMEM16 functioning as an anion/H+ cotransporter and therefore, as a putative pH sensor. GABA presence: (1) inhibited the overall currents, an effect that is abrogated in the almt12-/- and (2) reduced the current in AtALMT12 transfected COS-7 cells, strongly suggesting the direct interaction of GABA with AtALMT12. Our data show that AtSLAH3 and AtCCC activity is sufficient to explain the major component of extracellular anion fluxes, and unveils a possible regulatory system linking PT growth modulation by pH, GABA, and [Ca2+ ]cyt through anionic transporters.
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Arabidopsis/metabolismo , Calcio/metabolismo , Fenómenos Electrofisiológicos , Polen/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Aniones , Arabidopsis/efectos de los fármacos , Proteínas de Arabidopsis/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Cloruros/farmacología , Fenómenos Electrofisiológicos/efectos de los fármacos , Concentración de Iones de Hidrógeno , Canales Iónicos/metabolismo , Transporte Iónico/efectos de los fármacos , Modelos Biológicos , Mutación/genética , Nitratos/farmacología , Polen/efectos de los fármacos , Tubo Polínico/efectos de los fármacos , Tubo Polínico/metabolismo , Protoplastos/efectos de los fármacos , Protoplastos/metabolismo , Simportadores/metabolismoRESUMEN
Pollen tubes (PTs) are characterized by having tip-focused cytosolic calcium ion (Ca2+ ) concentration ([Ca2+ ]cyt ) gradients, which are believed to control PT growth. However, the mechanisms by which the apical [Ca2+ ]cyt orchestrates PT growth are not well understood. Here, we aimed to identify these mechanisms by combining reverse genetics, cell biology, electrophysiology, and live-cell Ca2+ and anion imaging. We triggered Ca2+ -channel activation by applying hyperpolarizing voltage pulses and observed that the evoked [Ca2+ ]cyt increases were paralleled by high anion channel activity and a decrease in the cytosolic anion concentration at the PT tip. We confirmed a functional correlation between these patterns by showing that inhibition of Ca2+ -permeable channels eliminated the [Ca2+ ]cyt increase, resulting in the abrogation of anion channel activity via Ca2+ -dependent protein kinases (CPKs). Functional characterization of CPK and anion-channel mutants revealed a CPK2/20/6-dependent activation of SLAH3 and ALMT12/13/14 anion channels. The impaired growth phenotypes of anion channel and CPK mutants support the physiological significance of a kinase- and Ca2+ -dependent pathway to control PT growth via anion channel activation. Other than unveiling this functional link, our membrane hyperpolarization method allows for unprecedented manipulation of the [Ca2+ ]cyt gradient or oscillations in the PT tips and opens an array of opportunities for channel screenings.
Asunto(s)
Arabidopsis/crecimiento & desarrollo , Canales de Calcio/metabolismo , Calcio/metabolismo , Nicotiana/crecimiento & desarrollo , Tubo Polínico/enzimología , Tubo Polínico/crecimiento & desarrollo , Proteínas Quinasas/metabolismo , Animales , Aniones , Arabidopsis/metabolismo , Membrana Celular/metabolismo , Activación Enzimática , Activación del Canal Iónico , Oocitos/metabolismo , Nicotiana/metabolismo , XenopusRESUMEN
Animal ionotropic glutamate receptors (iGluRs) are ligand-gated channels whose evolution is intimately linked to the one of the nervous system, where the agonist glutamate and co-agonists glycine/D-serine act as neuro-transmitters or -modulators. While iGluRs are specialized in neuronal communication, plant glutamate receptor-like (GLR) homologues have evolved many plant-specific physiological functions, such as sperm signaling in moss, pollen tube growth, root meristem proliferation, innate immune and wound responses. GLRs have been associated with Ca2+ signaling by directly channeling its extracellular influx into the cytosol. Nevertheless, very limited information on functional properties of GLRs is available, and we mostly rely on structure/function data obtained for animal iGluRs to interpret experimental results obtained for plant GLRs. Yet, a deeper characterization and better understanding of plant GLRs is progressively unveiling original and different mode of functions when compared to their mammalian counterparts. Here, we review the function of plant GLRs comparing their predicted structure and physiological roles to the well-documented ones of iGluRs. We conclude that interpreting GLR function based on comparison to their animal counterparts calls for caution, especially when presuming physiological roles and mode of action for plant GLRs from comparison to iGluRs in peripheral, non-neuronal tissues.
RESUMEN
Hydroxyproline O-arabinosyltransferases (HPATs) are members of a small, deeply conserved family of plant-specific glycosyltransferases that add arabinose sugars to diverse proteins including cell wall-associated extensins and small signaling peptides. Recent genetic studies in flowering plants suggest that different HPAT homologs have been co-opted to function in diverse species-specific developmental contexts. However, nothing is known about the roles of HPATs in basal plants. We show that complete loss of HPAT function in Arabidopsis thaliana and the moss Physcomitrella patens results in a shared defect in gametophytic tip cell growth. Arabidopsis hpat1/2/3 triple knockout mutants suffer from a strong male sterility defect as a consequence of pollen tubes that fail to fully elongate following pollination. Knocking out the two HPAT genes of Physcomitrella results in larger multicellular filamentous networks due to increased elongation of protonemal tip cells. Physcomitrella hpat mutants lack cell-wall associated hydroxyproline arabinosides and can be rescued with exogenous cellulose, while global expression profiling shows that cell wall-associated genes are severely misexpressed, implicating a defect in cell wall formation during tip growth. Our findings point to a major role for HPATs in influencing cell elongation during tip growth in plants.
Asunto(s)
Arabidopsis/enzimología , Bryopsida/enzimología , Pentosiltransferasa/genética , Proteínas de Plantas/genética , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Bryopsida/genética , Bryopsida/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Hidroxiprolina/metabolismo , Pentosiltransferasa/metabolismo , Proteínas de Plantas/metabolismoRESUMEN
Oscillations in pollen tubes have been reported for many cellular processes, including growth, extracellular ion fluxes, and cytosolic ion concentrations. However, there is a shortage of quantitative methods to measure and characterize the different dynamic regimes observed. Herein, a suite of open-source computational methods and original algorithms were integrated into an automated analysis pipeline that we employed to characterize specific oscillatory signatures in pollen tubes of Arabidopsis thaliana (Col-0). Importantly, it enabled us to detect and quantify a Ca2+ spiking behaviour upon growth arrest and synchronized oscillations involving growth, extracellular H+ fluxes, and cytosolic Ca2+, providing the basis for novel hypotheses. Our computational approach includes a new tip detection method with subpixel resolution using linear regression, showing improved ability to detect oscillations when compared to currently available methods. We named this data analysis pipeline 'Computational Heuristics for Understanding Kymographs and aNalysis of Oscillations Relying on Regression and Improved Statistics', or CHUKNORRIS. It can integrate diverse data types (imaging, electrophysiology), extract quantitative and time-explicit estimates of oscillatory characteristics from isolated time series (period and amplitude) or pairs (phase relationships and delays), and evaluate their synchronization state. Here, its performance is tested with ratiometric and single channel kymographs, ion flux data, and growth rate analysis.
Asunto(s)
Arabidopsis/crecimiento & desarrollo , Botánica/métodos , Biología Computacional/métodos , Tubo Polínico/crecimiento & desarrolloRESUMEN
Apical growth in pollen tubes (PTs) is associated with the presence of tip-focused ion gradients and fluxes, implying polar localization or regulation of the underlying transporters. The molecular identity and regulation of anion transporters in PTs is unknown. Here we report a negative gradient of cytosolic anion concentration focused on the tip, in negative correlation with the cytosolic Ca(2+) concentration. We hypothesized that a possible link between these two ions is based on the presence of Ca(2+)-dependent protein kinases (CPKs). We characterized anion channels and CPK transcripts in PTs and analyzed their localization. Yellow fluorescent protein (YFP) tagging of a homolog of SLOW ANION CHANNEL-ASSOCIATED1 (SLAH3:YFP) was widespread along PTs, but, in accordance with the anion efflux, CPK2/CPK20/CPK17/CPK34:YFP fluorescence was strictly localized at the tip plasma membrane. Expression of SLAH3 with either CPK2 or CPK20 (but not CPK17/CPK34) in Xenopus laevis oocytes elicited S-type anion channel currents. Interaction of SLAH3 with CPK2/CPK20 (but not CPK17/CPK34) was confirmed by Förster-resonance energy transfer fluorescence lifetime microscopy in Arabidopsis thaliana mesophyll protoplasts and bimolecular fluorescence complementation in living PTs. Compared with wild-type PTs, slah3-1 and slah3-2 as well as cpk2-1 cpk20-2 PTs had reduced anion currents. Double mutant cpk2-1 cpk20-2 and slah3-1 PTs had reduced extracellular anion fluxes at the tip. Our studies provide evidence for a Ca(2+)-dependent CPK2/CPK20 regulation of the anion channel SLAH3 to regulate PT growth.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Canales Iónicos/metabolismo , Tubo Polínico/crecimiento & desarrollo , Proteínas Quinasas/metabolismo , Animales , Aniones/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Calcio/metabolismo , Citosol/metabolismo , Femenino , Transferencia Resonante de Energía de Fluorescencia , Canales Iónicos/genética , Células del Mesófilo/metabolismo , Mutación , Oocitos/metabolismo , Plantas Modificadas Genéticamente , Tubo Polínico/metabolismo , Proteínas Quinasas/genética , Nicotiana/genética , Nicotiana/metabolismo , Xenopus laevisRESUMEN
PREMISE OF THE STUDY: Upon pollination, dehydrated pollen grains take water out of the stigma surface, an event that constitutes the first functional checkpoint of sexual reproduction in higher plants. Little is known about possible functional connections between rehydration speed and further steps of fertilization. Here we addressed the mechanisms of water uptake control by dehydrated pollen grains. Because dehydrated cells have no energy-driven active mechanism such as membrane-based osmoregulation for controlling water movement, we tested the hypothesis that another mechanism might exist, namely, the use of hydrogel-behaving molecules. METHODS: We developed an imaging protocol to visualize and quantify the rate of water entry into pollen grains of Eucalyptus globulus and tested the influence of different treatments linked to hydrogel-behaving molecules. We complemented these analyses by immunostaining pectins in the pollen grain with monoclonal antibodies JIM5 and JIM7. KEY RESULTS: Water entry seemed to proceed exclusively through the germination apertures of the pollen grain, and the changes observed in different hydration media are compatible with hydrogel behavior. When JIM5 and JIM7 were used to characterize pectins on the germination apertures during hydration, pectin localization and esterification changed during hydration and were affected by the hydration solutions. These results suggest that chemical modification of the pectins may change their hydrogel behavior, thus modifying the hydration speed. CONCLUSIONS: The hydrogel behavior of pectins and pectin localization on apertures strongly suggest that pectins act like "valves" for water entry, enabling a regulated process of water uptake into the dehydrated pollen grain. We propose that this regulation evolved in terms of achieving the correct self-organization of molecules and cellular components to resume metabolism and pollen tube growth, especially in species that are subject to demanding environmental water stress.
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Eucalyptus/fisiología , Hidrogel de Polietilenoglicol-Dimetacrilato/farmacología , Pectinas/metabolismo , Polen/fisiología , Agua/metabolismo , Eucalyptus/efectos de los fármacos , Germinación/efectos de los fármacos , Concentración de Iones de Hidrógeno , Modelos Biológicos , Polen/efectos de los fármacos , SolventesRESUMEN
BACKGROUND: Current views on the control of cell development are anchored on the notion that phenotypes are defined by networks of transcriptional activity. The large amounts of information brought about by transcriptomics should allow the definition of these networks through the analysis of cell-specific transcriptional signatures. Here we test this principle by applying an analogue to comparative anatomy at the cellular level, searching for conserved transcriptional signatures, or conserved small gene-regulatory networks (GRNs) on root hairs (RH) and pollen tubes (PT), two filamentous apical growing cells that are a striking example of conservation of structure and function in plants. RESULTS: We developed a new method for isolation of growing and mature root hair cells, analysed their transcriptome by microarray analysis, and further compared it with pollen and other single cell transcriptomics data. Principal component analysis shows a statistical relation between the datasets of RHs and PTs which is suggestive of a common transcriptional profile pattern for the apical growing cells in a plant, with overlapping profiles and clear similarities at the level of small GTPases, vesicle-mediated transport and various specific metabolic responses. Furthermore, cis-regulatory element analysis of co-regulated genes between RHs and PTs revealed conserved binding sequences that are likely required for the expression of genes comprising the apical signature. This included a significant occurrence of motifs associated to a defined transcriptional response upon anaerobiosis. CONCLUSIONS: Our results suggest that maintaining apical growth mechanisms synchronized with energy yielding might require a combinatorial network of transcriptional regulation. We propose that this study should constitute the foundation for further genetic and physiological dissection of the mechanisms underlying apical growth of plant cells.
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Arabidopsis/metabolismo , Raíces de Plantas/metabolismo , Polen/metabolismo , Arabidopsis/crecimiento & desarrollo , Aumento de la Célula , Perfilación de la Expresión Génica , Raíces de Plantas/crecimiento & desarrollo , Polen/crecimiento & desarrollo , Regiones Promotoras Genéticas , TranscriptomaRESUMEN
Pressurized cells with strong walls make up the hydrostatic skeleton of plants. Assembly and expansion of such stressed walls depend on a family of secreted RAPID ALKALINIZATION FACTOR (RALF) peptides, which bind both a membrane receptor complex and wall-localized LEUCINE-RICH REPEAT EXTENSIN (LRXs) in a mutually exclusive way. Here we show that, in root hairs, the RALF22 peptide has a dual structural and signalling role in cell expansion. Together with LRX1, it directs the compaction of charged pectin polymers at the root hair tip into periodic circumferential rings. Free RALF22 induces the formation of a complex with LORELEI-LIKE-GPI-ANCHORED PROTEIN 1 and FERONIA, triggering adaptive cellular responses. These findings show how a peptide simultaneously functions as a structural component organizing cell wall architecture and as a feedback signalling molecule that regulates this process depending on its interaction partners. This mechanism may also underlie wall assembly and expansion in other plant cell types.
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Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/química , Arabidopsis/metabolismo , Péptidos/metabolismo , Plantas/metabolismo , Pared Celular/metabolismo , Raíces de Plantas/metabolismoAsunto(s)
Señalización del Calcio , Iones/metabolismo , Magnoliopsida/fisiología , Tubo Polínico/fisiología , Transporte Biológico , Homeostasis , Magnoliopsida/genética , Magnoliopsida/crecimiento & desarrollo , Magnoliopsida/ultraestructura , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Modelos Biológicos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tubo Polínico/genética , Tubo Polínico/crecimiento & desarrollo , Tubo Polínico/ultraestructuraRESUMEN
Using the tip-growing pollen tube of Arabidopsis thaliana and Nicotiana tabacum as a model to investigate endocytosis mechanisms, we show that phosphatidylinositol-4-phosphate 5-kinase 6 (PIP5K6) regulates clathrin-dependent endocytosis in pollen tubes. Green fluorescent protein-tagged PIP5K6 was preferentially localized to the subapical plasma membrane (PM) in pollen tubes where it apparently converts phosphatidylinositol 4-phosphate (PI4P) to phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)]. RNA interference-induced suppression of PIP5K6 expression impaired tip growth and inhibited clathrin-dependent endocytosis in pollen tubes. By contrast, PIP5K6 overexpression induced massive aggregation of the PM in pollen tube tips. This PM abnormality was apparently due to excessive clathrin-dependent membrane invagination because this defect was suppressed by the expression of a dominant-negative mutant of clathrin heavy chain. These results support a role for PI(4,5)P(2) in promoting early stages of clathrin-dependent endocytosis (i.e., membrane invagination). Interestingly, the PIP5K6 overexpression-induced PM abnormality was partially suppressed not only by the overexpression of PLC2, which breaks down PI(4,5)P(2), but also by that of PI4Kß1, which increases the pool of PI4P. Based on these observations, we propose that a proper balance between PI4P and PI(4,5)P(2) is required for clathrin-dependent endocytosis in the tip of pollen tubes.